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Temperature and pressure adaptations of substrate and coenzyme binding by M4 lactate dehydrogenase Norberg, Carol Louise 1975

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TEMPERATURE AND PRESSURE ADAPTATIONS OF SUBSTRATE AND COENZYME BINDING BY M 4 LACTATE DEHYDROGENASE by C a r o l Louise Norberg B.'Sc, U n i v e r s i t y of C a l i f o r n i a , 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY UNIVERSITY OF BRITISH COLUMBIA We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1975 In p resent ing t h i s t he s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t ha t permiss ion for ex ten s i ve copying o f t h i s t he s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r ep re sen ta t i ve s . It i s understood that copying or p u b l i c a t i o n of t h i s t he s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n permi s s ion . Department of ^oo'lc* The U n i v e r s i t y of B r i t i s h Columbia 20 75 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date J 4 f I i ABSTRACT L a c t a t e dehydrogenases from an a b y s s a l f i s h , a d o g f i s h , a t i d e p o o l s c u l p i n , and a mammal have been found to d i f f e r i n t h e i r a b i l i t y to b i n d s u b s t r a t e analog and coenzyme a t v a r y i n g temperatures and p r e s s u r e s . A f f i n i t i e s f o r a s u b s t r a t e analog are q u i t e s i m i l a r f o r each l a c t a t e dehydrogenase at t h e i r r e s p e c t i v e b i o l o g i c a l temperatures, suggesting temperature-dependent m o d i f i c a t i o n of enzyme-substrate b i n d i n g f o r o p t i m a l f u n c t i o n . B i n d i n g of coenzyme by the three ectothermic enzymes i s l e s s a f f e c t e d by changes i n temperature than i s coenzyme b i n d i n g by the mammalian enzyme, and coenzyme b i n d i n g by the a b y s s a l f i s h enzyme i s c o n s i d e r a b l y l e s s s e n s i t i v e to h i g h h y d r o s t a t i c pressure than i t i s i n the case of the other three l a c t a t e dehydrogenases. The t o t a l f r e e energy change i n v o l v e d i n b i n d i n g coenzyme and s u b s t r a t e analog i s o n l y s l i g h t l y h i g h e r f o r the endothermic than f o r the three ectothermic enzymes, but the e n t h a l p i c and e n t r o p i c c o n t r i b u t i o n s are q u i t e d i f f e r e n t . The ectotherms appear to have minimized the e n t h a l p i c c o n t r i b u t i o n and hence minimized temperature e f f e c t s on b i n d i n g . The r e l a t i o n s h i p between enthalpy and entropy f o r each of the b i n d i n g i n t e r a c t i o n s s t u d i e d i s a s t r a i g h t l i n e of slope w i t h i n the l i m i t s found by other workers f o r water-s o l u t e i n t e r a c t i o n s and/or weak bond formation and i s presumed to be a r e s u l t of the c o n f o r m a t i o n a l changes accompanying l i g a n d b i n d i n g . i i The c o n t r i b u t i o n s to b i n d i n g of the AMP and n i c o t i n a m i d e s u b s i t e s of the coenzyme b i n d i n g s i t e g i v e a good estimate of many of the b i n d i n g i n t e r a c t i o n s of the coenzyme as a whole, and appear to compensate one another t o ma i n t a i n low AH and AS val u e s f o r coenzyme b i n d i n g to the ectothermic enzymes. T h i s same type of compensation i n volume change can be seen between the s u b s t r a t e and coenzyme b i n d i n g s i t e s f o r the a b y s s a l f i s h l a c t a t e dehydrogenase, r e s u l t i n g i n a net volume change very c l o s e to zero. The observed temperature and pre s s u r e e f f e c t s on b i n d i n g cannot be e x p l a i n e d s o l e l y i n terms of the types of weak bonds i n v o l v e d , and known homologies between d o g f i s h and p i g LDH make major d i f f e r e n c e s between the a c t i v e s i t e s u n l i k e l y . Conformational changes o c c u r r i n g s i m u l t a n e o u s l y w i t h b i n d i n g may be of c o n s i d e r a b l e importance i n modifying the observed responses to both temperature and p r e s s u r e . i i i TABLE OF CONTENTS Page I. I n t r o d u c t i o n 1 I I . Methods 17 A. Experimental animals 17 B. P u r i f i c a t i o n o f LDH from white muscle of s c u l p i n and d o g f i s h 17 C. E l e c t r o p h o r e s i s of d o g f i s h and s c u l p i n LDH 24 D. P r o t e i n d e t e r m i n a t i o n 2 8 E. NADH de t e r m i n a t i o n 28 F. L a c t a t e dehydrogenase assay 29 G. I n h i b i t o r s t u d i e s 30 I I I . R e s u l t s 34 A. Oxamate b i n d i n g to the LDH-NADH b i n a r y complex 34 B. NADH b i n d i n g to LDH 34 C. AMP b i n d i n g to LDH 48 D. Nicotinamide b i n d i n g to LDH 55 IV. D i s c u s s i o n 64 A. The AMP s u b s i t e 65 B. The ni c o t i n a m i d e s u b s i t e 67 C. NADH b i n d i n g to LDH 68 D. Oxamate b i n d i n g t o the LDH-NADH b i n a r y complex 73 E. LDH-NADH-oxamate i n t e r a c t i o n s 81 i v Page F. Enthalpy-entropy compensation 84 V. C o n c l u s i o n s 90 L i t e r a t u r e C i t e d 9 3 V LIST OF TABLES Page I. P u r i f i c a t i o n of LDH from white muscle of the s c u l p i n and d o g f i s h 25 I I . Temperature and pressure e f f e c t s on oxamate i n h i b i t i o n 35 I I I . Thermodynamic parameters f o r oxamate b i n d i n g to LDH-NADH b i n a r y complex 40 I V . Temperature and pre s s u r e e f f e c t s on NADH i n h i b i t i o n 42 V . Thermodynamic parameters f o r LDH-NADH a s s o c i a t i o n 47 V I . Temperature and pre s s u r e e f f e c t s on AMP i n h i b i t i o n 49 V I I . Thermodynamic parameters f o r LDH-AMP a s s o c i a t i o n 54 V I I I . Temperature and pre s s u r e e f f e c t s on n i c o t i n a -mide i n h i b i t i o n 56 I X . Thermodynamic parameters f o r LDH-nicotinamide a s s o c i a t i o n 6 3 X . A comparison of the sum of the thermodynamic parameters a s s o c i a t e d w i t h AMP and n i c o t i n a -mide b i n d i n g w i t h those f o r NADH b i n d i n g t o M 4 LDH 69 X I . R e l a t i o n s h i p between oxamate of M^ LDH and b i o l o g i c a l temperature 79 X I I . Sum of thermodynamic parameters f o r LDH-NADH and LDH-NADH-oxamate i n t e r a c t i o n s 82 v i LIST OF FIGURES Page 1 . Schematic diagram of the LDH b i n d i n g s i t e ... 9 2 . S t r u c t u r e of l a c t a t e , pyruvate, and oxamate 1 2 3 . Competitive i n h i b i t o r s of NADH and the regi o n s of the coenzyme molecule t o which they correspond 1 4 4 . P u r i f i c a t i o n of d o g f i s h white muscle LDH by oxamate a f f i n i t y chromatography 2 0 5 . P u r i f i c a t i o n of s c u l p i n white muscle LDH by oxamate a f f i n i t y chromatography 2 2 6 . Ln oxamate versus 1/temperature 3 6 7 . Ln oxamate versus pressure 3 8 8 . L a NADH versus 1/temperature 4 3 9 . Ln NADH versus p r e s s u r e 4 5 1 0 . Ln K-L AMP versus 1/temperature 5 0 1 1 . Ln AMP versus p r e s s u r e 5 2 1 2 . L n H j versus l n n i c o t i n a m i d e c o n c e n t r a t i o n .. 5 7 1 3 . Temperature e f f e c t s on NADH b i n d i n g to LDH .. 7 0 1 4 . Pressure e f f e c t s on NADH b i n d i n g to LDH 7 4 1 5 . Temperature e f f e c t s on oxamate b i n d i n g t o LDH 7 7 1 6 . Enthalpy-entropy compensation p l o t s f o r NADH and oxamate b i n d i n g to LDH 8 6 v i i ACKNOWLEDGEMENTS Thanks go f i r s t o f a l l to my s u p e r v i s o r , Peter Hochachka, f o r p r o v i d i n g i d e a s , chemicals, and p a t i e n c e ; t o members of my committee, p a r t i c u l a r l y John G o s l i n e f o r many h e l p f u l suggestions on the manuscript; and to The Lab, f o r c r e a t i n g a c h e e r f u l environment. S p e c i a l thanks a l s o t o M i c h a e l Guppy and C h r i s French f o r d i s c u s s i o n s r e l a t e d and u n r e l a t e d to t h i s work; to Wendy C r a i k f o r c a t c h i n g s c u l p i n s ; and to Derek f o r drawing the f i g u r e s and g i v i n g the o c c a s i o n a l needed prod of encouragement. I . INTRODUCTION I t has been c l e a r f o r some time t h a t organisms must adapt to temperature and pressure a t a b i o c h e m i c a l as w e l l as a t p h y s i o l o g i c a l and h i g h e r l e v e l s of o r g a n i z a t i o n . P a r t i c u l a r l y f o r ectothermic organisms, changes i n temperature c o u l d have p o t e n t i a l l y d r a s t i c e f f e c t s on r a t e s of enzyme a c t i v i t y , a f f e c t i n g both o v e r a l l r a t e s of metabolism and i t s c o n t r o l by a l t e r i n g the r e l a t i v e a c t i v i t i e s of d i f f e r e n t enzymes. S i m i l a r l y , p r e s s u r e has been shown to i n f l u e n c e c a t a l y t i c r a t e s of many enzymes, and i s -•.undoubtedly an impor-t a n t environmental parameter to organisms l i v i n g i n the a b y s s a l r e g i o n s of the ocean f l o o r , a t p r e s s u r e s of up to s e v e r a l hundred atmospheres, as w e l l as to organisms which migrate v e r t i c a l l y i n the water column. Temperature and pressure e f f e c t s on the a c t i v i t y of s e v e r a l e ctothermic enzymes have been looked a t i n the p a s t few years (e.g., Hochachka and Somero, 1968; Hochachka e t .al, 1972; Low e t .al, 1973; Hochachka, 1975) . The b a s i c parameters of enzyme f u n c t i o n which have been of i n t e r e s t w i t h regard t o temperature are (1) the a c t i v a t i o n energy (Ea) and f r e e energy of a c t i v a t i o n (AG£), and (2) the K m, or enzyme-substrate a f f i n i t y . In terms of e v o l u t i o n a r y a d a p t a t i o n to low temperatures, i t has been suggested (e.g. Somero, 1969) t h a t lowered a c t i v a -t i o n energy may be important i n improving c a t a l y t i c e f f i c i e n c y . Large d i f f e r e n c e s between Ea of ectotherms and endotherms have been found; Low e t a l (1973) summarize data f o r l a c t a t e 2 dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and glycogen phosphorylase which show ectothermic Ea v a l u e s averaging about 5000 cal/mole below the comparable endothermic v a l u e s . The corresponding AG$ v a l u e s , however, which d i f f e r i n t h a t entropy changes are taken i n t o account, show d i f f e r e n c e s of o n l y a few hundred cal/mole. The r e l a t i v e e n t h a l p i c and e n t r o p i c c o n t r i b u t i o n s to the AGt appear to be of more s i g -n i f i c a n c e ; the AH$ i s much h i g h e r , and the Ast a s m a l l e r n e gative v a l u e f o r the endothermic than f o r the e c t o t h e r m i c enzymes. These d i f f e r e n c e s may c o n s t i t u t e temperature adap-t a t i o n s ; the advantage of a low enthalpy of a c t i v a t i o n to ectotherms i s the r e s u l t a n t temperature-independence of the r e a c t i o n . Enzyme-substrate a f f i n i t y , however, i s b e l i e v e d t o be of g r e a t e r importance under u n s a t u r a t i n g s u b s t r a t e c o n d i t i o n s i n r a t e s t a b i l i z a t i o n w i t h f l u c t u a t i n g temperatures. A d i r e c t r e l a t i o n s h i p between K m and temperature has been found f o r numerous ect o t h e r m i c enzymes (Hochachka and Somero, 19 71). The net r e s u l t of t h i s K m-temperature r e l a t i o n s h i p i s to compensate r a t e decreases due to lowered temperature by an i n c r e a s e d a f f i n i t y of the s u b s t r a t e f o r the enzyme. Somero (1969) found the minimum K m value f o r l a c t a t e dehydrogenase and pyruvate kinase i n s e v e r a l p o i k i l o t h e r m s t o correspond wi t h t h e i r minimum h a b i t a t temperature, as do the K m's f o r NAD and NADH of o c t o p i n e dehydrogenase i n the s c a l l o p ( L u i s i e t a l , 1975). These data c o n f i r m t h a t the enzyme-substrate a f f i n i t y may be m o d i f i e d a c c o r d i n g to the b i o l o g i c a l temperature of the organism. 3 Analagous parameters have been of i n t e r e s t i n s t u d y i n g the e f f e c t s of p r e s s u r e on enzyme a c t i v i t y . Under s a t u r a t i n g c o n d i t i o n s , the volume change of a c t i v a t i o n (AVt) would be r a t e - d e t e r m i n i n g . T h i s volume change i s egual to the d i f f e r e n c e between the volume of the a c t i v a t e d complex and the volume of the r e a c t a n t s , and i t i s apparent t h a t any r e a c t i o n o c c u r r i n g with a p o s i t i v e volume change w i l l be i n h i b i t e d a t h i g h p r e s s u r e s , and one o c c u r r i n g w i t h a negative volume change w i l l be a c t i v a t e d . Hence enzymes from high p r e s s u r e or v a r y i n g p r e s s u r e organisms might be expected to minimize A v f , or to have a negative A V f . T h i s has not, i n f a c t , been found; data a v a i l a b l e f o r l a c t a t e dehydrogenase and pyruvate k i n a s e a c t i v a t i o n volumes (Low and Somero, 1975) show l i t t l e d i f f e r e n c e betweem the f o r shallow-water, mid-water, and a b y s s a l f i s h . As w i t h temperature e f f e c t s , enzyme-substrate a f f i n i t y r e l a t i v e to p r e s s u r e would be expected to be of s i g n i f i c a n c e under u n s a t u r a t i n g c o n d i t i o n s , and s e v e r a l examples c o n f i r m t h i s e x p e c t a t i o n . A c e t y l t h i o c h o l i n e b i n d i n g to a c e t y l c h o l i n -e s t e r a s e i s d i s r u p t e d by p r e s s u r e i n a s u r f a c e f i s h , but pressure-enhanced i n an a b y s s a l f i s h (Hochachka, 1974); the K m f o r pyruvate i s l e s s p ressure s e n s i t i v e f o r l a c t a t e dehydrogenase from an a b y s s a l f i s h than f o r t h a t enzyme i n two s u r f a c e s p e c i e s (Low and Somero, 19 75); and the K m f o r pyruvate f o r l a c t a t e dehydrogenase from another a b y s s a l s p e c i e s has been shown to be p r e s s u r e - i n s e n s i t i v e a t low temperatures (Baldwin e t a l , 19 75). 4 Since enzyme-ligand interactions are c l e a r l y of impor-tance i n both temperature and pressure adaptation, the obvious next question concerns the molecular basis for the observed inter-species differences. This question has been considered for f i s h - b r a i n acetylcholinesterases (Hochachka, 1974) and lactate dehydrogenases from an abyssal f i s h , a mammal, and several intermediate-temperature organisms (Hochachka, 19 75; Hochachka jei a l , 1975a) . Two mechanisms of compensation of substrate binding seem plausible at present: f i r s t , an adjust-ment i n the r e l a t i v e importance of d i f f e r e n t weak bonding contributions, which must occur somewhere i n the active (but not necessarily the catalytic) s i t e ; and secondly, an adjustment i n conformational changes and/or enzyme-solute interactions, which could presumably occur anywhere i n the enzyme molecule. Weak bond types important i n enzyme-ligand binding include hydrogen bonds, ion i c bonds, and hydrophobic interactions. Model compound studies of pressure and temperature e f f e c t s on weak-bond formation form the basis for speculation on the involvement of d i f f e r e n t bonding contributions i n homologous enzymes. Hydrogen and io n i c bonds have been found to be more stable at lower temperatures, whereas hydrophobic interac-tions are des t a b i l i z e d at low temperatures. The denaturation of hydrophobic bonds i s believed to be related to increased hydrogen bond formation between water- molecules surrounding non-polar groups which normally exclude water (Brandts, 1967). Disruption of hydrophobic interactions r e s u l t s i n a large decrease i n entropy because of the increased ordering of 5 water around the exposed hydrophobic r e s i d u e s . Suzuki and T a n i g u c h i (19 72) have summarized the known enthalpy changes f o r f ormation of these bonds; A h i s negative f o r hydrogen bonds, p o s i t i v e f o r hydrophobic bonds, and may be e i t h e r p o s i t i v e or negative f o r i o n i c bonds. They a l s o g i v e the known range of the a s s o c i a t e d volume change. Hydrophobic bonds g e n e r a l l y occur w i t h a l a r g e volume i n c r e a s e (up to 23 cm3/mole a t p r e s s u r e s of 0-1000 atmospheres, which encom-passes the b i o l o g i c a l range). Hydrophobic i n t e r a c t i o n s are s t r o n g l y i n f l u e n c e d by s o l u t e c o n d i t i o n s , however. (Brandts, 1969), and Low and Somero (1975) have p o i n t e d out t h a t they may i n f a c t occur w i t h a s m a l l volume decrease depending upon the types and c o n c e n t r a t i o n s of hydrophobic groups p r e s e n t . The A V f o r hydrophobic bond formation obtained from model compound s t u d i e s assumes i n f i n i t e d i l u t i o n , which i s a p p r o x i -mated f o r p r o t e i n - l i g a n d i n t e r a c t i o n s but not n e c e s s a r i l y f o r p r o t e i n - p r o t e i n i n t e r a c t i o n s . I o n i c bonds may occur w i t h a volume i n c r e a s e of up to 26 cm^/mole, whereas hydrogen bonds occur w i t h a s m a l l volume decrease (Suzuki and T a n i g u c h i , 1972). Hence as a r u l e , i n c r e a s i n g p r e s s u r e would be expected to d i s r u p t hydrophobic and i o n i c bonds, and f a v o r hydrogen bond formation, and i n c r e a s i n g temperature would be expected to d i s r u p t hydrogen bonds and i o n i c bonds, and f a v o r hydrophobic i n t e r a c t i o n s . P r e d i c t i o n s from model compound s t u d i e s have been confirmed, as f a r as temperature i s concerned, by Hochachka's (1974) s u b s t r a t e analog s t u d i e s on a c e t y l c h o l i n e s t e r a s e . B i n d i n g by an uncharged carbon analog of a c e t y l c h o l i n e , expected to 6 i n t e r a c t w i t h t h e s u b s t r a t e s i t e p r i m a r i l y by h y d r o p h o b i c ' b o n d s , was weakened a t l o w e r t e m p e r a t u r e s t o a c o n s i d e r a b l y g r e a t e r e x t e n t t h a n was b i n d i n g by a c h a r g e d c a r b o n a n a l o g . The a f f i n i t y o f a r e l a t i v e l y s i m p l e c h a r g e d i o n ( d i m e t h y l -ammonium) , on t h e o t h e r hand, was l o w e r a t h i g h t e m p e r a t u r e s . A t any g i v e n t e m p e r a t u r e a c e t y l c h o l i n e s t e r a s e s f r o m o r g a n i s m s o f l o w e r b i o l o g i c a l t e m p e r a t u r e s (an a b y s s a l f i s h , 2 C, v e r s u s a s u r f a c e f i s h , 15-30 C, and a mammal, 37 C) bound t h e s u b s t r a t e a n a l o g more t i g h t l y . T h i s d a t a i s c o n s i s t e n t w i t h t h e h y p o t h e s i s p r e s e n t e d t h a t h y d r o p h o b i c c o n t r i b u t i o n s t o b i n d i n g have b e e n d i m i n i s h e d , and t h e c o u l o m b i c c o n t r i b u t i o n e n h a n c e d i n t h e a b y s s a l o r g a n i s m . E f f o r t s t o a t t r i b u t e o b s e r v e d v olume c h a n g e s t o t h e k i n d s o f weak bonds i n v o l v e d i n e n z y m e - l i g a n d i n t e r a c t i o n s have b e e n l e s s s u c c e s s f u l . I n t h e same s t u d y c i t e d a b ove, p r e s s u r e was shown t o i n c r e a s e t h e a f f i n i t y o f a c e t y l c h o l i n e s t e r a s e f o r t h e dimethylammonium i o n ; t h i s i s c o n t r a r y t o what w o u l d be e x p e c t e d f r o m model compound s t u d i e s , where i o n i c i n t e r a c t i o n s a r e p r e s s u r e - i n h i b i t e d . Hence e n z y m e - s u b s t r a t e i n t e r a c t i o n s a t t h e a c t i v e s i t e a r e i n s u f f i c i e n t t o e x p l a i n t h e o b s e r v e d p r e s s u r e e f f e c t s on b i n d i n g . Low and Somero (1975) have s u g g e s t e d t h a t c o n f o r m a t i o n a l c h a n g e s e l s e w h e r e i n t h e p r o t e i n o c c u r s i m u l t a n e o u s l y w i t h b i n d i n g t o p r o d u c e c o m p e n s a t o r y volume c h a n g e s . T h i s may be i n v o l v e d i n t h e c a s e o f a c e t y l -c h o l i n e s t e r a s e (Hochachka gt. a l , 19 75) . I n t h i s c o n t e x t , t h e n , t e m p e r a t u r e and p r e s s u r e e f f e c t s on t h e b i n d i n g o f coenzyme and s u b s t r a t e a n a l o g have b e e n e x a m i n e d f o r l a c t a t e d e h y d r o g e n a s e s f r o m s e v e r a l d i f f e r e n t 7 organisms l i v i n g i n v a r y i n g temperature and p r e s s u r e e n v i r o n -ments. I t was hoped t h a t a c l e a r e r i d e a might be o b t a i n e d of the r e l a t i v e importance of a l t e r a t i o n s i n the a c t i v e s i t e and elsewhere i n the p r o t e i n i n modifying responses of homologous enzymes to temperature and p r e s s u r e . L a c t a t e dehydrogenase was chosen because of the l a r g e amount of i n f o r m a t i o n which has been a c q u i r e d about i t s t h r e e - d i m e n s i o n a l s t r u c t u r e , amino a c i d sequence, s u b s t r a t e and coenzyme b i n d i n g i n t e r a c -t i o n s , and accompanying c o n f o r m a t i o n a l changes. The r e a c t i o n i t c a t a l y z e s , Pyruvate + NADH + H + \ > L a c t a t e + NAD i s important as the t e r m i n a l step i n anaerobic g l y c o l y s i s , i n gluconeogenesis, and i n the o x i d a t i o n of l a c t a t e i n a e r o b i c t i s s u e s . In v e r t e b r a t e s , i t has been found to e x i s t i n d i f f e r e n t isozymic forms, depending on the t i s s u e , w i t h i n a s i n g l e organism. The b a s i c mammalian p a t t e r n i s one of f i v e isozymes ( e x c l u d i n g the "c" gene), which have been accounted f o r on the b a s i s of two gene products H(B) and M(A) which are capable of b i n d i n g together to form a c t i v e tetramers of the form H4, H^M, H2M2, HM3, and M4. The d i s t r i -b u t i o n of these forms i s such t h a t H subunits tend to predom-i n a t e i n a e r o b i c t i s s u e s (e.g. heart) and M subunits are more numerous i n anaerobic t i s s u e s (e.g. muscle) (Markert, 19 68). The b a s i s f o r t h i s d i s t r i b u t i o n has been e x p l a i n e d i n terms of comparative r e g u l a t o r y p r o p e r t i e s of the isozymes (Everse and Kaplan, 1975). Although the mammalian isozyme p a t t e r n s are r e l a t i v e l y c o n s i s t e n t , those of f i s h vary i n the number of isozymes 8 p r e s e n t and t h e i r d i s t r i b u t i o n (Markert and Faulhaber, 1965); i n many f i s h some combinations of the two gene products i n t o tetramers are a p p a r e n t l y not p o s s i b l e even when both H and M subunits are p r e s e n t . The o r i g i n of the isozymic forms i s b e l i e v e d to be by gene d u p l i c a t i o n and subsequent divergence f o r t h e i r p a r t i c u l a r p h y s i o l o g i c a l f u n c t i o n ; c o r r e s p o n d i n g isozymes from d i f f e r e n t s p e c i e s are more a l i k e than are the H and M forms w i t h i n the same s p e c i e s (Markert e i a l , 19 75). Recent e x t e n s i v e c r y s t a l l o g r a p h i c and sequence data f o r the d o g f i s h M4 isozyme, i n a d d i t i o n to k i n e t i c and b i n d i n g s t u d i e s , have enabled the e l u c i d a t i o n of the c a t a l y t i c process of l a c t a t e dehydrogenase. The probable i n t e r a c t i o n s of the enzyme w i t h the coenzyme and s u b s t r a t e have been d e s c r i b e d by Adams e t a l (19 73) and Holbrook e t a l (19 75). These i n c l u d e s p e c i f i c weak bonds formed on l i g a n d b i n d i n g and a s s o c i a t e d c o n f o r m a t i o n a l changes. The d o g f i s h M4 enzyme has been completely sequenced (Taylo r ejfc a l , 1973) , and some pe p t i d e sequences from p i g M4 and H4 and other l a c t a t e dehydrogenases are a v a i l a b l e f o r comparison (Taylo r and Oxley, 19 75). A schematic diagram of the l a c t a t e dehydrogenase b i n d i n g s i t e i s shown i n F i g u r e 1. A s i n g l e subunit can be d i v i d e d i n t o (1) the N - t e r m i n a l 20 r e s i d u e s , i n v o l v e d i n s u b u n i t i n t e r a c t i o n s (2) r e s i d u e s 21-115, the coenzyme b i n d i n g r e g i o n , (3) the s u b s t r a t e b i n d i n g and c a t a l y t i c r e g i o n , r e s i d u e s 134-205, and (4) the c a r b o x y - t e r m i n a l r e g i o n , r e s i d u e s 253-331. I t i s apparent t h a t a good d e a l of the molecule, a p p r o x i -mately t w o - t h i r d s , i s somehow i n v o l v e d i n coenzyme and s u b s t r a t e 9 F i g u r e 1. Schematic diagram of the l a c t a t e dehydrogenase b i n d i n g s i t e ( a f t e r Holbrook e t a l , 19 75) . AMP, adenosine monophosphate; NMN, n i c o t i n -amide mononucleotide. Br, AMP B 2 NMN C SUBSTRATE 11 b i n d i n g . Region ( 2 ) , the f i r s t t h i r d , i s d i v i d e d i n t o two mononucleotide b i n d i n g r e g i o n s , and B 2; B^ i s i n v o l v e d i n AMP b i n d i n g and B 2 w i t h n i c o t i n a m i d e mononucleotide b i n d i n g . These s i m i l a r r e g i o n s may be the r e s u l t of gene d u p l i c a t i o n . A "loop" ( r e s i d u e s 9 8-114) found i n the second domain i s i n v o l v e d i n pyrophosphate b i n d i n g , and, when NADH bi n d s , f o l d s over to encl o s e the a c t i v e c e n t e r . AMP b i n d i n g may cre a t e a b i n d i n g s i t e f o r ni c o t i n a m i d e mononucleotide (McPherson, 19 70) and pyruvate w i l l b i n d o n l y to the b i n a r y complex (LDH-NADH or LDH-NAD). An o b l i g a t o r y b i n d i n g sequence has thus been e s t a b l i s h e d : adenine f i r s t binds i n a hydrophobic pocket, the phosphates are c o r r e c t l y o r i e n t e d so t h a t one of them may b i n d to a r g i n i n e 1 0 1 , c o l l a p s i n g the loop over the. a c t i v e c e n t e r ; t h i s i n c r e a s e s the number of charged and h y d r o p h i l i c groups i n the a c t i v e s i t e , so t h a t the s u b s t r a t e may b i n d , v i a one or more charge i n t e r a c t i o n s . A convenient way to study enzyme-ligand i n t e r a c t i o n s i s to use co m p e t i t i v e i n h i b i t o r s of the s u b s t r a t e and coenzyme, which i n h i b i t LDH by b i n d i n g a t the a c t i v e s i t e i t s e l f . The i n h i b i t o r s used here are: (1) oxamate, a c l o s e s t r u c t u r a l analog of pyruvate (Figure 2) which has been used i n c r y s t a l -l o g r a p h i c s t u d i e s of the t e r n a r y s t r u c t u r e of LDH; (2) AMP, which corresponds to the r e g i o n of the NADH molecule b i n d i n g to domain B]_; (3) n i c o t i n a m i d e , which corresponds t o p a r t of the n i c o t i n a m i d e mononucleotide molecule b i n d i n g a t domain B 2 (Figure 3 ) ; and (4) NADH, the tr u e coenzyme, which was used to i n h i b i t the re v e r s e r e a c t i o n ( l a c t a t e — * pyruvate) . The organisms chosen f o r comparison were three f i s h , 12 F i g u r e 2. S t r u c t u r e s o f l a c t a t e a n d p y r u v a t e , s u b s t r a t e s o f l a c t a t e d e h y d r o g e n a s e , a n d o x a m a t e , a s t r u c t u r a l a n a l o g o f p y r u v a t e . 13 14 F i g u r e 3. I n h i b i t o r s o f NADH and t h e r e g i o n s o f t h e coenzyme m o l e c u l e t o w h i c h t h e y c o r r e s p o n d . OH OK Oi- o c^-o-pfo-p-o-01 OH •CONH 2 (NICOTINAMIDE) NICOTINAMIDE ADENINE DINUCLEOTIDE (OXIDIZED) 16 Antimora r o s t r a t a f an a b y s s a l d w e l l e r ; Squalus a c a n t h i a s , the d o g f i s h ; O l i g o c o t t u s maculosus, an i n t e r t i d a l s c u l p i n . and a mammal, the ox. Antimora r o s t r a t a i s u s u a l l y found a t depths from about 800-1800 meters (although i t has been found as deep as 2900 meters), corresponding to h y d r o s t a t i c p r e s s u r e s up to 300 atmospheres and temperatures averaging 2 C (Iwamoto, 1975). The d o g f i s h used here i s the same s p e c i e s as t h a t f o r which LDH has been sequenced and c r y s t a l l i z e d , although i t i s from the P a c i f i c r a t h e r than the A t l a n t i c . I t may be found anywhere between the s u r f a c e and 950 meters, e x p e r i e n c i n g p r e s s u r e s up to 100 atmospheres and temperatures of 6-15 C. (Perlmotter, 1961). O l i g o c o t t u s maculosus p r e f e r s r e l a t i v e l y h i g h t i d e p o o l s , which are s u b j e c t t o l a r g e thermal f l u c t u a t i o n s (Nakamura, 19 70). The mean temperature of these t i d e p o o l s on the west c o a s t of Vancouver I s l a n d , where these s c u l p i n s were obtained, ranges from 5 to 20 C throughout the year, and the a b s o l u t e temperature may go from very c l o s e t o 0 C to 25 C. Although t h i s s p e c i e s i s able to t o l e r a t e 25 C f o r a c o n s i d e r a b l e l e n g t h of time when t r a n s f e r r e d from 10 C (Nakamura, 1970), i t seems to p r e f e r c o o l e r temperatures and migrates to the bottom of the t i d e p o o l when s u r f a c e temperatures are h i g h (Green, 19 67). The mammalian enzyme was used f o r comparison because of i t s h i g h and s t a b l e (37 C) body temperature and low environmental p r e s s u r e . 17 I I . METHODS A. Experimental animals Muscle LDH from four organisms was used i n t h i s study. Squalus a c a n t h i a s , p r o v i d e d by Dr. Don C l a r k of the Chemistry Department was caught l o c a l l y . O l i g o c o t t u s maculosus was captured i n t i d e p o o l s i n the v i c i n i t y of Ba m f i e l d Marine S t a t i o n and t r a n s p o r t e d f r o z e n t o Vancouver. L a c t a t e dehy-drogenase from Antimora r o s t r a t a , caught o f f the Kona coa s t of Hawaii from a depth of approximately 1100 fathoms, was prov i d e d a l r e a d y p u r i f i e d (Baldwin e£ aJL, 1975) . Beef M^ LDH was obtained from Sigma Chemical Company. B. P u r i f i c a t i o n of LDH from white muscle of s c u l p i n and d o g f i s h P u r i f i c a t i o n was performed by use of a f f i n i t y chromato-graphy. Sepharose 4B s u b s t i t u t e d w i t h aminohexyl groups and coupled to o x a l a t e , producing a d e r i v a t i v e of oxamate, was prov i d e d by Uwe Borgmann. A d e s c r i p t i o n of t h i s s y n t h e s i s i s g i v e n by Cuatrecasas. " (1970) . The method used here was d e v i s e d by O'Carra and Barry (1972) and i s s p e c i f i c enough t h a t s e p a r a t i o n of LDH isozymes wi t h s l i g h t l y v a r y i n g a f f i n i t i e s i s p o s s i b l e (Spielmann .e_t a l , 1973). LDH w i l l not b i n d to the column i n the absence of NADH (as a r e s u l t of the o b l i g a t o r y b i n d i n g sequence of pyruvate, or i n t h i s case oxamate, to the b i n a r y complex). LDH does b i n d s t r o n g l y to the column i n the presence of .2 mM NADH (lower c o n c e n t r a t i o n s have been used elsewhere) even when .5 M NaCI i s p r e s e n t . These p r o p e r t i e s of LDH 18 have enabled the f o l l o w i n g procedure f o r a one-step p u r i f i -c a t i o n to be used: the homogenate, i n the presence of .2 mM NADH and .5 M NaCI i s a p p l i e d to the column and the column washed wi t h s e v e r a l volumes of the buffer-salt-NADH mixture. The presence of NADH ensures t h a t LDH w i l l b i n d , and the high s a l t c o n c e n t r a t i o n prevents n o n - s p e c i f i c b i n d i n g of other p r o t e i n s to the column. Once LDH i s bound and the other p r o t e i n s removed, NADH i s omitted from the washing s o l u t i o n , and i n the presence of b u f f e r and s a l t alone the bound LDH comes o f f the column. P r i o r t o a p p l i c a t i o n of the sample to the column, white muscle i n b u f f e r a t a r a t i o of approximately 1:5 (weight/ volume) was ground i n a mortar and p e s t l e and c e n t r i f u g e d f o r ten minutes a t 10,000 RPM. The p r e c i p i t a t e was d i s c a r d e d , and the supernatant made 30% i n Ammonium S u l f a t e , f o l l o w e d by c e n t r i f u g a t i o n f o r 20 minutes a t 12,000 RPM. Again the p r e c i p i t a t e was d i s c a r d e d (assay of the p r e c i p i t a t e showed ver y low l e v e l s of LDH a c t i v i t y ) and the supernatant made 70% i n Ammonium S u l f a t e . C e n t r i f u g a t i o n a t 12,000 RPM f o r 20 minutes was repeated, and t h i s time the p r e c i p i t a t e was r e t a i n e d and the supernatant, w i t h l i t t l e or no LDH a c t i v i t y , d i s c a r d e d . The p u r i f i c a t i o n o b t a i n e d by t h i s procedure was s m a l l , but i t d i d serve to b r i n g the homogenate down to a sma l l e r volume r e q u i r e d f o r a p p l i c a t i o n t o the column. The Ammonium S u l f a t e p r e c i p i t a t e was d i a l y z e d twice f o r one hour a g a i n s t one l i t e r of 100 mM T r i s - H C l pH 7.5. Approximately 5 ml of the oxamate-sepharose column was suspended i n 100 mM T r i s HC1 b u f f e r , pH 7.5, i n a 5 ml g l a s s 19 s y r i n g e . The column was then e q u i l i b r a t e d w i t h .5 M NaCI and .2 mM NADH i n the same b u f f e r . A f t e r s e v e r a l volumes had passed through, the column was checked f o r e q u i l i b r a t i o n by measuring c o n d u c t i v i t y and o p t i c a l d e n s i t y a t 340 nm (the a b s o r p t i o n peak f o r NADH) of the e l u e n t t o see t h a t they corresponded to t h a t of the o r i g i n a l wash added t o the column. Up to 1 ml of the homogenate, prepared as d e s c r i b e d above, was a p p l i e d t o the column by means of a pasteur p i p e t t e , t a k i n g care not to d i s r u p t the s u r f a c e . In some cases, a s l i g h t d i l u t i o n of the homogenate was found to be necessary s i n c e high p r o t e i n c o n c e n t r a t i o n s appeared to impede the b i n d i n g of the LDH to the column. The s i d e s of the column were washed twice w i t h the Tris-NaCl-NADH mixture, a f t e r which the column was connected t o the r e s e r v o i r and f o u r to f i v e column volumes were allowed to run through. A LKB perspex p e r i s t a l t i c pump and LKB u l t r o r a c f r a c t i o n c o l l e c t o r were used to c o l l e c t the f r a c t i o n s , which were u s u a l l y 2 ml. The r e s e r v o i r s o l u t i o n was then changed t o T r i s - N a C l , and another f o u r to f i v e column volumes allowed t o pass through. The c o l l e c t e d f r a c t i o n s were then assayed f o r p r o t e i n , LDH a c t i v i t y , and NADH as d e s c r i b e d below. F i g u r e s 4 and 5 show the e l u t i o n p a t t e r n of p r o t e i n and LDH o f f the oxamate column f o r d o g f i s h and s c u l p i n r e s p e c t i v e l y . I t can be seen t h a t the p r o t e i n came o f f the column over a f a i r l y wide range w i t h i n the f i r s t two column volumes of buffer-NADH-NaCl. In the case of the d o g f i s h LDH p u r i f i c a t i o n , a second, s m a l l e r peak of p r o t e i n came o f f a f t e r the f i r s t . T h i s might be due to d i f f e r e n t i a l e x c l u s i o n of p r o t e i n s of 20 Figure 4. P u r i f i c a t i o n of dogfish white muscle l ac ta te dehydrogenase by oxamate a f f i n i t y chromatography. 2 4 6 8 10 12 14 16 FRACTION NUMBER 22 Figure 5. P u r i f i c a t i o n of scu lp in white muscle lactate dehydrogenase by oxamate a f f i n i t y chromatography. NADH (mM) 23 ro rv) —UL LDH ACTIVITY fcOD./min/ml)* O 00 co . (|W/6uu) NBlOdd 24 d i f f e r e n t s i z e s from the pores i n the sepharose to which the oxamate i s l i n k e d , or perhaps to some n o n - s p e c i f i c b i n d i n g , although t h i s seems u n l i k e l y i n the h i g h NaCI con-c e n t r a t i o n s p r e s e n t . High p r o t e i n c o n c e n t r a t i o n s were found to i n t e r f e r e w i t h LDH b i n d i n g to the column, and the p r o t e i n c o n c e n t r a t i o n i n the d o g f i s h muscle homogenate was c o n s i d e r -a b l y higher than t h a t of the s c u l p i n muscle. In both cases a d e f i n i t e peak of LDH a c t i v i t y occurs i n a s i n g l e f r a c t i o n , w i t h one to two other f r a c t i o n s having a lower amount of a c t i v i t y . A l s o , the peak of LDH a c t i v i t y corresponds w e l l w i t h the drop i n NADH c o n c e n t r a t i o n , making i t c l e a r t h a t a NADH c o n c e n t r a t i o n of g r e a t e r than .1 mM i s e s s e n t i a l f o r the b i n d i n g of these LDH 1s t o the oxamate. Table I shows the p u r i f i c a t i o n , y i e l d , and s p e c i f i c a c t i v i t y o b tained f o r each of the enzymes. A l l of these were c o n s i d e r a b l y h i g h e r f o r the d o g f i s h than the s c u l p i n LDH. A sm a l l amount of LDH d i d come o f f wi t h the i n i t i a l p r o t e i n peak i n the case of the s c u l p i n , but t h i s amount of a c t i v i t y i s not s u f f i c i e n t t o account f o r the d i f f e r e n c e s . C. E l e c t r o p h o r e s i s of d o g f i s h and s c u l p i n LDH St a r c h g e l e l e c t r o p h o r e s i s was performed by m o d i f i c a t i o n s of the method used by Markert and Faulhaber (1965) u s i n g a v e r t i c a l g e l apparatus. A Tris-Borate-EDTA b u f f e r (0.9 M T r i s , 0.5 M B o r i c A c i d , .02 M EDTA, pH 8.7) was d i l u t e d 1:20 to make a 12-13% s t a r c h s o l u t i o n (Connaught, T o r o n t o ) . The same b u f f e r was used i n a 1:7 d i l u t i o n i n the lower, anodal in Table I. P u r i f i c a t i o n of LDH from white muscle of the s c u l p i n and d o g f i s h . S c u l p i n D o g f i s h S p e c i f i c S p e c i f i c A c t i v i t y Y i e l d P u r i f i c a t i o n A c t i v i t y Y i e l d P u r i f i c a t i o n (jumoles/min/mg) (%) (pmoles/min/mg) (%) Crude Homogenate 2.2 100 l x 3.8 100 l x Ammonium S u l f a t e 5.1 91 2.3x 5.3 82 1.4x OXclITlcl t G Column 180 67 82x 1565 82 412x 26 chamber and a 1:5 d i l u t i o n i n the upper, c a t h o d a l chamber. Samples were a p p l i e d by means of a pasteur p i p e t t e , and s e a l e d i n t o the g e l s l o t s w i t h melted v a s o l i n e . The g e l was covered w i t h a sheet of p l a s t i c wrap, and run i n the coldroom (around 5 C) w i t h the v o l t a g e r e g u l a t e d a t 400 V (amperage was u s u a l l y about 35 mA) f o r 3-5 hours. The g e l was then removed from the t r a y , s l i c e d i n two and incubated i n the dark w i t h the i n n e r s u r f a c e up f o r a t l e a s t 30 minutes w i t h the f o l l o w i n g s t a i n : T r i s - H C l , 0.1 M, pH 8 50 ml L a c t a t e , 85% 0.5 ml N i t r o Blue T e t r a z o l i u m 15 mg Phenazine M e t h o s u l f a t e 1 mg NAD 25 mg When s t a i n i n g was complete, the g e l was washed w i t h a mixture of a c e t i c acid-ethanol-water and the p o s i t i o n s of the bands i n d i c a t i n g a c t i v i t y noted. Both the p u r i f i e d samples of LDH from the oxamate column and the crude homogenates of s e v e r a l t i s s u e s (white muscle, red muscle, h e a r t , l i v e r ) , which were g e n e r a l l y simply homogenized i n approximately 10 volumes of b u f f e r and c e n t r i -fuged when l a r g e enough, were run on s t a r c h g e l s . A n a l y s i s of d o g f i s h t i s s u e s done p r e v i o u s l y (Markert e t a l , 1975) has shown the presence of a l l f i v e p o s s i b l e isozymes formed from the H and M s u b u n i t s , w i t h the H4 m i g r a t i n g f u r t h e r a n o d a l l y than the M4. Under the c o n d i t i o n s 27 used here, a t o t a l of a t l e a s t f o u r isozymes were c l e a r l y v i s i b l e i n the v a r i o u s t i s s u e s looked a t ; some s t r e a k i n e s s may have obscured the f i f t h . Homogenized white muscle showed a very i n t e n s e band a t approximately 0.8 cm towards the cathode, and some a c t i v i t y a t 0.2-0.3 cm towards the cathode. The LDH p u r i f i e d from t h i s homogenate showed onl y the 0.8 cm band, which i s b e l i e v e d t o r e p r e s e n t the M 4 isozyme. L i v e r c o ntained two c l e a r bands, at approximately 0.2 and 0.9 cm towards the cathode. Heart and red muscle had these same two bands and i n a d d i t i o n , a l a r g e band a t approximately 1.4 cm towards the anode. The h e a r t a l s o had a f o u r t h band m i g r a t i n g about 0.2 cm a n o d a l l y . Since l i v e r i n f i s h u s u a l l y c o n t a i n s the M 4 isozyme, and the H 4 isozyme i n d o g f i s h i s known to migrate f u r t h e r a n o d a l l y than the M 4 , i t was concluded t h a t the f u r t h e s t c a t h o d a l l y - m i g r a t i n g band found i n the p u r i f i e d LDH was . Two bands of a c t i v i t y were found i n the s c u l p i n muscle, one ( f a i n t ) m i g r a t i n g about 0.3 cm a n o d a l l y , and a darker band 2.2 cm towards the anode. P u r i f i e d f r a c t i o n s showed only the l a t t e r band. L i v e r showed the 0.3 cm band o n l y , and h e a r t o n l y a band at 2.0 cm. T h i s i s not the more u s u a l case i n which l i v e r and muscle isozymes are s i m i l a r , and d i f f e r e n t from those of the h e a r t . However, Markert e t a l (19 75) have summarized a v a i l a b l e data on isozymic p a t t e r n s of LDH i n f i s h , and found t h a t a l l members of the orders P l e u r o n e c t i f o r m e s and T e t r a o d o n t i f o r m e s , and s e v e r a l s p e c i e s i n the order Perciformes show reduced a c t i v i t y of 28 the H gene. In a d d i t i o n , the u s u a l order of m i g r a t i o n i s re v e r s e d f o r some P e r c i f o r m e s , w i t h the M4 band m i g r a t i n g f u r t h e r a n o d a l l y than the H4 band. The s c u l p i n belongs to the order P e r c i f o r m e s , and i n t h i s c ontext the observed isozymic p a t t e r n seems p l a u s i b l e . A t e n t a t i v e e x p l a n a t i o n i s t h a t the M4 isozyme predominates i n both h e a r t and muscle and migrates a n o d a l l y r e l a t i v e t o the H4 form. That t h i s i s i n f a c t the case would r e q u i r e more c a r e f u l i d e n t i f i c a t i o n of the H4 and M4 forms, s i n c e i t remains a p o s s i b i l i t y t h a t the H4 LDH i s the major isozyme, although t h i s has not been found f o r any r e l a t e d f i s h . In s p i t e of the f a c t t h a t the s c u l p i n LDH isozyme used i n t h i s study i s not d e f i n i t e l y i d e n t i f i e d as the M 4 form, i t i s c e r t a i n l y the predominant muscle isozyme and comparisons can be made on t h a t b a s i s w i t h the M4 isozymes from the other s p e c i e s . D. P r o t e i n Determination P r o t e i n was determined u s i n g the Lowry method (Layne, 1957) . The OD-ygQ obtained was compared t o t h a t of standard s o l u t i o n s of Bovine Serum Albumin, which were found to g i v e a l i n e a r r e l a t i o n s h i p w i t h c o n c e n t r a t i o n i n the range of .05-1.0 mg/ml p r o t e i n . E. NADH Determination The amount of NADH presen t was determined by measuring the OD34Q and u s i n g the formula (Lowry and Passonneau, 19 72): 29 C o n c e n t r a t i o n (mM) = P.P. 6.27 (molar e x t i n c t i o n c o e f f i c i e n t of NADH = 6270 a t 340 nm). F. L a c t a t e Dehydrogenase Assay LDH was assayed s p e c t r o p h o t o m e t r i c a l l y u s i n g a Unicam SP 1800 r e c o r d i n g spectrophotometer and f o l l o w i n g the change i n o p t i c a l d e n s i t y a t 340 nm due to NADH o x i d a t i o n (or NAD r e d u c t i o n when the r e a c t i o n was c a r r i e d out i n the r e v e r s e d i r e c t i o n ) . A c t i v i t y was g e n e r a l l y recorded i n AO.D./min, which can be expressed as mmoles/min of s u b s t r a t e converted by u s i n g the above formula. S p e c i f i c a c t i v i t y i s g i v e n i n jajnoles/min/mg p r o t e i n . Most experiments were performed i n 1 ml cu v e t t e s of 1 cm l i g h t path, except f o r those under v a r y i n g p r e s s u r e s , i n which case the volume was 5 ml and the l i g h t path 1 cm. Temperature was c o n t r o l l e d by a Lauda constant temperature bath and c i r c u l a t o r and measured immediately p r i o r to assay by a Model 46 TUC Tele-thermometer (Yellow Springs Instruments). For experiments under v a r y i n g h y d r o s t a t i c p r e s s u r e s , a high p r e s s u r e c e l l i n c o r p o r a t e d i n t o an SP 1800 spectrophotometer was used. A l l assays were performed i n 100 mM T r i s - H C l , pH 7.5, which was ad j u s t e d a c c o r d i n g to temperature t o m a i n t a i n a constant pH. No adjustment of pH f o r pre s s u r e was made, but t h i s i s not b e l i e v e d to be a problem. Phosphate b u f f e r , pH 7 a t atmospheric p r e s s u r e , w i l l drop 0.4 pH u n i t s a t 10,000 PSI (Johnson e t a l , 19 74) and the volume change of i o n i z a t i o n of T r i s i s l e s s than t h a t of phosphate b u f f e r 30 (Disteche, 19 72). C o n c e n t r a t i o n s of s u b s t r a t e and coenzyme used i n the assay v a r i e d depending on the p a r t i c u l a r i n h i b i t o r b e i n g s t u d i e d , but u s u a l l y ranged from 0.5-1 mM pyruvate and .025-0.2 mM NADH, and i n the r e v e r s e d i r e c t i o n 10 mM l a c t a t e and 1-4 mM NAD. Reagents and i n h i b i t o r s used were from Sigma Chemical Company. G . I n h i b i t o r S t u d i e s I n h i b i t i o n of LDH i n the pyruvate-»lactate d i r e c t i o n by oxamate, AMP and ni c o t i n a m i d e and i n the lactate-*pyruvate d i r e c t i o n by NADH was s t u d i e d under v a r y i n g temperatures and pr e s s u r e s . A t l e a s t s i x i n h i b i t o r c o n c e n t r a t i o n s and, with the e x c e p t i o n of n i c o t i n a m i d e , two or three competing s u b s t r a t e c o n c e n t r a t i o n s were used. In the case o f oxamate, AMP, and NADH, i n h i b i t i o n was found t o be co m p e t i t i v e a c c o r d i n g to Dixon p l o t s . The i n h i b i t o r d i s s o c i a t i o n c onstants were determined by p l o t t i n g 1 / v e l o c i t y versus i n h i b i t o r c o n c e n t r a t i o n f o r competing s u b s t r a t e c o n c e n t r a t i o n s ; t h i s y i e l d e d i n t e r s e c t i n g l i n e s (one f o r each s u b s t r a t e c o n c e n t r a t i o n ) , the slopes of which were determined by r e g r e s s i o n a n a l y s i s . The i n t e r s e c t i o n p o i n t was then c a l c u l a t e d , .the x value of which corresponds to -K\ (Webb, 1963) . The K i ' s determined r e p r e s e n t the d i s s o c i a t i o n constant of the i n h i b i t o r from the en z y m e - i n h i b i t o r complex: K ± = (E) (I) (EI) The f r e e energy of d i s s o c i a t i o n , A G , can be determined f o r 31 a g i v e n temperature by u s i n g the e q u a t i o n AG = RT l n K. where R, the gas constant, = 1.9 8 cal/mole/deg and T i s the a b s o l u t e temperature, i n degrees K e l v i n . Determination of the enthalpy of d i s s o c i a t i o n , A H , r e q u i r e s versus temperature data, u s i n g the equation AH = -(R) (3ln K i/S.(l/T) ) . T h i s was taken as the o p p o s i t e of the slope of l n K-^  v e rsus 1/T (T i n degrees K e l v i n ) , determined by r e g r e s s i o n a n a l y s i s , m u l t i p l i e d by R. Once AG and AH are known, AS can be c a l c u l a t e d from A S = (AH - AG) /T. Volume change, AV, of d i s s o c i a t i o n , was found by p l o t t i n g l n K^ a g a i n s t p r e s s u r e , i n an analagous manner to AH determina-t i o n . A V = -RT ( l n K i ( p i ) - In p 2 " ?! where R i s the gas constant (82.07 cm^/mole), T i s the temperature i n degrees K e l v i n , and K^p.^-' and Kj_ ^ p ^ are the i n h i b i t o r d i s s o c i a t i o n constants a t p r e s s u r e s P2 and P l atmospheres. The f r e e energy, enthalpy, entropy and volume change of a s s o c i a t i o n f o r each of the i n h i b i t o r s was taken as the o p p o s i t e of each of those parameters c a l c u l a t e d f o r the d i s s o c i a t i o n c o n s t a n t . A note should be made here on the r e l a t i v e accuracy of the k i n e t i c and thermodynamic data o b t a i n e d . Since 32 experiments were not r e p l i c a t e d , s t a t i s t i c a l d e t e r m i n a t i o n of s i g n i f i c a n c e c o u l d not be made. However, the and A G v a l u e s were determined d i r e c t l y by p l o t t i n g the raw data, and are b e l i e v e d to be more accurate than the other thermo-dynamic parameters, which were c a l c u l a t e d from a r e p l o t t i n g of the Kj_ v a l u e s . For t h i s reason, the l a t t e r are rounded o f f t o the n e a r e s t whole number, whereas A G and are g i v e n to one decimal p l a c e . Nicotinamide i n h i b i t i o n was more d i f f i c u l t t o a n a l y z e . In s p i t e of the f a c t t h a t i t r e p r e s e n t s a p o r t i o n of the coenzyme molecule, i n h i b i t i o n a c c o r d i n g to Dixon p l o t s was not c o m p e t i t i v e and was n o n - l i n e a r , i n h i b i t i o n becoming p r o p o r t i o n a t e l y g r e a t e r a t h i g h e r n i c o t i n a m i d e c o n c e n t r a t i o n s . I n i t i a l l y I ^ Q ' S , the c o n c e n t r a t i o n r e q u i r e d to produce 50% of the u n i n h i b i t e d a c t i v i t y , were determined. T h i s gave some i n d i c a t i o n of the r e l a t i v e a f f i n i t i e s of the enzymes f o r i n h i b i t o r a t d i f f e r e n t p r e s s u r e s and temperatures, but was not u s e f u l f o r the d e t e r m i n a t i o n of thermodynamic c h a r a c t e r i s t i c s . The data was then p l o t t e d a c c o r d i n g to Johnson e t a l (1974) u s i n g the i n h i b i t i o n c onstant T^: f ] _ = r a t e i n absence of i n h i b i t o r - 1 r a t e i n presence of i n h i b i t o r A l i n e a r r e l a t i o n s h i p between logPi and l o g X, where X i s the molar c o n c e n t r a t i o n of i n h i b i t o r , i s i n d i c a t i v e of a r e v e r s i b l e i n h i b i t i o n and was o b t a i n e d . The f r e e energy of d i s s o c i a t i o n can then be found: l n r x = r l n (X) - AG . RT where r , the r a t i o of i n h i b i t o r molecules to enzyme molecules, 33 i s t h e s l o p e o f lnr± v e r s u s I n X. The e n t h a l p y o f d i s s o c i a t i o n c a n be d e t e r m i n e d b y p l o t t i n g l n T i a g a i n s t l n 1/T, AH = (R) ( l n f V I n (1/T) ) . E n t r o p y c a n t h e n be c a l c u l a t e d f r o m t h e known f r e e e n e r g y and e n t h a l p y o f d i s s o c i a t i o n . Volume change c a n be d e t e r m i n e d by a p l o t o f lnT]_ v e r s u s p r e s s u r e , AV = RT d n r i ( p i ) - l n T 1 ( p 2 ) ) P 2 - P Thermodynamic p a r a m e t e r s c a l c u l a t e d by t h i s method f o r t h e o t h e r i n h i b i t o r s c o r r e s p o n d e d c l o s e l y i n t h e c a s e o f A G w i t h t h e v a l u e s o b t a i n e d f r o m D i x o n p l o t s , b u t l e s s w e l l i n t h e c a s e o f AH, A S, a n d A V . A g a i n , t h e A G v a l u e s a r e t a k e n t o be more a c c u r a t e f o r t h e n i c o t i n a m i d e d a t a t h a n a r e t h e o t h e r t h e rmodynamic v a l u e s . 34 I I I . RESULTS A. Oxamate b i n d i n g to the LDH-NADH b i n a r y complex The e f f e c t s of temperature and pressure on the oxamate f o r the fou r l a c t a t e dehyrogenases s t u d i e d are shown i n Table I I . In a l l cases the i n c r e a s e d w i t h temperature, t h a t i s , the a f f i n i t y f o r oxamate decreased. The g r e a t e s t change i n a f f i n i t y was found f o r the beef LDH, the l e a s t f o r Antimora, w i t h s c u l p i n and d o g f i s h f a l l i n g i n the middle. Pressure had v i r t u a l l y no e f f e c t on the (25 C) oxamate f o r Antimora and d o g f i s h LDH, but i n c r e a s e d the s l i g h t l y f o r the s c u l p i n and to a g r e a t e r e x t e n t f o r the beef enzyme. F i g u r e s 6 and 7 show the r e l a t i o n s h i p between l n R\ and 1/temperature and pressure used f o r the c a l c u l a t i o n of the AH and A V of a s s o c i a t i o n . Table I I I g i v e s the c a l c u l a t e d thermodynamic parameters f o r the oxamate i n t e r a c t i o n w i t h the LDH-NADH b i n a r y complex. The f r e e e n e r g i e s of a s s o c i a t i o n are q u i t e s i m i l a r , the A G f o r beef LDH being o n l y s l i g h t l y l a r g e r than the o t h e r s . The enthalpy and entropy of a s s o c i a -t i o n , however, are q u i t e d i f f e r e n t and are more h i g h l y n e g a t i v e f o r the mammalian enzyme than f o r Antimora, d o g f i s h , and s c u l p i n LDH. The volume change i s low and p o s i t i v e f o r the l a t t e r t h r e e , and hig h and p o s i t i v e f o r the beef enzyme. B. NADH b i n d i n g to LDH Temperature and pressure e f f e c t s on the Kj_ NADH are 35 Table I I . Temperature and pr e s s u r e e f f e c t s on oxamate i n h i -b i t i o n . K i I s , i n mMoles, were determined by Dixon p l o t s . C o n c e n t r a t i o n s of r e a c t a n t s used were: 0.1-0.2 vate , 0-2.0 mM a t 25 C. mM NADH, 0.5, oxamate. A l l 0.7 and pre s s u r e 1.0 mM pyru-data o b t a i n e d Temperature K i (C) Antimora-'- D o g f i s h S c u l p i n B e e f 1 7 .22 10 .13 .11 15 .27 .15 .12 .025 25 .34 .18 .18 .08 35 .34 .28 38 .63 .18 45 .67 .57 Pressure (PSI) Antimora-'-K i D o g f i s h S c u l p i n B e e f 1 14.7 .34 .18 .18 .08 4000 .17 .20 5000 .32 .17 8000 .16 .23 10000 .35 .28 1. From Hochachka, 19 75. 36 F i g u r e 6. L n K i oxamate f o r A n t i m o r a , d o g f i s h , s c u l p i n and b e e f M 4 LDH v e r s u s 1 / t e m p e r a t u r e ( i n d e g r e e s K e l v i n ) . S l o p e s a r e p r o p o r t i o n a l t o A H. 38 F i g u r e 7. Ln K, oxamate f o r Antimora, d o g f i s h , s c u l p i n and beef M4 LDH versus p r e s s u r e ( i n atmospheres). Slopes are p r o p o r t i o n a l t o AV. 39 40 Table I I I . Thermodynamic parameters f o r oxamate b i n d i n g to LDH-NADH b i n a r y complex. , A S , and A V v a l u e s o btained a t 25 C. Source of AG AH AS AV LDH (kcal/mole) (kcal/mole) (cal/mole-deg) (cm^/mole) Antimora -4.7 -6 -4 10 Do g f i s h -5.1 -9 -12 -5 S c u l p i n -5.1 -8 -11 11 Beef -5.6 -15 -32 46 41 shown i n Tabl e IV. A f f i n i t y f o r NADH decreased w i t h i n c r e a s i n g temperature f o r a l l f o u r enzymes, and wit h i n c r e a s i n g p r essure (25 C) except i n the case of Antimora, where the R\ was l i t t l e a f f e c t e d . The beef LDH-NADH b i n d i n g i n t e r a c t i o n was most i n f l u e n c e d by temperature and p r e s s u r e . The K-L f o r the d o g f i s h enzyme changed s l i g h t l y more wi t h temperature than those of the other two f i s h . With regard t o p r e s s u r e , the s c u l p i n and d o g f i s h LDH 1s were i n t e r m e d i a t e i n t h e i r responses, between the beef and Antimora enzymes. F i g u r e s 8 and 9 g i v e the p l o t s of l n versus 1/temper-atu r e and pre s s u r e used to determine AH and A V r e s p e c t i v e l y . T able V summarizes the thermodynamic parameters f o r LDH-NADH a s s o c i a t i o n . A G ' s were again s i m i l a r f o r a l l f o u r enzymes, whereas the AH's and A S ' s were more h i g h l y n e g a t i v e f o r beef than f o r d o g f i s h , s c u l p i n , and Antimora LDH. In f a c t , a s l i g h t p o s i t i v e A S of a s s o c i a t i o n was found f o r the l a t t e r two enzymes. L u i s i e t a l (1975) have a l s o found a p o s i t i v e entropy change on b i n d i n g of NADH to octo p i n e dehydrogenase i n the s c a l l o p . The A V ranged from a s m a l l n e g a t i v e f o r Antimora, t o p r o g r e s s i v e l y higher v a l u e s f o r d o g f i s h , s c u l p i n , and beef LDH. A few comparable values of NADH b i n d i n g to LDH are a v a i l a b l e i n the l i t e r a t u r e . S t i n s o n and Holbrook (19 73) determined a K D f o r NADH of 1.4-2.0 uM f o r the ox enzyme, and 3.6 pM f o r the d o g f i s h enzyme, u s i n g d i r e c t f l o u r e s c e n t measurements of NADH b i n d i n g . Although these v a l u e s are lower than those obtained here f o r the NADH, the r a t i o s 42 Table IV. Temperature and pre s s u r e e f f e c t s on NADH i n h i b i t i o n . K-j^'s, in-pmoles, were determined by Dixon p l o t s . C o n c e n t r a t i o n s of r e a c t a n t s used were: 10 mM l a c t a t e , 1.0, 2.0, and 4.0 mM NAD, 0-30 juM NADH. A l l p r e ssure data were obtained a t 25 C. Temperature (C) Antimora D o g f i s h S c u l p i n Beef 15 4.0 5.7 5.3 2.5 25 6.0 11.8 6.4 4.6 35 7.5 19.2 7.0 13.7 45 10.0 28.5 11.4 27.8 Pressure (PSI) Antimora D o g f i s h S c u l p i n Beef 14.7 4.5 11.8 6.4 7.0 6000 3.8 15.2 13.3 15.0 10000 4.0 26.0 19.5 44.0 43 F i g u r e 8. Ln K.^  NADH f o r Antimora, d o g f i s h , s c u l p i n , and beef M4 LDH versus 1/temperature ( i n degrees K e l v i n ) . Slopes are p r o p o r t i o n a l to AH. 45 Figure 9. Ln K i NADH for Antimora, dogfish, sculpin, and beef M4 LDH versus pressure (in atmospheres). Slopes are proportional to AV. 46 -9. 10. 11 BEEF DOGFISH .^SCULPIN 12 13. ANTIMORA 408 680 PRESSURE (aim) 47 Table V. Thermodynamic parameters f o r LDH-NADH a s s o c i a t i o n . AG, AS, and AV v a l u e s o b t a i n e d a t 25 C. Source of AG AH AS A V LDH (kcal/mole) (kcal/mole) (cal/mole«deg) (cirr/mole) Antimora -7.1 -6 3 -8 D o g f i s h -6.7 -10 -10 31 S c u l p i n -7.1 -5 5 41 Beef -7.2 -17 -32 68 48 between the d i s s o c i a t i o n constants f o r the two enzymes are n e a r l y i d e n t i c a l i n both cases. Hinz and J a e n i c k e (1973), on the other hand, obtained a K d of 11.16 uM f o r NADH-pig M4 LDH a s s o c i a t i o n based on c a l o r i m e t r i c measurements. The Kj^ o b t a i n e d here f o r the mammalian enzyme a t 25 C, 4.6 (oM, f a l l s between the found by S t i n s o n and Holbrook f o r beef M4 and by Hinz and Ja e n i c k e f o r p i g M4. Values of AH f o r NADH d i s s o c i a t i o n range from 8.1 k c a l / mole (Stinson and Holbrook, 1973) to 32.5 kcal/mole (Hinz and J a e n i c k e , 1973) f o r p i g M4 LDH. The AH found here f o r the beef M4 i s 17 kcal/mole. C l e a r l y the method used f o r determina-t i o n of and enthalpy changes i s an important v a r i a b l e . C. AMP b i n d i n g t o LDH Table VI summarizes temperature and pressure e f f e c t s on the AMP f o r the f o u r LDH's s t u d i e d . Again, a f f i n i t y decreased w i t h temperature and pressure i n a l l cases. A s l i g h t l y more marked temperature e f f e c t was observed f o r the Antimora, d o g f i s h , and s c u l p i n enzymes than f o r beef. Pressure (at 25 C) i n c r e a s e d the beef LDH R\ AMP to a g r e a t e r e x t e n t than i t d i d the other t h r e e . F i g u r e 10 shows the l n K^-temperature r e l a t i o n s h i p , and F i g u r e 11 the K^-pressure r e l a t i o n s h i p f o r each of the enzymes. Thermodynamic c h a r a c t e r i s t i c s are l i s t e d i n Table VII.AG shows a s l i g h t i n c r e a s e , i n the sequence Antimora, d o g f i s h , s c u l p i n , beef. AH and A S were approximately the same f o r the Antimora, d o g f i s h , and s c u l p i n enzymes, and had a s m a l l e r negative value f o r the beef enzyme. The volume change f o r the former three enzymes was a l s o of approximately 49 Table VI. Temperature and pr e s s u r e e f f e c t s on AMP i n h i b i t i o n . K i ' s , i n mMoles, were determined by Dixon p l o t s . C o n c e n t r a t i o n s of r e a c t a n t s used were: 1.0 mM pyruvate, 0-20 mM AMP, .025-0.1 mM NADH. A l l pre s s u r e data were obtained a t 25 C. Temperature (C) Antimora D o g f i s h S c u l p i n Beef 15 3.0 2.1 1.7 1.5 25 4.9 3.1 2.9 2.2 35 8.1 5.1 4.9 2.4 45 10.2 7.3 6.0 3.2 Pressure (PSI) Antimora D o g f i s h S c u l p i n Beef 14.7 4.9 3.1 2.9 2.2 5000 8.5 3.8 3.8 6.4 10000 9.7 5.3 6.2 11.5 50 F i g u r e 10. Ln R\ AMP f o r Antimora, d o g f i s h , s c u l p i n and beef M 4 LDH versus 1/temperature ( i n degrees K e l v i n ) . Slopes are p r o p o r t i o n a l to AH. 4 5. 6. .ANTIMORA .DOGFISH SCULPIN BEEF 7. 320 330 340 1/T X 1 0 5 350 52 F i g u r e 1 1 . Ln AMP f o r Antimora, d o g f i s h , s c u l p i n , and beef M 4 LDH versus p r e s s u r e ( i n atmospheres). Slopes are p r o p o r t i o n a l to A V . 1 340 680 953 PRESSURE (atm) 54 Table V I I . Thermodynamic parameters f o r LDH-AMP a s s o c i a -t i o n . AG, AS, and AV v a l u e s obtained a t 25 C. Source of AG A H AS AV LDH (kcal/mole) (kcal/mole) (cal/mole-deg) (cm3/mole) Antimora -3.1 -7 -15 25 Dogfish -3.4 -8 -14 20 S c u l p i n -3.4 -8 -14 27 Beef -3.6 -4 -2 60 55 the same v a l u e , about h a l f t h a t of the beef LDH-AMP a s s o c i a -t i o n . McPherson (19 70), u s i n g the same k i n e t i c methods, found a K± AMP f o r d o g f i s h M 4 LDH equal to 5.5 mM, with a AG value of -3.2, not f a r from the AG of 3.4 kcal/mole ob-t a i n e d here. D. Nicotinamide b i n d i n g to LDH The r e s u l t s f o r n i c o t i n a m i d e are more complex. ^ g ' s versus temperature and p r e s s u r e , shown i n Table V I I I , i n d i c a t e t h a t the a f f i n i t y of a l l f o u r LDH's f o r the i n h i b i t o r becomes g r e a t e r a t h i g h e r p r e s s u r e s and temperatures. The temperature e f f e c t i s l e a s t marked f o r the s c u l p i n enzyme, f o r which the b i n d i n g i s n e a r l y temperature independent, and i s s l i g h t l y g r e a t e r f o r Antimora and l a r g e f o r d o g f i s h and beef. The p r e s s u r e e f f e c t i s l a r g e s t f o r the beef LDH-nicotinamide a s s o c i a t i o n . P l o t s of lnTj versus l n n i c o t i n a m i d e c o n c e n t r a t i o n a t 25 C y i e l d good l i n e a r i t y (Figure 12); however, the slope (r) f o r a l l f o u r enzymes was equal to 1.4, which by d e f i n i t i o n i n d i c a t e s a r a t i o of 1.4 i n h i b i t o r molecules f o r each enzyme molecule. T h i s i s not compatible with a simple c o m p e t i t i v e mechanism, and the n i c o t i n a m i d e may be combining w i t h the enzyme or s u b s t r a t e s i n some other way, a d i s t i n c t p o s s i b i l i t y c o n s i d e r i n g the high (.5 M) c o n c e n t r a t i o n s used). AG's were c a l c u l a t e d from the r of 1.4 obtained; the s e r i e s of v a l u e s f o r each c o n c e n t r a t i o n used were q u i t e 56 Table V I I I . Temperature and pr e s s u r e e f f e c t s on n i c o t i n a m i d e i n h i b i t i o n . I ^ Q ' S , i n moles, were determined by p l o t t i n g p ercent of u n i n h i b i t e d a c t i v i t y a g a i n s t i n h i b i t o r c o n c e n t r a t i o n . C o n c e n t r a t i o n s of r e a c t a n t s used were: .05 mM NADH, 1 mM pyru-vate, 0-0.6 M n i c o t i n a m i d e . A l l pressure data were obtained a t 25 C. Temperature I50 (C) Antimora D o g f i s h S c u l p i n Beef 15 .18 .28 .14 .31 25 .16 .20 .16 .29 35 .14 .20 .13 .24 45 .12 .15 .13 .14 Pressure I ^ Q (PSI) Antimora D o g f i s h S c u l p i n Beef 14.7 .16 .20 .16 .29 5000 .16 .20 .16 .24 10000 .10 .15 .14 .16 57 F i g u r e 12. L n r j versus l n n i c o t i n a m i d e c o n c e n t r a t i o n f o r (a) Antimora, (b) d o g f i s h , (c) s c u l p i n and (d) beef M 4 LDH. !"*•]_ determined as d e s c r i b e d under Methods; slope = r , the r a t i o of i n h i b i t o r molecules to enzyme molecules. 58 -2.5 -2.0 -1.5 -1.0 -0.5 0 LN CONC 'N NICOTINAMIDE 59 0.2, (b) 2 5 °C DOGFISH 0.1 0 0.1 -2.5 r =1.4 2.0 -1.5 -1.0 -0 .5 LN CONC'N NICOTINAMIDE 6 0 + 3 r (c) 25 °C SCULPIN + 2. +1 0 - 1 . r=1.4 2.5 -2.0 -1.5 -1.0 LN CONC'N NICOTINAMIDE -0.5 61 + 2.0, (d) 2 5 °C BEEF +1.0 L 0 _ i -1.0 r = 1.4 -2.5 -2.0 -1.5 -1.0 -0.5 L N CONC 'N NICOTINAMIDE 62 c o n s i s t e n t and are shown i n Table I X . , T h e A G was low f o r a l l f o u r enzymes; t h a t of beef was s l i g h t l y l e s s than the o t h e r s . McPherson ( 1 9 7 0 ) found a A G of - 1 . 8 kcal/mole f o r n i c o t i n a m i d e mononucleotide b i n d i n g to the d o g f i s h M 4 L D H complex, s l i g h t l y h i g h e r than the value of - 1 . 3 kcal/mole f o r n i c o t i n a m i d e b i n d i n g alone to the d o g f i s h enzyme ob t a i n e d here. P l o t s of lnp-^ versus 1/temperature and p r e s s u r e were used to o b t a i n estimates of the A H and A V of n i c o t i n a m i d e b i n d i n g . In both cases, the s l o p e s were dependent upon the c o n c e n t r a t i o n of i n h i b i t o r used. In an attempt to compensate f o r these anomalous c o n c e n t r a t i o n e f f e c t s , A H and A V were c a l c u l a t e d both from an average of the s l o p e s f o r 0 . 1 - 0 . 5 M n i c o t i n a m i d e , and from the slope f o r 0 . 1 M n i c o t i n a m i d e alone, where i t was thought these e f f e c t s would be minimized. These v a l u e s are compared f o r A H i n Table IX, where they are q u i t e s i m i l a r except i n the case of the beef enzyme. The A V v a l u e s were too i n c o n s i s t e n t t o be u s e f u l , and so are not i n c l u d e d . 63 Table IX. Thermodynamic parameters f o r LDH-nicotinamide a s s o c i a t i o n . Values i n parentheses c a l c u l a t e d from data f o r lowest c o n c e n t r a t i o n of n i c o t i n a m i d e alone; others are an average of data f o r 0.1-0.5 M n i c o t i n a m i d e . AG and AS valu e s obtained a t 25 C. Source of LDH AG AH (kcal/mole) (kcal/mole) A S (cal/mole-deg) Antimora D o g f i s h S c u l p i n Beef •1.4 •1.3 •1.5 •1.0 5 (5) 5 (5) 5 (5) 12 (5) 11 ( I D 11 (11) 13 (13) 38 (20) 64 IV. DISCUSSION The r e s u l t s show that there are clear differences i n the behavior of the binding properties of lactate dehydrogenase from organisms l i v i n g i n varying physical environments. These differences are p a r t i c u l a r l y marked between the abyssal f i s h and mammalian enzymes, as might be expected since they experience the most extreme and opposite temperature and pressure regimes. The data obtained can be discussed on at least two l e v e l s : f i r s t , at an adaptational l e v e l , i n terms of how the p a r t i c u l a r binding properties of each LDH may improve the functioning of the enzyme i n vivo, and secondly, at a mechanistic l e v e l , the kinds of differences i n enzyme structure which could account for the temperature and pressure responses. In discussing the former, the data for NADH and oxamate binding are of most relevance, since i t i s the t o t a l of the interacitons occurring which w i l l be of importance to the enzyme's function and hence to the organism. NADH, as the true coenzyme, and oxamate, a very close analog of the substrate, should give a good in d i c a t i o n of the magnitude and sign of temperature and pressure e f f e c t s on LDH binding functions. In looking at the possible basis of these e f f e c t s , however, the nicotinamide and AMP interactions become of int e r e s t . Since they e s s e n t i a l l y "dissect" the coenzyme binding s i t e , t h e i r binding should give an in d i c a t i o n of the kinds of contributions involved i n NADH binding to each of the LDH's studied. 65 A. The AMP s u b s i t e The b i n d i n g i n t e r a c t i o n s of AMP wit h d o g f i s h M4 LDH have been summarized by Holbrook e t aj, (1975) . Adenosine binds i n a hydrophobic c r e v i c e , c o n s i s t i n g of r e s i d u e s such as v a l i n e , g l y c i n e , a l a n i n e , methionine, and t h r e o n i n e . Known i n t e r a c t i o n s i n c l u d e seven hydrophobic and two hydrogen bonds; one of the l a t t e r may be presen t o n l y i n the b i n a r y complex (Adams e t a l , 19 73). The adenine r i b o s e forms two hydrogen bonds between the 03' and 02' hy d r o x y l groups and r e s i d u e s 29 and 53; the l a t t e r , a s p a r t a t e , i s b e l i e v e d to move to make room f o r the 02' hyd r o x y l group on b i n d i n g . The pyrophosphate, i n the t e r n a r y complex, has one neg a t i v e charge balanced by a r g i n i n e 101 and the other s o l v a t e d . On formation of the t e r n a r y complex, the guanidinium group of a r g i n i n e 101 (part of the "loop" sequence) moves 13 A to bi n d t o the phosphate ( i n t o a p o s i t i o n which would p r o h i b i t pyrophosphate b i n d i n g were i t the normal conformation). Adams s i a l (19 73) have suggested t h a t the c o r r e c t o r i e n t a t i o n of the pyrophosphate i s a p r e r e q u i s i t e f o r a r g i n i n e 101 p o s i t i o n i n g and the subsequent c o l l a p s e of the loop. I f changes i n b i n d i n g c o n t r i b u t i o n s a t the a c t i v e s i t e are the b a s i s f o r the d i f f e r i n g AMP b i n d i n g c h a r a c t e r i s t i c s of the fou r l a c t a t e dehydrogenases, then i t should be p o s s i b l e to i n t e r p r e t the d i f f e r i n g temperature and pressure e f f e c t s i n terms of a change i n emphasis on the type of bonding i n v o l v e d . A predominance of hydrophobic i n t e r a c t i o n s would be expected t o r e s u l t i n t i g h t e r b i n d i n g of the l i g a n d (a decrease i n K^) a t hi g h e r temperatures and lower p r e s s u r e s . 66 The r e v e r s e should be the case i f hydrogen bonds are more important. A look a t Table VI shows t h a t high temperatures and high pressure both i n c r e a s e the f o r AMP f o r a l l f o u r enzymes. AMP b i n d i n g to the three f i s h LDH's, however, i s more s e n s i t i v e t o temperature changes and l e s s s e n s i t i v e to p r e ssure changes than i s AMP b i n d i n g to the beef LDH. T h i s i s r e f l e c t e d by the AH v a l u e s , which are n e a r l y twice as l a r g e f o r the f i s h LDH-AMP b i n d i n g i n t e r a c t i o n as f o r the beef, and by the volume changes of a s s o c i a t i o n , which are about h a l f as g r e a t f o r the f i s h enzymes. These responses would be c o n s i s t e n t w i t h an i n c r e a s e d hydrophobic c o n t r i b u t i o n t o AMP b i n d i n g i n the beef enzyme r e l a t i v e to the other three,but are not c o n s i s t e n t with a simple predominance of e i t h e r type of bond. Hochachka (1975) found f o r ADP b i n d i n g t h a t the f o r beef LDH v a r i e d i n v e r s e l y w i t h temperature and d i r e c t l y w i t h p r e s s u r e , and the K i f o r the Antimora LDH-ADP i n t e r a c t i o n e x h i b i t e d e x a c t l y o p p o s i t e b e h a v i o r . These o b s e r v a t i o n s imply an emphasis of hydrophobic c o n t r i b u t i o n s i n the beef enzyme, and of hydrogen bonds i n the Antimora enzyme f o r ADP b i n d i n g . Although t h i s corresponds t o the types of d i f f e r e n c e s found f o r the two enzymes f o r AMP b i n d i n g , the d i f f e r e n c e s i n temperature and pressure e f f e c t s on two l i g a n d s which are so s i m i l a r i s somewhat p u z z l i n g . ADP i s not as e f f e c t i v e an i n h i b i t o r of LDH as i s AMP, presumably because of some i n t e r f e r e n c e i n b i n d i n g by the a d d i t i o n a l phosphate group. 67 B. The ni c o t i n a m i d e s u b s i t e In d o g f i s h M 4 LDH, the u n r e a c t i v e s i d e of the n i c o -tinamide r i n g i s supported p r i m a r i l y by hydrophobic i n t e r -a c t i o n s ; i n a d d i t i o n there i s one hydrogen bond and, i n the o x i d i z e d coenzyme, a charge i n t e r a c t i o n between the N;j_ and glutamate 140. The p o s i t i o n of the n i c o t i n a m i d e r i n g i n the t e r n a r y complex i s s l i g h t l y a l t e r e d from t h a t i n the b i n a r y complex (Holbrook e t a l , 19 75). Nicotinamide mononucleotide a t 3.5 mM was found by McPherson (19 70) not to i n h i b i t d o g f i s h M 4 u n l e s s TAMP was pre s e n t , and methyl-and p r o p y l - n i c o t i n a m i d e a t 1 0 - 2 d i d not i n h i b i t the enzyme e i t h e r i n the presence or absence of 7AMP. At the conc e n t r a -t i o n s of n i c o t i n a m i d e used here (0.1-0.6 M) AMP d i d not have any e f f e c t on the degree of i n h i b i t i o n ; hence under these c o n d i t i o n s McPherson's c o n c l u s i o n t h a t AMP b i n d i n g i s a p r e r e q u i s i t e f o r b i n d i n g of the n i c o t i n a m i d e p o r t i o n of the coenzyme does not seem a p p l i c a b l e . The temperature data f o r a l l f o u r l a c t a t e dehydrogenases, showing an i n c r e a s e ; i n a f f i n i t y a t hi g h e r temperatures, would be c o n s i s t e n t w i t h a predominance of hydrophobic i n t e r a c t i o n s . T h i s would seem to be reasonable, c o n s i d e r i n g the known bonds i n v o l v e d i n ni c o t i n a m i d e b i n d i n g mentioned above. However, hydrophobic i n t e r a c t i o n s are known to occur w i t h a s i z a b l e volume i n c r e a s e , and i t can be seen from Table V I I I t h a t the nicotinamide-LDH a s s o c i a t i o n i n each case i s enhanced by i n c r e a s e d p r e s s u r e . Thus any simple hypothesis u s i n g b i n d i n g i n t e r a c t i o n s of n i c o t i n a m i d e to 68 t h e enzyme t o e x p l a i n t h i s d a t a i s c l e a r l y u n t e n a b l e . C. NADH b i n d i n g t o l a c t a t e dehydrogenase NADH b i n d i n g i n v o l v e s , i n a d d i t i o n t o the i n t e r a c t i o n s d i s c u s s e d above f o r n i c o t i n a m i d e and AMP b i n d i n g , t h r e e p r o b a b l e h y d r o p h o b i c i n t e r a c t i o n s and hydrogen bonds t o the n i c o t i n a m i d e r i b o s e , b e l i e v e d t o be o f importance i n o r i e n t i n g t h e n i c o t i n a m i d e m o n o n u c l e o t i d e p o r t i o n o f the coenzyme (Holbrook e t aJL, 19 75) . The K i NADH v a r i e s d i r e c t l y w i t h t e m p e r a t u r e and p r e s s u r e f o r each LDH e x c e p t t h a t o f A n t i m o r a , f o r w h i c h NADH b i n d i n g appears t o be s l i g h t l y p r e s s u r e - e n h a n c e d . Beef LDH-coenzyme b i n d i n g i s most s e n s i t i v e t o b o t h temper-a t u r e and p r e s s u r e . T h e i b a s i s o f t h e l a r g e i n c r e a s e o f Kj_ w i t h t emperature i s n o t apparent from the n i c o t i n a m i d e and AMP d a t a (Table X) w h i c h t o g e t h e r would p r e d i c t a de c r e a s e o f Kj_ a t h i g h e r t e m p e r a t u r e s , s i n c e n i c o t i n a m i d e b i n d i n g proceeds w i t h a h i g h p o s i t i v e AH, and AMP b i n d i n g w i t h a s m a l l n e g a t i v e AH. Hence e i t h e r t h e b i n d i n g o f t h e n i c o t i n a m i d e r i b o s e o r the c o n f o r m a t i o n a l changes known t o oc c u r w i t h NADH b i n d i n g , o r b o t h , must p r o v i d e an i m p o r t a n t c o n t r i b u t i o n t o the observ e d e n t h a l p y change. NADH b i n d i n g t o t h e d o g f i s h LDH o c c u r s w i t h a s l i g h t l y h i g h e r A H t h a n does t h a t t o the s c u l p i n and A n t i m o r a enzymes. I n the case o f t h e l a t t e r two, the b i n d i n g o f NADH i s v i r t u a l l y t e m p e r a t u r e - i n s e n s i t i v e ( F i g u r e 1 3 ) . The t o t a l A H c o n t r i b u t i o n s o f the AMP and n i c o t i n a m i d e b i n d i n g (Table X) a r e s l i g h t l y s m a l l e r n e g a t i v e v a l u e s t h a n f o r NADH 69 T a b l e X. A c o m p a r i s o n o f t h e sum o f t h e t h e r m o d y n a m i c p a r a m e t e r s a s s o c i a t e d w i t h AMP and n i c o t i n a m i d e b i n d i n g w i t h t h o s e f o r NADH b i n d i n g t o M. LDH. AG and AS d a t a o b t a i n e d a t 25 C. A n t i m o r a D o g f i s h S c u l p i n B e e f AG ( k c a l / m o l e ) AMP + n i c o t i n a m i d e -4.5 NADH -7.1 -4.7 -6.7 -4.9 -7.1 -4.6 -7.2 A H ( k c a l / m o l e ) AMP + n i c o t i n a m i d e NADH -2 -6 -3 -10 •3 •5 1 •17 AS ( c a l / m o l e - d e g ) AMP + n i c o t i n a m i d e NADH -4 3 -3 10 •1 5 18 •32 70 F i g u r e 13. Temperature e f f e c t s on NADH b i n d i n g t o Antimora, d o g f i s h , s c u l p i n , and beef M 4 LDH; 71 ^ C\J (lAirV) HQVN M 72 b i n d i n g , and are very c l o s e f o r a l l three f i s h . Again, the c o n t r i b u t i o n s of these two s u b s i t e s do not account completely f o r the observed coenzyme-binding v a l u e s , but are i n the same d i r e c t i o n and of s i m i l a r magnitude. In the case of Antimora, d o g f i s h , and s c u l p i n LDH, a p o s i t i v e enthalpy change o c c u r r i n g w i t h n i c o t i n a m i d e b i n d i n g i s compensated by a neg a t i v e enthalpy of AMP b i n d i n g , making the t o t a l enthalpy change s m a l l and n e g a t i v e . T h i s type of compensation c o u l d be of importance i n re d u c i n g the t e m p e r a t u r e - s e n s i t i v i t y of coenzyme b i n d i n g by the ectothermic enzymes. The AH and AS va l u e s f o r NADH b i n d i n g to the mammalian enzyme do not correspond to the sum of the va l u e s f o r AMP and n i c o t i n a m i d e b i n d i n g . The f r e e e n e r g i e s of b i n d i n g are s i m i l a r f o r a l l f o u r enzymes, both i n the case of AMP + ni c o t i n a m i d e and NADH, but are c o n s i d e r a b l y h i g h e r f o r NADH b i n d i n g . T h i s c o u l d be a t t r i b u t e d to (1) the absence of the ni c o t i n a m i d e r i b o s e , (2) the d i f f e r e n c e s between the co n f o r m a t i o n a l changes o c c u r r i n g w i t h AMP and NADH b i n d i n g , and presumably wi t h n i c o t i n a m i d e b i n d i n g , or (3) the 200-400 f o l d g r e a t e r a f f i n i t y of the LDH-NADH b i n a r y complex over the LDH-NAD complex ( o x i d i z e d n i c o t i n a m i d e was used i n these experiments). In view of McPherson's (1970) v a l u e of -1.8 kcal/mole f o r the f r e e energy of b i n d i n g of n i c o t i n a m i d e mononucleotide, on l y ..5 kcal/mole h i g h e r than t h a t found f o r n i c o t i n a m i d e here, the l a t t e r e x p l a n a t i o n seems most l i k e l y . 73 In the case of p r e s s u r e , as F i g u r e 14 makes apparent, the d o g f i s h and s c u l p i n enzymes behave more l i k e the mam-malian enzyme than l i k e the Antimora LDH. While the former have l a r g e p o s i t i v e volume changes a s s o c i a t e d w i t h coenzyme b i n d i n g , the l a t t e r i s e s s e n t i a l l y p r e s s u r e - i n s e n s i t i v e . T h i s s m a l l volume change may c o n s t i t u t e an a d a p t a t i o n of the a b y s s a l enzyme f o r improved f u n c t i o n a t h i g h (or varying) p r e s s u r e s , and has been found f o r the volume change of a c t i v a t i o n of the Antimora LDH r e a c t i o n as w e l l (Baldwin e t a l , 19 75). In the other three organisms, w i t h the p o s s i b l e e x c e p t i o n of the d o g f i s h , which experiences minor p r e s s u r e v a r i a t i o n s , volume change would not be expected to be s e l e c t e d f o r one way or another. D. Oxamate b i n d i n g to the LDH-NADH b i n a r y complex In the t e r n a r y complex, the p o s i t i o n of the "loop" s t r u c t u r e excludes bulk water from the a c t i v e s i t e , and the number of charged and h y d r o p h i l i c groups pres e n t i n the c a t a l y t i c r e g i o n i s i n c r e a s e d . On pyruvate b i n d i n g , the c a r b o x y l group i s n e u t r a l i z e d , probably by the formation of an i o n p a i r w i t h a r g i n i n e 171, and the keto group of pyruvate forms a hydrogen bond wi t h h i s t i d i n e 19 5. T h i s stage of the b i n d i n g sequence i s b e l i e v e d to be r e p r e s e n t e d by the i n h i b i t o r complex LDH-NADH-oxamate (Holbrook .e_t a l , 1975). I f the charge i n t e r a c t i o n i s assumed to be predominant i n oxamate b i n d i n g , i t would be expected to be s t a b i l i z e d 74 F i g u r e 14. Pressure e f f e c t s on NADH b i n d i n g to Antimora, d o g f i s h , s c u l p i n , and beef M 4 LDH. 0 6000 10000 PRESSURE (PSI) 76 a t low t e m p e r a t u r e s . T h i s i s the' c a s e f o r e a c h o f t h e LDH 1 s s t u d i e d , as was shown i n T a b l e I I , i n w h i c h t h e v a r i e s d i r e c t l y w i t h t e m p e r a t u r e . However, as a p l o t o f K-L v e r s u s t e m p e r a t u r e ( F i g u r e 15) shows, a l t h o u g h t h e g e n e r a l e f f e c t o f t e m p e r a t u r e on oxamate b i n d i n g i s s i m -i l a r , t h e c u r v e s a r e e a c h l o c a t e d a t d i f f e r e n t p o s i t i o n s a l o n g t h e t e m p e r a t u r e a x i s . Thus t h e a f f i n i t y o f t h e b e e f LDH-NADH complex f o r oxamate i s h i g h e r a t any g i v e n temper-a t u r e t h a n t h e a f f i n i t i e s o f t h e t h r e e e c t o t h e r m i c L D H 1 s , and s i m i l a r l y t h e a f f i n i t i e s o f t h e s c u l p i n and d o g f i s h LDH's f o r oxamate a r e h i g h e r t h a n t h a t o f t h e A n t i m o r a LDH. The s i g n i f i c a n c e o f t h i s c a n be s e e n by l o o k i n g a t t h e LDH-NADH-oxamate a f f i n i t y a t t h e e n v i r o n m e n t a l t e m p e r a t u r e o f e a c h o f t h e o r g a n i s m s ( T a b l e X I ) ; t h e K^'s f a l l w i t h i n a r e l a t i v e l y n a r r o w r a n g e w h i c h s u g g e s t s t h a t t h e b i n d i n g i s s p e c i f i c a l l y m o d i f i e d i n e a c h one so as t o be optimum f o r t h e i r p a r t i c u l a r b i o l o g i c a l t e m p e r a -t u r e . T h i s i s a n a l a g o u s t o s t u d i e s o f p y r u v a t e b i n d i n g (K m) done f o r s e v e r a l e c t o t h e r m i c L D H 1 s i n w h i c h t h e minimum K M v a l u e c o r r e s p o n d e d w i t h t h e minimum h a b i t a t t e m p e r a t u r e (Somero, 19 6 9 ) , and i s n o t s u r p r i s i n g i n v i e w o f t h e c l o s e s t r u c t u r a l s i m i l a r i t y between oxamate and p y r u v a t e . The r e l a t i o n s h i p o f t h e oxamate t o t e m p e r a t u r e has a l s o b e en d e t e r m i n e d f o r s e v e r a l o t h e r o r g a n i s m s o f i n t e r m e d i a t e body t e m p e r a t u r e s (a m a r s u p i a l , two monotremes, and an A u s t r a l i a n l i z a r d ) , and t h e K ^ - t e m p e r a t u r e c u r v e s c o n f i r m t h e p a t t e r n shown h e r e (Hochachka e t a l , 19 7 5 a ) . A c h a r g e i n t e r a c t i o n w o u l d a l s o be e x p e c t e d t o be 77 F i g u r e 15. Temperature e f f e c t s on oxamate b i n d i n g to Antimora, d o g f i s h , s c u l p i n , and beef M4 LDH-NADH b i n a r y complex. TEMPERATURE C O 79 Table XI. R e l a t i o n s h i p between oxamate of M4 LDH and b i o l o g i c a l temperature. Source of LDH B i o l o g i c a l Temperature Range Oxamate a t B i o l o g i c a l Temperatures Antimora Dogfish S c u l p i n Beef 2-4 C 6-15 C 5-20 C 37 C .20-.21 mM .12-.15 mM .10-.15 mM .16 mM 80 s t a b i l i z e d by low p r e s s u r e s , and the Kj_ oxamate does decrease a t high p r e s s u r e s f o r each of the LDH's. However, the volume change a s s o c i a t e d w i t h b i n d i n g f o r Antimora, d o g f i s h and s c u l p i n LDH i s much lower than would be expected f o r charge n e u t r a l i z a t i o n . Hochachka (19 75) has c a l c u l a t e d from Suzuki and T a n i g u c h i 1 s (19 72) v a l u e s f o r n e u t r a l i z a -t i o n of CH3COOH and of l y s i n e an estimated t o t a l A V of 38 cm3/mole. T h i s value i s c l o s e to t h a t o b t a i n e d f o r beef LDH, but h i g h e r than t h a t f o r the other three enzymes. That t h i s charge i n t e r a c t i o n occurs i n each LDH i s c e r t a i n ; comparisons of amino a c i d sequence i n the r e g i o n of h i s t i -dine 19 5 (191-20 3) show d o g f i s h M4 and p i g H 4 to be i d e n t i -c a l w i t h the e x c e p t i o n of the s u b s t i t u t i o n of l e u c i n e f o r i s o l e u c i n e 19 2 i n the l a t t e r , and a r g i n i n e p e p t i d e s 101-115 are i d e n t i c a l i n d o g f i s h M 4, c h i c k e n M 4, and p i g M 4 and H 4 (Holbrook e_£ aJL, 1975) . These homologies leave l i t t l e room f o r m o d i f i c a t i o n i n these p a r t i c u l a r ^ i n t e r a c -t i o n s , and i n d i c a t e t h a t compensatory volume changes must occur elsewhere i n the three f i s h l a c t a t e dehydrogenases. U n f o r t u n a t e l y complete sequences are not a v a i l a b l e f o r comparison, but Everse and Kaplan (1973) have estimated a 20% d i f f e r e n c e between the amino a c i d composition of d o g f i s h and c h i c k e n l a c t a t e dehydrogenases. While i t appears t h a t the key substrate-LDH i n t e r a c t i o n s have not been a l t e r e d , the f a c t t h a t the s u b s t r a t e and coenzyme b i n d i n g s i t e s together comprise tw o - t h i r d s of the molecule make some m o d i f i c a t i o n s i n these r e g i o n s probable. Whether 81 or not these might be s p e c i f i c a l l y i n v o l v e d i n b i n d i n g e i t h e r of the l i g a n d s cannot be determined from the a v a i l -able data. Hence the o r i g i n of "compensatory volume changes" c o u l d be v i r t u a l l y anywhere i n the enzyme molecule, w i t h i n or o u t s i d e of the a c t i v e s i t e . E. LDH-NADH-oxamate i n t e r a c t i o n s The t e r n a r y complex LDH-NADH-oxamate i s isomorphous w i t h the LDH-NAD-pyruvate complex (Adams e_£ al., 19 73) with the d i f f e r e n c e t h a t i n the former, there i s no coenzyme-i n h i b i t o r bond. Hence i t i s b e l i e v e d t h a t the oxamate complex may r e p r e s e n t the t r a n s i t i o n s t a t e of LDH. I t was t h e r e f o r e thought t h a t a summation of the thermodynamic parameters i n v o l v e d i n b i n d i n g of the two l i g a n d s would be of i n t e r e s t . The t o t a l s g i v e n i n Table XII f o r the A G , A S , A H , and A V of the two i n t e r a c t i o n s emphasize t h a t the observed d i f f e r e n c e s f o r both l i g a n d s are i n the same d i r e c t i o n , and t h a t the magnitude of the t o t a l d i f f e r e n c e s between the A H , A S , a n d A V of beef and Antimora LDH are, indeed, q u i t e l a r g e (2.7x f o r A H , 81x f o r A S , 67x f o r A V ) . In the case of the Antimora enzyme, i t can be seen t h a t both f o r entropy and volume change, a sm a l l p o s i t i v e change i n b i n d i n g of one l i g a n d i s almost e x a c t l y compensated by a s m a l l n e g a t i v e change i n b i n d i n g of the o t h e r , making the t o t a l A S and A V very c l o s e to zero. I f t h i s i s an a d a p t a t i o n to h i g h (or varying) p r e s s u r e s , as seems l i k e l y , i t might be expected 82 Table X I I . Sum of thermodynamic parameters f o r LDH-NADH and LDH-NADH-oxamate i n t e r a c t i o n s . A G , AS, and A V values obtained a t 25 C. Antimora D o g f i s h S c u l p i n Beef A G (kcal/mole) NADH -7.1 -6.7 -7.1 -7.2 Oxamate -4.7 -5.1 +5.1 -5.6 T o t a l -11.8 -11.8 -12.2 -12.8 A H (kcal/mole) NADH -6 -10 -5 -17 Oxamate -6 -9 -8 -15 T o t a l -12 -19 -13 -32 AS (cal/mole-deg) NADH +3 -10 +5 -32 Oxamate -4 -12 -11 -32 T o t a l -1 -22 -6 -64 A V (cm^/mole) NADH -8 +31 +41 +68 Oxamate +10 -5 +11 +46 T o t a l +2 +26 +52 +114 83 t h a t the d o g f i s h , s c u l p i n and beef enzymes would have A V s of more or l e s s s i m i l a r v a l u e s . D o g f i s h and s c u l p i n LDH appear to be i n t e r m e d i a t e i n t h e i r ^V's, but i f these valu e s are c o n s i d e r e d p r o p o r t i o n a t e l y , i t i s seen t h a t the AV f o r these two f i s h enzymes i s many-fold l a r g e r than t h a t f o r the Antimora, whereas the beef AV i s o n l y 2-4 times as l a r g e as the d o g f i s h and s c u l p i n A^'s. T h i s d i f f e r e n c e c o u l d of course be a r e f l e c t i o n of something other than p r e s s u r e - a d a p t a t i o n . Another o b s e r v a t i o n which can be made on the LDH-NADH-oxamate summed data i s t h a t the AG v a l u e s are q u i t e s i m i l a r , showing a s l i g h t i n c r e a s e i n the case of the mammalian enzyme. R e l a t i v e l y s t a b l e A GF v a l u e s f o r r e a c t i o n s i n v o l v i n g homologous enzymes have been noted p r e v i o u s l y . Somero and Low (19 75), comparing the f r e e energy of a c t i v a t i o n f o r three s e t s of homologous enzymes ( i n c l u d i n g LDH) i n d i f f e r e n t organisms found a s l i g h t decrease i n AG^ f o r e c t o -therms r e l a t i v e to endotherms. T h e i r s u g g e s t i o n t h a t l a r g e changes i n the AG* of a r e a c t i o n may not be f e a s i b l e seems a p p l i c a b l e i n t h i s case to the b i n d i n g of s u b s t r a t e and coenzyme. I t i s c l e a r from the data, however, t h a t l a r g e changes i n the e n t h a l p i c and e n t r o p i c c o n t r i b u t i o n s to b i n d i n g are p o s s i b l e . For NADH and oxamate b i n d i n g , both the A H and A s are much g r e a t e r (more negative) f o r the mammalian than f o r the three f i s h enzymes. The advantage of a low A^H f o r ectotherms, p a r t i c u l a r l y those s u b j e c t to l a r g e 84 thermal v a r i a t i o n s , i s t h a t of r a t e s t a b i l i z a t i o n under f l u c t u a t i n g temperature c o n d i t i o n s . For mammalian enzymes, a high AH c o u l d be the r e s u l t of an absence of s e l e c t i o n f o r temperature independence, or a r e f l e c t i o n of i n c r e a s e d enzyme-ligand bond s t r e n g t h r e q u i r e d to prevent i n s t a b i l i t y at h igh environmental temperatures. I t has a l s o been sug-gested (Low and Somero, 1974) t h a t the d i f f e r e n c e s i n AH* between endothermic and e c t o t h e r m i c enzymes might be caused by i n c r e a s e s i n the " r i g i d i t y " of the p r o t e i n i t s e l f i n high temperature organisms, i n order to minimize thermal l a b i l i t y . As a consequence of the r e l a t i o n s h i p between A G , A S, and A H , an i n c r e a s e i n A H , when A G i s maintained c o n s t a n t , must r e s u l t i n an i n c r e a s e i n A S . T h i s i s observed f o r the LDH-NADH and LDH-NADH-oxamate b i n d i n g . F. Enthalpy-entropy compensation The "enthalpy-entropy compensation p l o t " , A H versus A S , has been found to be l i n e a r f o r s e v e r a l types of homol-ogous r e a c t i o n s , i n c l u d i n g r e a c t i o n s c a t a l y z e d by homologous enzymes. The slope of t h i s l i n e , c a l l e d the compensation temperature (T c) f a l l s w i t h i n the range of 250 K-315 K f o r a number of r e a c t i o n s known to i n v o l v e w a t e r - s o l u t e i n t e r a c t i o n s (Lumry and Rajender, 19 70) as w e l l as f o r s e v e r a l enzyme-catalyzed r e a c t i o n s (Lumry and Rajender, 19 70; Borgmann e t aJL_, 19 75; Cohen _et _ a l , 19 70; Subramian e t a l , 19 75). T h i s l i m i t e d range i n compensation temper-atures i s not simply a consequence of thermodynamic laws, 85 and i t has been suggested t h a t the observed T c ' s f o r enzymic r e a c t i o n s are i n d i c a t i v e of the involvement o f water. The i d e a t h a t w a t e r - p r o t e i n i n t e r a c t i o n s are impor-t a n t i s not a new one, and i s i m p l i c i t i n mechanisms i n -v o l v i n g c o n f o r m a t i o n a l changes (Lumry and B i l t o n e n , 1969). Conformational changes have been shown to occur d u r i n g c a t a l y s i s f o r many enzymes, i n c l u d i n g LDH, and a l t e r a t i o n s i n weak bonds of the p r o t e i n w i l l almost c e r t a i n l y r e s u l t i n a l t e r a t i o n s of the s t r u c t u r e of the surrounding water. In a d d i t i o n , b i n d i n g of some s u b s t r a t e s "(e.g. pyruvate to LDH) i s thought to i n v o l v e the e x c l u s i o n of water. The compensation temperatures obtained f o r NADH and oxamate b i n d i n g here are shown i n F i g u r e 16, where i t can be seen t h a t i n each case the three e c t o t h e r m i c enzymes are grouped together, and a t the op p o s i t e end of the s c a l e t o the mammalian enzyme, as i s i m p l i c i t i n the AH and AS values presented e a r l i e r . Both i n the case of NADH and oxamate, the compensation temperatures (320 and 330 K r e s p e c t i v e l y ) f a l l s l i g h t l y above the giv e n range f o r water - s o l u t e i n t e r a c t i o n s . Low and Somero (19 74) have a l s o found compensation temperatures near 330 K f o r the a c t i v a -t i o n e n t h a l p i e s and e n t r o p i e s of s e v e r a l homologous l a c t a t e dehydrogenases, glyceraldehyde-3-phosphate dehydrogenases, and glycogen phosphorylases. They have suggested t h a t v a l u e s i n t h i s range are too hig h to f i t i n w e l l w i t h Lumry and Rajender's data, and may i n s t e a d be r e l a t e d to the breakage and r e f o r m a t i o n of weak bonds i n the p r o t e i n 86 F i g u r e 16. E n t h a l p y - e n t r o p y c o m p e n s a t i o n p l o t s f o r (a) NADH b i n d i n g and (b) oxamate b i n d i n g t o M 4 LDH f r o m A n t i m o r a , d o g f i s h , s c u l p i n and b e e f . L9 - 4 , ( b ) Tc = 330°K -6 ^ANTIMORA -8 10 12 SCULPIN DOGFISH 14. •16. BEEF - 3 5 - 2 5 - 1 5 A S (cal/mole/°C) - 5 89 during c a t a l y s i s . They use Steam's (1949) estimates for breaking the weakest intra-protein bonds i n the a c t i -vation of protein denaturation, which are a AH of 4000 cal/mole and a AS of 12 cal/mole•deg. This gives a r a t i o of AH/AS equal to 333 K. There i s no way of knowing, at present, whether or not the values found here for the T c of NADH and oxamate binding, and by Somero and Low (1974) are s i g n i f i c a n t l y d i f f e r e n t from the values summarized by Lumry and Ra.jender (19 70). Because of the known conformational changes occurring with NADH binding, breakage and reformation of weak bonds would not be unexpected. I t seems l i k e l y that i f changes i n weak bonds are involved, water-solute i n t e r -actions w i l l be as well!, and that there i s no way to d i f -ferentiate between the two. 90 V. CONCLUSIONS The f i r s t g e n e r a l c o n c l u s i o n from t h i s work i s one which has been made befo r e ; t h a t homologous enzymes, c a t a l y z i n g i d e n t i c a l r e a c t i o n s , can be s p e c i f i c a l l y m o d i f i e d so t h a t t h e i r f u n c t i o n i s op t i m i z e d under d i f f e r e n t temperature and pressure environments. That t h i s type of a d a p t a t i o n i s expected, c o n s i d e r i n g the wide range of environments which organisms are capable of i n h a b i t i n g , makes no l e s s i n t e r e s t i n g the q u e s t i o n of how e v o l u t i o n has produced such d i v e r s i t y of p r o p e r t i e s i n p r o t e i n s r e q u i r e d to c a r r y out the same c a t a l y t i c f u n c t i o n s . Numerous s t u d i e s of e l e c t r o p h o r e t i c v a r i a b i l i t y and amino a c i d sequence com-p a r i s i o n s have demonstrated the e x i s t e n c e of d i f f e r e n t s t r u c t u r a l forms of homologous enzymes, and a t a f u n c t i o n a l l e v e l homologous enzymes have been shown to have d i f f e r e n t k i n e t i c and thermodynamic c h a r a c t e r i s t i c s . What remains i s to t i e the two together: what kinds of s t r u c t u r a l a l t e r a t i o n s form the b a s i s f o r m o d i f y i n g enzyme f u n c t i o n and, i n t u r n , how i s t h i s of advantage to the organism? The gap between the p r o p e r t i e s of a s i n g l e enzyme and the " f i t n e s s " of an e n t i r e organism makes the l a t t e r q u e s t i o n d i f f i c u l t t o answer except a t a very s i m p l i s t i c l e v e l , as has been done here f o r enzyme f u n c t i o n under d i f f e r e n t temperature and pressure regimes. I t should be remembered t h a t there are undoubtedly a myriad of s p e c i f i c c o n d i t i o n s to which an enzyme must be s u i t e d , which i n c l u d e the 91 environment inside the organism — the entire complex metabolism into which i t must f i t — as well as physical conditions to which the organism i s exposed. Hence though the conclusions given here, that modifications i n enthalpy and volume change of binding are important i n temperature and pressure adaptation, are reasonable ones, they are not conclusive, and the observed properties could well be the r e s u l t of some selection factor which i s not im-mediately apparent. The other question of i n t e r e s t , the s t r u c t u r a l basis of modified enzyme function, may ultimately be easier to answer as knowledge of c a t a l y t i c mechanisms and enzyme structure advance. The primary conclusion reached here i s that more i s involved than alterations i n binding of enzyme to ligand i n producing d i f f e r e n t k i n e t i c and thermo-dynamic c h a r a c t e r i s t i c s . Differences have a l t e r n a t i v e l y been attributed to "conformational" changes i n the protein accompanying c a t a l y s i s , a vague statement at best, which may encompass protein-protein or protein-water interactions v i r t u a l l y anywhere i n the protein. With the determination of the structure of lactate dehydrogenase showing two-thirds of the protein to be involved i n the substrate and coenzyme binding s i t e s , and the elucidation of the complex conformational changes accompanying the binding of these ligands, the simple lock and key hypothesis i s no longer an adequate description of enzymic c a t a l y s i s . 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