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Mechanistic studies on [alpha]-glycosyl transferases

University of British Columbia

In order to provide insight into the mechanism of glycogen phosphorylase (phos b) and cyclodextrin glycosyltransferase (CGTase) the modified substrates, 4-deoxy-α-maltotriosyl fluoride (4DαG3F), 4-deoxymaltopentaose (4DG5), and 4-deoxymaltohexaose (4DG6) have been synthesized using chemical and enzymatic means. These types of incompetent substrate analogues can be used to "trick" each enzyme into catalyzing only the first half of its normal reaction in order to trap a glycosyl-enzyme intermediate. Glycosyl fluorides such as α-maltotriosyl fluoride (αG3F) are good substrates for Bacillus circulans 251 CGTase (kcat = 275 s⁻¹, Km = 2.5 mM) providing a convenient method of measurement. Reaction of the acid/base catalyst mutant of CGTase, Glu257Gln, with 4DαG3F, yielded a stable glycosyl-enzyme intermediate. Using neutral loss mass spectrometry, Asp229 was identified as the catalytic nucleophile. An X-ray structure of the 4- deoxymaltotriosyl moiety bound to Glu257Gln provided the first structural insights into such a covalent intermediate for any a-1,4 glycosyl transferase or α-glycosidase. The binding of active site mutants of CGTase to the inhibitor acarbose was investigated through measurement of Ki values. Kinetic parameters for αG3F and α-glucosyl fluoride were determined and reasonable correlations were observed in logarithmic plots relating the Ki value for acarbose with each mutant and both kcat/Km and Km for the hydrolysis of either substrate by the corresponding mutants. The dependence was greater in the plot of log (kcat/Km) vs log Ki than in the plot of log Km indicating that the binding mode of acarbose more closely resembles that of the reaction transition state than of the ground state. Kinetic results on phos b indicated that 4DG6 was a competitive inhibitor with respect to maltopentaose, but binds noncompetitively with respect to orthophosphate or glucose-1- phosphate. Two trapping experiments in the presence of 4DG6, a radiolabelling study using ¹⁴C-glucose-1-phosphate and an electrospray mass spectrometry investigation, provided preliminary evidence for a glycosyl-enzyme intermediate. Analogous experiments were performed with potato phosphorylase using 4DG5 yielding similar results. Positional isotope exchange experiments were attempted with both phosphorylases. Glucose-1-phosphate selectively labelled with ¹⁸O at the anomeric position was chemically synthesized for this purpose and ³¹PNMR was used to follow the extent of isotope exchange.

Thesis/Dissertation

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2009-06-24

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http://hdl.handle.net/2429/9566

Chemistry

University of British Columbia

UBCV

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