UBC Theses and Dissertations
Covalent catalyst in the UDP-glucose dehydrogenase Ge, Xue
UDP-glucose dehydrogenase from Streptococcus pyogenes (EC 220.127.116.11) is a NAD ⁺- dependent enzyme that catalyzes the 4-electron oxidation of UDP-glucose to UDPglucuronic acid without the release of an aldehyde intermediate. This enzyme is interesting because its single active site carries out two sequential oxidations, converting an alcohol to a carboxylic acid, whereas most NAD+-dependent dehydrogenases catalyze only a single step of oxidation. The recombinant dehydrogenase was purified to homogeneity and determined to have a k[sub cal] of 1.8 ± 0.1 s⁻¹ and an apparent K[sub m] of 20 ± 4 μM for UDP-glucose (in 50 mM Trien- HC1 buffer, pH 8.7, 30 °C, with 0.5 mM NAD ⁺ and 2 mM DTT). The studies on the enzymatic reaction in H₂¹⁸0 showed that only a single solvent-derived oxygen atom is incorporated into the product UDP-glucuronic acid, suggesting that an aldehyde intermediate is involved in the reaction mechanism as opposed to an imine intermediate linked via a lysine residue. The role of the conserved cysteine residue was explored using the two mutant enzymes, Cys260Ser and Cys260Ala. The dramatic loss of activity with the mutant enzymes indicates that Cys260 is catalytically important. The direct observation of a covalent adduct generated from the incubation of the Cys260Ser mutant with NAD+ and either UDP-glucose or UDP-gluco-hexodialdose (UDPGlc- 6-CHO) is the best evidence to date for covalent catalysis in the mechanism of UDPglucose dehydrogenase. Ser260 was identified as the covalently labeled amino acid residue by peptic digestion of the enzyme-adduct and analysis of the peptide mixture using neutral loss mass spectrometry coupled to HPLC. The formation of an ester intermediate from the attachment of a UDP-sugar to the serine residue of the Cys260Ser mutant convincingly supports the involvement of a thioester intermediate in the mechanism employed by the wild type enzyme. UDP-(6,6-di-²H)glucose was synthesized to investigate the two hydride transfer steps in the reaction catalyzed by the wild type enzyme. No primary kinetic isotope effect was observed indicating that neither of the two hydride transfer steps is rate-limiting. The best candidate for the rate-limiting step is the hydrolysis of the thioester intermediate. The fact that the Cys260Ala mutant readily oxidizes UDP-Glc-6-CHO indicates that in the absence of an active site nucleophile the mutated dehydrogenase is capable of catalyzing the second oxidation step without the involvement of covalent catalysis. The oxidation is very likely to occur through the hydrated form of the aldehyde that resembles the thiohemiacetal intermediate. The observation that the Cys260Ala mutant does not appreciably catalyze the oxidation of UDP-glucose could be explained if the aldehyde intermediate is tightly bound in the mutant active site and there is no mechanism by which it can be hydrated and proceed forward in the second oxidation step. NADH formed from the first oxidation step is proposed to be released after the formation of the (thio)hemiacetal intermediate.
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