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Properties and kinetic analysis of UDP-glucose dehydrogenase from group A streptococci. Irreversible inhibition by UDP-chloroacetol. 1997

R E Campbell, and R F Sala, and I van de Rijn, and M E Tanner
Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.

UDP-glucuronic acid is used by many pathogenic bacteria in the construction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD+-dependent 2-fold oxidation of UDP-glucose and provides a source of the acid. In the present study the recombinant dehydrogenase from group A streptococci has been purified and found to be active as a monomer. The enzyme contains no chromophoric cofactors, and its activity is unaffected by the presence of EDTA or carbonyl-trapping reagents. Initial velocity and product inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDP-glucuronate is released last. UDP-xylose was found to be a competitive inhibitor (Ki, 2.7 microM) of the enzyme. The enzyme is irreversibly inactivated by uridine 5'-diphosphate-chloroacetol due to the alkylation of an active site cysteine thiol. The apparent second order rate constant for the inhibition (ki/Ki) was found to be 2 x 10(3) mM-1 min-1. Incubation with the truncated compound, chloroacetol phosphate, resulted in no detectable inactivation when tested under comparable conditions. This supports the notion that uridine 5'-diphosphate-chloroacetol is bound in the place of UDP-glucose and is not simply acting as a nonspecific alkylating agent.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008956 Models, Chemical Theoretical representations that simulate the behavior or activity of chemical processes or phenomena; includes the use of mathematical equations, computers, and other electronic equipment. Chemical Models,Chemical Model,Model, Chemical
D009243 NAD A coenzyme composed of ribosylnicotinamide 5'-diphosphate coupled to adenosine 5'-phosphate by pyrophosphate linkage. It is found widely in nature and is involved in numerous enzymatic reactions in which it serves as an electron carrier by being alternately oxidized (NAD+) and reduced (NADH). (Dorland, 27th ed) Coenzyme I,DPN,Diphosphopyridine Nucleotide,Nadide,Nicotinamide-Adenine Dinucleotide,Dihydronicotinamide Adenine Dinucleotide,NADH,Adenine Dinucleotide, Dihydronicotinamide,Dinucleotide, Dihydronicotinamide Adenine,Dinucleotide, Nicotinamide-Adenine,Nicotinamide Adenine Dinucleotide,Nucleotide, Diphosphopyridine
D002850 Chromatography, Gel Chromatography on non-ionic gels without regard to the mechanism of solute discrimination. Chromatography, Exclusion,Chromatography, Gel Permeation,Chromatography, Molecular Sieve,Gel Filtration,Gel Filtration Chromatography,Chromatography, Size Exclusion,Exclusion Chromatography,Gel Chromatography,Gel Permeation Chromatography,Molecular Sieve Chromatography,Chromatography, Gel Filtration,Exclusion Chromatography, Size,Filtration Chromatography, Gel,Filtration, Gel,Sieve Chromatography, Molecular,Size Exclusion Chromatography
D004791 Enzyme Inhibitors Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction. Enzyme Inhibitor,Inhibitor, Enzyme,Inhibitors, Enzyme
D013056 Spectrophotometry, Ultraviolet Determination of the spectra of ultraviolet absorption by specific molecules in gases or liquids, for example Cl2, SO2, NO2, CS2, ozone, mercury vapor, and various unsaturated compounds. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Ultraviolet Spectrophotometry
D014530 Uridine Diphosphate A uracil nucleotide containing a pyrophosphate group esterified to C5 of the sugar moiety. UDP,Uridine Pyrophosphate,Diphosphate, Uridine,Pyrophosphate, Uridine
D014533 Uridine Diphosphate Glucose Dehydrogenase An enzyme that catalyzes the oxidation of UDPglucose to UDPglucuronate in the presence of NAD+. EC 1.1.1.22. UDP Glucose Dehydrogenase,UDPG Dehydrogenase,Dehydrogenase, UDP Glucose,Dehydrogenase, UDPG,Glucose Dehydrogenase, UDP
D014534 UDPglucose 4-Epimerase A necessary enzyme in the metabolism of galactose. It reversibly catalyzes the conversion of UDPglucose to UDPgalactose. NAD+ is an essential component for enzymatic activity. EC 5.1.3.2. UDP Galactose Epimerase,UDP Glucose Epimerase,UDPgalactose 4-Epimerase,Uridine Diphosphate Glucose Epimerase,UDP-Galactose 4-Epimerase,UDP-Glucose 4-Epimerase,4-Epimerase, UDP-Galactose,4-Epimerase, UDP-Glucose,4-Epimerase, UDPgalactose,4-Epimerase, UDPglucose,Epimerase, UDP Galactose,Epimerase, UDP Glucose,Galactose Epimerase, UDP,Glucose Epimerase, UDP,UDP Galactose 4 Epimerase,UDP Glucose 4 Epimerase,UDPgalactose 4 Epimerase,UDPglucose 4 Epimerase
D014540 Uridine Diphosphate Xylose The decarboxylation product of UDPglucuronic acid, which is used for formation of the xylosides of seryl hydroxyl groups in mucoprotein synthesis. Also forms plant xylans. UDP Xylose,Diphosphate Xylose, Uridine,Xylose, UDP,Xylose, Uridine Diphosphate

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