Properties and kinetic analysis of UDP-glucose dehydrogenase from group A streptococci. Irreversible inhibition by UDP-chloroacetol.
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.
Campbell
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RE
Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
Sala
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RF
van de Rijn
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Tanner
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ME
D002850:Chromatography, Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014534:UDPglucose 4-Epimerase / Q000037:antagonists & inhibitors*; D014530:Uridine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000378:metabolism*; D014540:Uridine Diphosphate Xylose / Q000378:metabolism
D004791:Enzyme Inhibitors; D009243:NAD; C064064:uridine 5'-diphosphate chloroacetol; D014540:Uridine Diphosphate Xylose; D014530:Uridine Diphosphate; D014533:Uridine Diphosphate Glucose Dehydrogenase; D014534:UDPglucose 4-Epimerase