Properties and Kinetic Analysis of UDP-glucose Dehydrogenase from Group A Streptococci

Robert E. Campbell, Rafael F. Sala, Ivo van de Rijn, Martin E. Tanner
1997 Journal of Biological Chemistry  
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
more » ... 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 UDPglucuronate is released last. UDP-xylose was found to be a competitive inhibitor (K i , 2.7 M) 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 (k i /K i ) was found to be 2 ؋ 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.
doi:10.1074/jbc.272.6.3416 pmid:9013585 fatcat:lu5kxvm7y5c75lpt5sshiza7he