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 (K-i, 2.7 mu 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 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.