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 constru ction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD(+)-dependent 2-fol d oxidation of UDP-glucose and provides a source of the acid. In the p resent study the recombinant dehydrogenase from group A streptococci h as been purified and found to be active as a monomer. The enzyme conta ins no chromophoric cofactors, and its activity is unaffected by the p resence of EDTA or carbonyl-trapping reagents. Initial velocity and pr oduct inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDP-glucur onate is released last. UDP-xylose was found to be a competitive inhib itor (K-i, 2.7 mu M) of the enzyme. The enzyme is irreversibly inactiv ated by uridine 5'-diphosphate chloroacetol due to the alkylation of a n 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, resu lted in no detectable inactivation when tested under comparable condit ions. This supports the notion that uridine 5'-diphosphate-chloroaceto l is bound in the place of UDP-glucose and is not simply acting as a n onspecific alkylating agent.