Investigation of the kinetic mechanism of cytidine 5 '-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi with new insights onrate-limiting steps from product inhibition analysis

The presence of sialic acid as a component of cell surface lipooligosacchar ides or capsular polysaccharides has been shown to be correlated with the v irulence of a number of Gram-negative mucosal pathogens, including several Haemophilus and Neisseria spp. As part of our efforts to evaluate the role of sialic acid in the pathobiology of these organisms, we have initiated st udies of the enzymes from Haemophilus ducreyi (the infectious agent of chan croid) responsible for the activation and attachment of sialic acid to the lipooligosaccharide. In this report, we describe results of an investigatio n of the steady-state kinetic mechanism of the activating enzyme, cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase. Using a co mbination of initial velocity, product inhibition, and dead-end inhibition studies, the reaction is shown to be freely reversible and to proceed throu gh an ordered bi-bi kinetic mechanism in which CTP binds first and CMP-NeuA c dissociates last. In addition, a detailed analysis of the kinetic express ions for the observable constants is presented showing how the variation in apparent product inhibition constants (K-ii) can be used to predict the ra te-limiting step in k(cat) which appears to be dissociation of CMP-NeuAc in this enzyme. To our knowledge, this relationship has not been previously r ecognized.