Enzymological Characterization of thePasteurella multocidaHyaluronic Acid Synthase

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Abstract
Hyaluronic acid (HA), a linear polysaccharide composed of alternating glucuronic acid and N-acetylglucosamine residues, is an essential molecule of higher vertebrates. The fowl cholera pathogen Pasteurella multocida Carter Type A also produces HA in the form of an extracellular capsule in order to evade host defenses. HA synthase activity could be obtained from cell-free membrane preparations of P. multocida. The enzyme utilized UDP-sugar precursors of HA in the presence of Mg2+ or Mn2+ at neutral pH. Mn2+ at 1 mM stimulated ∼2-fold more incorporation than Mg2+ at 10 mM. On the other hand, the analogous enzyme from group A Streptococcus, HasA, is stimulated more by Mg2+ than Mn2+. The apparent Michaelis constants, KM, of the P. multocida HA synthase for UDP-N-acetylglucosamine and UDP-glucuronic acid were estimated to be ∼75 and ∼20 μM, respectively, in the presence of Mg2+, which suggests that the substrates are bound with 2−3-fold higher affinity than by the HasA enzyme. The rate enhancement observed with Mn2+ is apparently not due to better binding of the sugar nucleotide precursors complexed to Mn ion because the KM value, a measure of substrate affinity, increases by 25−50% in comparison to Mg2+. In summary, the HA synthase from P. multocida, a Gram-negative bacterium, has kinetic optima distinct from those of HasA, the analog from the Gram-positive group A Streptococcus.