Kinetic characterization of the recombinant hyaluronan syntheses from Streptococcus pyogenes and Streptococcus equisimilis

The two hyaluronan synthases (HASs) from Streptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia colt as re combinant proteins containing His(6) tails, The accompanying paper has desc ribed the purification and lipid dependence of both HASs, their preference for cardiolipin, and their stability during storage (Tlapak-Simmons, V. L., Baggenstoss, B. A. Clyne, T., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4239-4245). Kinetic characterization of the enzymes in isolated membranes gave K-m values for UDP-GlcUA of 40 +/- 4 mu M for spHAS and 51 +/- 5 mu M for seHAS, In both cases, the V-max profiles at various concentrations of U DP-GlcNAc were hyperbolic, with no evidence of cooperativity. In contrast, membrane-bound spHAS, but not seHAS, showed sigmoidal behavior as the UDP-G lcNAc concentration was increased, with a Hill number of similar to 2, indi cating significant cooperativity, The Hill number for UDP-GlcNAc utilizatio n by seHAS was I, confirming the lack of cooperativity for UDP-GlcNAc in th is enzyme. The K-m values for UDP-GlcNAc were 60 +/- 7 mu M for seHAS and 1 49 +/- 3 mu M for spHAS in the isolated membranes. The kinetic characterist ics of the two affinity-purified HAS enzymes were assessed in the presence of cardiolipin after 8-9 days of storage at -80 degrees C without cardiolip in. With increasing storage time, the enzymes showed a gradual increase in their K-m values for both substrates and a decrease in V-max. Even in the p resence of cardiolipin, the detergent-solubilized; purified HASs had substa ntially higher K-m values for both substrates than the membrane-bound enzym es, The KUDP-GlcUA for purified spHAS and seHAS increased 2-4-fold. The KUD P-GlcNAc for spHAS and seHAS increased 4- and 5-fold, respectively. Despite the higher K-m values, the V-max values for the purified HASs were only si milar to 50% lower than those for the membrane-bound enzymes. Significantly , purified spHAS displayed the same cooperative interaction with UDP-GlcNAc (n(H) similar to 2), whereas purified seHAS showed no cooperativity.