Kinetic Characterization of the Recombinant Hyaluronan Synthases from Streptococcus pyogenes and Streptococcus equisimilis *

The two hyaluronan synthases (HASs) fromStreptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia coli as recombinant proteins containing His6 tails. The accompanying paper has described 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 μm for spHAS and 51 ± 5 μm for seHAS. In both cases, theV max profiles at various concentrations of UDP-GlcNAc were hyperbolic, with no evidence of cooperativity. In contrast, membrane-bound spHAS, but not seHAS, showed sigmoidal behavior as the UDP-GlcNAc concentration was increased, with a Hill number of ∼2, indicating significant cooperativity. The Hill number for UDP-GlcNAc utilization by seHAS was 1, confirming the lack of cooperativity for UDP-GlcNAc in this enzyme. The K m values for UDP-GlcNAc were 60 ± 7 μm for seHAS and 149 ± 3 μm for spHAS in the isolated membranes. The kinetic characteristics of the two affinity-purified HAS enzymes were assessed in the presence of cardiolipin after 8–9 days of storage at –80 °C without cardiolipin. With increasing storage time, the enzymes showed a gradual increase in their K m values for both substrates and a decrease inV max. Even in the presence of cardiolipin, the detergent-solubilized, purified HASs had substantially higherK m values for both substrates than the membrane-bound enzymes. The K UDP-GlcUA for purified spHAS and seHAS increased 2–4-fold. TheK UDP-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 ∼50% lower than those for the membrane-bound enzymes. Significantly, purified spHAS displayed the same cooperative interaction with UDP-GlcNAc (n H ∼ 2), whereas purified seHAS showed no cooperativity.