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J Biol Chem, Vol. 274, Issue 7, 4246-4253, February 12, 1999
From the Department of Biochemistry and Molecular Biology,
University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma 73190
The two hyaluronan synthases (HASs) from
Streptococcus 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 Km values for
UDP-GlcUA of 40 ± 4 µM for spHAS and 51 ± 5 µM for seHAS. In both cases, the
Vmax 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 Km
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 Km values for both substrates and a decrease in
Vmax. Even in the presence of cardiolipin, the
detergent-solubilized, purified HASs had substantially higher
Km values for both substrates than the
membrane-bound enzymes. The KUDP-GlcUA for
purified spHAS and seHAS increased 2-4-fold. The
KUDP-GlcNAc for spHAS and seHAS increased 4- and 5-fold, respectively. Despite the higher Km values, the Vmax 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 (nH ~ 2), whereas
purified seHAS showed no cooperativity.
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