Characterization of the Purified Hyaluronan Synthase from Streptococcus equisimilis

Abstract

Hyaluronan synthase (HAS) utilizes UDP-GlcUA and UDP-GlcNAc in the presence of Mg²⁺ to form the GAG hyaluronan (HA). The purified HAS from Streptococcus equisimilis (seHAS) shows high fidelity in that it only polymerizes the native substrates, UDP-GlcNAc and UDP-GlcUA. However, other uridinyl nucleotides and UDP-sugars inhibited enzyme activity, including UDP-GalNAc, UDP-Glc, UDP-Gal, UDP-GalUA, UMP, UDP, and UTP. Purified seHAS was ∼40% more active in 25 mM, compared to 50 mM, PO₄ in the presence of either 50 mM NaCl or KCl, and displayed a slight preference for KCl over NaCl. The pH profile was surprisingly broad, with an effective range of pH 6.5−11.5 and the optimum between pH 9 and 10. SeHAS displayed two apparent pK_a values at pH 6.6 and 11.8. As the pH was increased from ∼6.5, both K_m and V_max increased until pH ∼ 10.5, above which the kinetic constants gradually declined. Nonetheless, the overall catalytic constant (120/s) was essentially unchanged from pH 6.5 to 10.5. The enzyme is temperature labile, but more stable in the presence of substrate and cardiolipin. Purified seHAS requires exogenous cardiolipin for activity and is very sensitive to the fatty acyl composition of the phospholipid. The enzyme was inactive or highly activated by synthetic cardiolipins containing, respectively, C14:0 or C18:1(Δ9) fatty acids. The apparent E_act for HA synthesis is 40 kJ (9.5 kcal/mol) disaccharide. Increasing the viscosity by increasing concentrations of PEG, ethylene glycol, glycerol, or sucrose inhibited seHAS activity. For PEGs, the extent of inhibition was proportional to their molecular mass. PEGs with average masses of 2.7, 11.7, and 20 kg/mol caused 50% inhibition of V_max at 21, 6.5, and 3.5 mM, respectively. The apparent K_i values for ethylene glycol, glycerol, and sucrose were, respectively, 4.5, 3.3, and 1.2 mM.