Clustered Conserved Cysteines in Hyaluronan Synthase Mediate Cooperative Activation by Mg Ions and Severe Inhibitory Effects of Divalent Cations.

Hyaluronan synthase (HAS) uses UDP-GlcUA and UDP-GlcNAc to make hyaluronan (HA). HAS (SeHAS) contains four conserved cysteines clustered near the membrane, and requires phospholipids and Mg for activity. Activity of membrane-bound or purified enzyme displayed a sigmoidal saturation profile for Mg with a Hill coefficient of 2. To assess if Cys residues are important for cooperativity we examined the Mg dependence of mutants with various combinations of Cys-to-Ala mutations. All Cys-mutants lost the cooperative response to Mg. In the presence of Mg, other divalent cations inhibited SeHAS with different potencies (Cu~Zn >Co >Ni >Mn >Ba Sr Ca). Some divalent metal ions likely inhibit by displacement of Mg-UDP-Sugar complexes (e.g. Ca, Sr and Ba had apparent K values of 2-5 mM). In contrast, Zn and Cu inhibited more potently (apparent K ≤ 0.2 mM). Inhibition of Cys-null SeHAS by Cu, but not Zn, was greatly attenuated compared to wildtype. Double and triple Cys-mutants showed differing sensitivities to Zn or Cu. Wildtype SeHAS allowed to make HA prior to exposure to Zn or Cu was protected from inhibition, indicating that access of metal ions to sensitive functional groups was hindered in processively acting HA•HAS complexes. We conclude that clustered Cys residues mediate cooperative interactions with Mg and that transition metal ions inhibit SeHAS very potently by interacting with one or more of these -SH groups.