Clustered Conserved Cysteines in Hyaluronan Synthase Mediate Cooperative Activation by Mg2+ Ions and Severe Inhibitory Effects of Divalent Cations.

Abstract

Hyaluronan synthase (HAS) uses UDP-GlcUA and UDP-GlcNAc to make hyaluronan (HA). Streptococcus equisimilis HAS (SeHAS) contains four conserved cysteines clustered near the membrane, and requires phospholipids and Mg²⁺ for activity. Activity of membrane-bound or purified enzyme displayed a sigmoidal saturation profile for Mg²⁺ with a Hill coefficient of 2. To assess if Cys residues are important for cooperativity we examined the Mg²⁺ dependence of mutants with various combinations of Cys-to-Ala mutations. All Cys-mutants lost the cooperative response to Mg²⁺. In the presence of Mg²⁺, other divalent cations inhibited SeHAS with different potencies (Cu²⁺~Zn²⁺ >Co²⁺ >Ni²⁺ >Mn²⁺ >Ba²⁺ Sr²⁺ Ca²⁺). Some divalent metal ions likely inhibit by displacement of Mg²⁺-UDP-Sugar complexes (e.g. Ca²⁺, Sr²⁺ and Ba²⁺ had apparent K_i values of 2-5 mM). In contrast, Zn²⁺ and Cu²⁺ inhibited more potently (apparent K_i ≤ 0.2 mM). Inhibition of Cys-null SeHAS by Cu²⁺, but not Zn²⁺, was greatly attenuated compared to wildtype. Double and triple Cys-mutants showed differing sensitivities to Zn²⁺ or Cu²⁺. Wildtype SeHAS allowed to make HA prior to exposure to Zn²⁺ or Cu²⁺ 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 Mg²⁺ and that transition metal ions inhibit SeHAS very potently by interacting with one or more of these -SH groups.