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

Valarie L. Tlapak-Simmons,

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 Ki values of 2-5 mM). In contrast, Zn2+ and Cu2+ inhibited more potently (apparent Ki ≤ 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.