Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates

Abstract

Metal cofactors are necessary for the activity of alkylation by prenyl transfer in enzyme-catalyzed reactions. Rubber transferase (RuT, a cis-prenyl transferase) associated with purified rubber particles from Hevea brasiliensis, Parthenium argentatum and Ficus elastica can use magnesium and manganese interchangably to achieve maximum velocity. We define the concentration of activator required for maximum velocity as [A]max. The AmaxMg2+ in F. elastica (100 mM) is 10 times the AmaxMg2+ for either H. brasiliensis (10 mM) or P. argentatum (8 mM). The AmaxMn2+ in F. elastica (11 mM), H. brasiliensis (3.8 mM) and P. argentatum (6.8 mM) and the AmaxMg2+ in H. brasiliensis (10 mM) and P. argentatum (8 mM) are similar. The differences in AmaxMg2+ correlate with the actual endogenous Mg2+ concentrations in the latex of living plants. Extremely low Mn2+ levels in vivo indicate that Mg2+ is the RuT cofactor in living H. brasiliensis and F. elastica trees. Kinetic analyses demonstrate that FPP–Mg2+ and FPP–Mn2+ are active substrates for rubber molecule initiation, although free FPP and metal cations, Mg2+ and Mn2+, can interact independently at the active site with the following relative dissociation constants KdFPP <KdFPP–Metal <KdE–Metal. Similarly, IPP–Mg2+ and IPP–Mn2+ are active substrates for rubber molecule polymerization. Although metal cations can interact independently at the active site with the relative dissociation constant KdIPP–Metal <KdE–Metal, unlike FPP, IPP alone does not interact independently. All three RuTs have similar characteristics—indeterminate sized products, high KmIPP, high metal [A]max, metal cofactor requirements, and are membrane-bound enzymes.