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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Crop Improvement and Genetics Research » Research » Publications at this Location » Publication #151122
Title: ACTIVATION AND INHIBITION OF RUBBER TRANSFERASES BY METAL COFACTORS AND PYROPHOSPHATE SUBSTRATES

Author
item Scott, Deborah
item DA COSTA, BERNARDO - UC BERKELEY
item ESPY, STEPHANIE - UC BERKELEY
item KEASLING, JAY - UC BERKELEY
item Cornish, Katrina

Submitted to: Phytochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/28/2003
Publication Date: 9/1/2003
Citation: Scott, D.J., Da Costa, B.M., Espy, S.C., Keasling, J.D., Cornish, K. 2003. Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates. Phytochemistry. 64:123-124.

Interpretive Summary: A thorough understanding of rubber biosynthesis is important to the design of new rubber-producing crop plants so that relevant parameters can be altered to improved rubber yield and rubber quality. In this paper, we have clarified, for the first time, the role of the metal cations that are needed for enzyme activity. We find that magnesium is the cation used by the plant although the enzyme can also use mangenese. The monomer for rubber production must complex with the metal before binding to the active site of the enzyme. The initiating substrate can bind with or without complexing with the metal. However, the formation of the initiator-metal complex within the active site is still needed before catalysis can occur. High concentrations of metal and initiator inhibit rubber biosynthesis, but the monomer does not inhibit activity.

Technical 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 [A]maxMg2+ in F. elastica (100 mM) is 10 times the [A]maxMg2+ for either H. brasiliensis (10 mM) or P. argentatum (8 mM). The [A]maxMn2+ in F. elastica (11 mM), H. brasiliensis (3.8 mM) and P. argentatum (6.8 mM) and the [A]maxMg2+ in H. brasiliensis (10 mM) and P. argentatum (8 mM) are similar. The differences in [A]maxMg2+ 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.