Detection of covalent and noncovalent intermediates in the polymerization reaction catalyzed by a C149S class III polyhydroxybutyrate synthase.

Ping Li, Sumit Chakraborty, JoAnne Stubbe

文献索引：Biochemistry 48 , 9202-9211, (2009)

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摘要

Polyhydroxybutyrate (PHB) synthases catalyze the conversion of 3-hydroxybutyryl coenzyme A (HBCoA) to PHB with a molecular mass of 1.5 MDa. The class III synthase from Allochromatium vinosum is a tetramer of PhaEPhaC (each 40 kDa). The polymerization involves covalent catalysis using C149 of PhaC with one PHB chain per PhaEC dimer. Two mechanisms for elongation have been proposed. The first involves an active site composed of two monomers in which the growing hydroxybutyrate (HB) chain alternates between C149 on each monomer. The second involves C149 and covalent and noncovalent (HB)(n)CoA intermediates. Two approaches were investigated to distinguish between these models. The first involved the wild-type (wt) PhaEC primed with sTCoA [a CoA ester of (HB)(3) in which the terminal HO group is replaced with an H] which uniformly loads the enzyme. The primed synthase was reacted with [1-(14)C]HBCoA by a rapid chemical quench method and analyzed for covalent and noncovalent intermediates. Radiolabel was found only with the protein. The second approach used C149S-PhaEC which catalyzes polymer formation at (1)/(2200) of the rate of wt-PhaEC (1.79 min(-1) vs 3900 min(-1)). C149S-PhaEC was incubated with [1-(14)C]HBCoA and chemically quenched on the minute time scale to reveal noncovalently bound [1-(14)C](HB)(2)CoA and (HB)(3)CoA as well as covalently labeled protein. Synthesized (HB)(n)CoA (n = 2 or 3) was shown to acylate PhaEC with rate constants of 1-2 min(-1), and these species were converted into polymer. Thus, the (HB)(n)CoA analogues function as kinetically and chemically competent intermediates. These results support the mechanism involving covalently and noncovalently bound intermediates.