Kinetic and mechanistic characterization of the polyhydroxybutyrate synthase from Ralstonia eutropha.
Purified Ralstonia eutropha polyhydroxybutyrate (PHB) synthase from recombinant cells can exist as monomer and dimer. The polymerization reaction catalyzed by this enzyme displays a lag phase, which causes difficulties for kinetic and mechanistic characterization of the enzymatic polymerization reaction. In this study, we developed a method to eliminate the lag phase of PHB synthase by physical means, i.e., adding multihydroxyl compounds to the enzyme solution. This method allows us to recognize the nature of the lag phase as a physical rather than a chemical process. With such lag-phase-free-enzyme, the kinetic properties of the enzyme were investigated. The results indicate that 3-hydroxybutyryl-CoA (3HBCoA) is the optimal substrate for the enzyme. A slower catalytic rate and lower binding ability account for a lower reactivity of 3-hydroxyvaleryl-CoA (3HVCoA) compared to that of 3HBCoA. The change of hydroxyl group from the beta to the gamma position causes dramatic decreases in the binding ability of 4-hydroxybutyryl-CoA (4HBCoA). By using a dilution strategy and size exclusion chromatographic technique, the active form of the enzyme was identified to be the dimeric form. The number of catalytic sites in the dimeric form of the enzyme was examined by comparing the molecular weight of polyhydroxybutyrate as a function of substrate-to-enzyme ratio. The results suggest that the dimeric enzyme has only one catalytic site. A revised model of polymerization reaction catalyzed by R. eutropha PHB synthase is described.
Zhang
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S
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Yasuo
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T
Lenz
R W
RW
Goodwin
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S
D000217:Acyltransferases / Q000737:chemistry*; D002384:Catalysis; D002850:Chromatography, Gel; D003065:Coenzyme A / Q000737:chemistry; D003470:Culture Media; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D020560:Proteobacteria / Q000201:enzymology* / Q000254:growth & development
D003470:Culture Media; D000217:Acyltransferases; C060970:poly-beta-hydroxybutyrate polymerase; D003065:Coenzyme A