1887

Abstract

To better understand the reasons for the hyper-accumulation of polyhydroxyalkanoate (PHA) in mutant UWD, the kinetic properties of 3-ketothiolase, acetoacetyl-CoA reductase, and -hydroxybutyrate dehydrogenase were examined. The regulation of the condensation of acetyl-CoA mediated by 3-ketothiolase was normal, in that it was negatively regulated by free CoA, but inhibition was overcome by higher concentrations of acetyl-CoA. Acetoacetyl-CoA from this reaction was reduced to 3-hydroxybutyryl-CoA by an NADPH-specific acetoacetyl-CoA reductase. This enzyme also reduced 3-ketovaleryl-CoA derived from the -oxidation of C, C or C -alkanoates, but at only 16% of the rate found with the C-substrate. The acetoacetyl-CoA reductase was determined to be an allosteric enzyme that bound NADPH and acetoacetyl-CoA at multiple binding sites in a general hybrid Ping-Pong random mechanism. The enzyme was negatively regulated by acetoacetyl-CoA, but this was overcome at high concentrations of NADPH. The activity of pyridine nucleotide transhydrogenase was determined to be important for the conversion of NADH in these mutant cells to NADPH and for decreasing the availability of NADP, which was a negative regulator of the acetoacetyl-CoA reductase. The combination of high acetoacetyl-CoA, the UWD mutation, transhydrogenase activity, and high NADPH appeared to be the conditions promoting PHA formation by strain UWD during active growth on glucose. Degradation of PHA in strain UWD did not appear to be regulated at the level of -hydroxybutyrate dehydrogenase. This enzyme was unaffected by NADH, was inhibited only 13% by pyruvate and its activity was enhanced by NADPH. The thiolysis of acetoacetyl-CoA also was unusual, in that 3-ketothiolase was not inhibited by acetoacetyl-CoA, but free CoA was a competitive inhibitor in a bireactant Ping-Pong mechanism. This inhibition was overcome by higher concentrations of the normal first substrate, acetoacetyl-CoA. Thus a single thiolase was used for the condensation of acetyl-CoA and the thiolysis of acetoacetyl-CoA, derived from PHA depolymerization or from the -oxidation of -alkanoates.

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1994-04-01
2023-01-27
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