Pyruvate carboxylase from Thiobacillus novellus: properties and possible function

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Abstract
Pyruvate carboxylase (EC 6.4.1.1) from Thiobacillus novellus (ATCC 8093) was highly purified and found to have a pH optimum of 7.6, a temperature optimum of 25–35 °C, and a requirement for a monovalent and a divalent cation, as well as a CoA derivative, for maximum activity. These were best served by K+, Mg2+, and acetyl-CoA. Km values for pyruvate, ATP, HCO3− and Mg2+ were 0.25, 0.04, 0.27, and 0.44 mM, respectively. Initial velocity plots of increasing acetyl-CoA concentrations gave a sigmoidal curve with Ka of 4.2 μM, and Hill coefficients of 2.2. Plots of fixed acetyl-CoA concentrations against varying concentrations of pyruvate, ATP, or CO2 all gave rectangular hyperbolae. Apart from end products, only hydroxypyruvic acid was found to be inhibitory. The enzyme was very sensitive to mercurials. This enzyme is not believed to serve an anaplerotic function, because of the simultaneous presence of the highly regulated phosphoenolpyruvate carboxylase in the organism. Instead, it may function either to supply oxaloacetate to the citrate cycle or as part of the system that provides reduced NADP+.