Pyruvate carboxylase from Arthrobacter globiformis has been purified approximately 300-fold. The highly purified enzyme has a specific activity of 27.0 nmoles of oxaloacetate formed per mg of protein and gave two bands on SDS-polyacrylamide gel electrophoresis. The pyruvate caxboxylase containing band constituted approximately 80% of the total protein. The enzyme-catalysed reaction requires pyruvate, MgATP and HCO-3 and is activated by Mg2+ and K+ ions. The enzyme is inhibited by preincubation with avidin and by oxalate. The enzyme shows only about of its maximum activity in the absence of acetyl-CoA, which activates the enzyme with an A0.5 value of 25 muM. Activation by this effector is sigmoidal, with a Hill coefficient of about 3.0, and it is antagonised by L-aspartate, which has a Hill coefficient of between 2.0 and 4.0 depending on the concentration of acetyl-CoA. CoA is able to replace acetyl-CoA, with an A0.5 value of 1.3muM and a Hill coefficient of 3.0. Kinetic analysis of the reaction has shown its mechanism to be of the non-classical Ping Pong Bi Bi Uni Uni type with kinetically significant abortive complexes, enzyme-HCO-3-MgADP and enzyme-HCO-3-Pi. Apparent Km values and inhibition constants were determined for the substrates and product inhibitors of the enzyme. The enzyme was inactivated on incubation at 0°C or at pH 8.0, 15°C, with a change in s20,W value in each case which indicated that a halving of molecular weight was occurring. The molecular weight of the inactive form of the enzyme found at pH 8.0 was shown to be 290 000, implying a value of 580 000 for the molecular weight of the native enzyme. The subunit molecular weight of the enzyme was shown by SDS-polyacrylamide gel electrophoresis to be 130 000, suggesting that the native enzyme is tetrameric. This suggestion is supported by the fact that the sedimentation similar to that found for other pyruvate carboxylases which have been shown to be tetrameric. It has been shown that pyruvate carboxylase can exist in biotin-deficient A. globiformis in both apoenzyme and holoenzyme forms. The apoenzyme is converted to holoenzyme on addition of biotin to biotin-deficient cells, in the absence of protein synthesis. The total amount of pyruvate carboxylase in cells of A. globiformis appears to be determined largely by the nature of the carbon source used for growth. During growth at biotin concentrations of between 40 pg/ml and 1 ng/ml, the amount of pyruvate carboxylase holoenzyme appears to be directly dependent upon the concentration of biotin in the medium in cells grown on lactate, glycine and glucose. The results have been discussed by comparison with other pyruvate carboxylases. Kinetic and regulatory properties are broadly similar to those of other pyruvate carboxylases but the structural characteristics are markedly different from those of any other pyruvate carboxylases so far studied.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:674084 |
| Date | January 1973 |
| Creators | Gurr, James Alan |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/35237 |
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