| Abstract: | The mechanism of oxaloacetate decarboxylase of Klebsiella aerogenes was investigated by enzyme kinetic methods. The activity of the decarboxylase was strictly dependent on the presence of Na+ or Li+ ions. For Li+ the Km was about 17 times higher and the Vmax about 4 times lower than for Na+. No activity was detectable at Na+ concentrations less than 5 microM. The curve for initial velocity versus Na+ concentration was hyperbolic. Initial velocity patterns with oxaloacetate or Na+ as the varied substrate at various fixed concentrations of the cosubstrate produced a pattern of parallel lines which is characteristic for a ping-pong mechanism. Product inhibition by pyruvate was competitive versus oxaloacetate and noncompetitive versus Na+. Oxalate, a dead-end inhibitor, was competitive versus oxaloacetate and uncompetitive versus Na+. The inhibition patterns are not consistent with a ping-pong mechanism comprising a single catalytic site but are analogous to kinetic patterns observed with the related biotin enzyme transcarboxylase, for which a catalytic mechanism at two different and independent sites has been demonstrated. The kinetic and other data support an oxaloacetate decarboxylase mechanism at two different sites of the enzyme with the intermediate formation of a carboxybiotin-enzyme complex. The first site is the carboxyltransferase which is localized on the alpha chain and the second site is the carboxybiotin-enzyme decarboxylase which is probably localized on the beta and/or gamma subunit. Binding studies with oxalate indicated that this is bound with high affinity to the alpha chain. The affinity was not affected by Na+ or by complex formation with the beta and gamma subunits. Oxalate protected the decarboxylase from heat inactivation but not from tryptic hydrolysis. The carboxybiotin-enzyme intermediate prepared from oxaloacetate decarboxylase with high specific activity was rapidly decarboxylated in the presence of Na+ ions alone. The effect of pyruvate on this reaction, noted previously, probably results from inhomogeneity of the enzyme preparation used which contained a considerable amount of free alpha subunits. |
