Pyruvate carboxylase : aspects of the reaction mechanism

Abstract

I. The interaction between pyruvate carboxylase and Mg2+ and acetyl CoA has been investigated. Using indirect binding studies the ka and nH values for acetyl coÀ have been determined in the presence and absence of Mg2. The results have shown that the activator which is present in the enzyme assay solution is Mg2+-acetyl CoA. This allosteric effector binds to the enzyme in a Michaelis-Menten manner. The site of interaction of Mg2 with acetyl CoA is the 3' ribose phosphate on the adenosine moiety. It has also been shown that acetyl CoA also binds to the enzyme non-cooperatively and thus the observed Hill coefficient has been attributed to be due to a function of the non-saturation of substrates and dilution inactivation of the enzyme at low concentrations of acetyl CoA. 2. The kinetics of oxalate inhibition have been characterized in order to gain further insight on the possible role of the bound manganese metal ion in the catalytic reaction. There is evidence to suggest that biotin-CO2 binding sites exist and that the bound metal ís in close proximity to both the pyruvate binding site and the biotin-CO2 binding region within the transcarboxylation site. Since oxalate exhibited competitive kinetics with respect to pyruvate and biotin-CO2, it was postulated that oxalate was in fact a transitíon state analogue of the carboxyl transfer intermediate. 3. The biotin carboxylation reaction has been reviewed in an attempt to explain the enzyme specific ATP/ADP exchange and the facile reactivity of the biotin cofactor. Phosphointermediates have subsequently been isolated by trapping with diazomethane. These products have been identified as phosphoserine and phosphobiotin. A new reaction mechanism for pyruvate carboxylase has therefore been proposed to incorporate this additional information.