The positional isotope exchange technique as a probe of enzymatic mechanisms

Abstract

The positional isotope exchange (PIX) technique has been used to investigate the mechanisms of five enzymes: uridine diphosphoglucose pyrophosphorylase, galactose 1-phosphate uridyltransferase, sucrose synthetase, D -alanyl-D -alanine ligase and carbamyl phosphate synthetase. Applications of the technique have been expanded to include determination of kinetic mechanisms (ordered or random), partitioning ratios of binary and ternary enzyme complexes, and individual rate constants. Combination of the PIX technique and traditional steady state kinetics allowed the determination of the lower limit of release of substrates from the binary and ternary enzyme complexes in the reaction catalyzed by uridine diphosphoglucose pyrophosphorylase. Because it was possible to follow a positional isotope exchange in both the forward and reverse direction, values for all of the individual rate constants were estimated. A positional isotope exchange method was developed for the analysis of enzyme catalyzed reactions which have ping-pong kinetic mechanisms. The application involves the recyclization of the modified enzyme form by inclusion of the unlabeled product. The methodology was applied to galactose-1-phosphate uridyltransferase. The relative rate of product dissociation from the enzyme-product complex was determined. The timing of intermediate formation was probed in the reactions catalyzed by sucrose synthetase and D-alanyl-D-alanine ligase. For both enzymes, the formation of an intermediate prior to the binding of the last substrate was investigated. The reaction of sucrose synthetase has been proposed to proceed by a two step mechanism in which the scissile bond of UDP-glucose is cleaved to form an intermediate which would then react with fructose..