The positional isotope exchange technique as a probe of enzymatic mechanisms

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. No positional isotope exchange in the absence of fructose could be detected for sucrose synthetase. It can thus be concluded that either the bond cleavage is not catalyzed in the absence of fructose or the phosphoryl group of the newly formed UDP is rotationally immobilized.;The investigation of the positional isotope exchange catalyzed by D-alanyl-D-alanine ligase verified the reaction follows an ordered kinetic mechanism as was determined in the steady state analysis. It was demonstrated that the formation of the D-alanine-phosphate intermediate must proceed by a two step mechanism involving either two chemical steps or a chemical and conformational step.;The energetics of the ATPase partial reaction catalyzed by site directed mutants of carbamyl phosphate synthetase have been determined. In the absence of added glutamine, the increased hydrolysis of ATP caused by site-directed mutational changes in the small subunit was due to greater access of solvent to the carboxy phosphate intermediate. The inclusion of glutamine further increased the hydrolysis by destabilization of the enzyme-carboxy phosphate complex.