Progress curve analysis in enzyme kinetics: model discrimination and parameter estimation.

Abstract

The method of progress curve analysis for enzyme-catalyzed reactions (Duggleby, R.G. and Morrison, J.F. (1977) Biochim. Biophys. acta 481, 297--312) has been extended to a two substrate, reversible reaction through the use of enzyme-catalyzed recycling of one of the products. The reaction investigated was that catalyzed by aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) and the product, alpha-ketoglutarate was recycled to glutamate using NADH and NH4Cl in the presence of glutamate dehydrogenase. The values determined for the kinetic parameters of the aminotransferase were found to agree well with those obtained from steady-state velocity measurements. The standard errors of the parameters, as calculated by the procedure originally described, were found to underestimate the observed variation between different experiments. Therefore, a procedure of data compression was devised which leads to more realistic values for standard errors. The compressed data obtained with aspartate aminotransferase have been fitted to the integrated rate equations that describe a variety of kinetic mechanisms. The best fit was obtained with the Ping-Pong model which is applicable to the aspartate aminotransferase reaction. Thus, progress curve analysis may be used to determine the kinetic mechanism of, and values of the kinetic parameters associated with, an enyzme-catalyzed reaction.