Model discrimination and nonlinear parameter estimation in the analysis of the mechanism of action of β-hydroxybutyrate dehydrogenase from Rhodopseudomon as spheroides

Abstract

1. 1. β-Hydroxybutyrate dehydrogenase ( d-3-hydroxybutyrate:NAD + oxide-reductase, EC 1.1.1.30) from Rhodopseudomonas spheroides was prepared and initial velocity studies in the presence and absence of product at zero time were performed. 2. 2. The product inhibition pattern was found to be consistent with an ordered sequential mechanism. 3. 3. Nonlinear regression analyses were applied to selected combinations of the velocity data to provide estimation of the parameters, their standard deviations, and confidence limits. 4. 4. Analysis of variance provided a basis for determination of the acceptability of the model and for discrimination of points that showed extreme lack of fit. 5. 5. A data-blocking procedure has been introduced that permits the estimation of parameters using combinations of data sets that were obtained with different concentrations of enzyme. 6. 6. The lack of one-to-one correspondence between the set of kinetic constants and the set of rate constants imposed difficulties in the initial attempt to apply the nonlinear regression analysis to the full rate equation in rate constant form. 7. 7. A reformulation of the kinetic constants based on the requirement for a non-zero Jacobian for the transformation has been developed that provides a one-to-one correspondence with the set of rate constants. 8. 8. A method for the evaluation of the approximate 95% confidence regions of the parameters and for the assessment of the experimental design by means of a determination of the eigenvalues of the solution matrix ( X′ X) is introduced.