Date of Award:

5-1988

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Chemistry and Biochemistry

Advisor/Chair:

Elizabeth Boeker

Abstract

The usual procedure employed in enzyme kinetic analysis is the method of initial rates. However, it has been appreciated for years that the analysis of enzyme-catalyzed reactions could, in principle, be more efficiently performed by examining the entire time course. There is much more information contained in a progress curve than in a simple initial rate. With the appearance of the computer, the formidable computations necessary for the use of integrated-rate equations are quite possible. The intention of this research was to develop the analytical and statistical methodology for applying an integrated-rate equation to a two-substrate reaction. I have analyzed the kinetics of pyruvate reduction, as catalyzed by the rabbit M4 isoenzyme of lactate dehydrogenase. Time courses were carried out, in sextuplicate, by observing the disappearance of NADH. Initial concentrations were: NADH, .026 to 1.7 mM; pyruvate, .016 to .29 mM; NAD+, 0 to 7 mM; and lactate, 0 to 40 mM. The concentrations of pyruvate and/or NAO+ were such that measurable enzyme inactivation did not occur.

For each progress curve, values of Cf, Cs, C1, and C2 in the integrated equation were obtained by nonlinear regression; variances were calculated using replicate observations. Multiple regression, weighting each coefficient according to its variance, then gave 8 of the 11 J coefficients that characterize an ordered ternary-complex mechanism. The values obtained are comparable to previously published initial-rate values and predict progress curves that are consistent with the observed curves. The analysis required as few as nine experiments. A similar initial-rate study would require perhaps 10 times this number.

This research shows that the computations necessary to apply progress curve methods can be routinely computerized; these methods are potentially a very powerful tool when used with the correct analytical techniques and experimental design.

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