Application of progress curve analysis to in situ enzyme kinetics using 1H NMR spectroscopy

Abstract

The steady-state kinetics of enzymes in tissues, cells, and concentrated lysates can be characterized using high-resolution nuclear magnetic resonance spectroscopy; this is possible because almost invariably there are differences in the spectra of substrates and products of a reaction and these spectra are obtainable even from optically opaque samples. We used 1H spin-echo NMR spectroscopy to study the hydrolysis of α- l-glutamyl- l-alanine by cytosolic peptidases of lysed human erythrocytes. Nonlinear regression of the integrated Michaelis-Menten expression onto the progress-curve data yielded, directly, estimates of V max and K m for the hydrolase; a procedure for analyzing progress curves in this manner was adapted and compared with a commonly used procedure which employs the Newton-Raphson algorithm. We also performed a sensitivity analysis of the integrated Michaelis-Menten expression; this yielded equations that indicate under what conditions estimates of K m and V max are most sensitive to variations in experimental observables. Specifically, we showed that the most accurate estimates of the steady-state parameters from analysis of progress curves are obtained when the initial substrate concentration is much greater than K m . Furthermore, estimates of these parameters obtained by such an analysis are most sensitive to data obtained when the reaction is 60–80% complete, having started with the highest practicable initial substrate concentration.