Quantitative full time course analysis of nonlinear enzyme cycling kinetics

Abstract

Enzyme inhibition due to the reversible binding of reaction products is common and underlies the origins of negative feedback inhibition in many metabolic and signaling pathways. Product inhibition generates non-linearity in steady-state time courses of enzyme activity, which limits the utility of well-established enzymology approaches developed under the assumption of irreversible product release. For more than a century, numerous attempts to find a mathematical solution for analysis of kinetic time courses with product inhibition have been put forth. However, no practical general method capable of extracting common enzymatic parameters from such non-linear time courses has been successfully developed. Here we present a simple and practical method of analysis capable of efficiently extracting steady-state enzyme kinetic parameters and product binding constants from non-linear kinetic time courses with product inhibition and/or substrate depletion. The method is general and applicable to all enzyme systems, independent of reaction schemes and pathways.

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“…5). These results can be interpreted in terms of product inhibition and/or substrate depletion, mimicking what has been observed for enzymatic reactions [26,27]. The initial velocity decreases as the pH of the incubation medium increases.…”

“…Using this approach, we have observed that the relaxation rate constant decreases with 5-HIAA concentration, suggesting that substrate depletion may account for non-linearity in the kinetic data [26]. The substrate concentration-dependence of the initial velocities obtained from fitting data to Eq.…”

“…(1)) according to a model recently developed to extract steady-state enzyme kinetic parameters [26]:…”

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“…Time courses were fit to the nonlinear function [P] = v 0 /h(1 À e Àht ), where P is the product, v 0 is the initial velocity, and h is the relaxation constant of v 0 that describes nonlinearity in progress curves (Cao and De La Cruz, 2013). Initial velocities were plotted against UDP-glucose concentrations and fit to the Michaelis-Menten equation v = V max [S]/K m + [S], to determine the kinetic constants K m and V max for each concentration of phloretin.…”

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“…Extraneous inhibitors present in unpurified enzyme solutions or in cell extracts systematically affect the quality of kinetic measurements [6]. Product inhibition can usually be ignored in initial-rate measurements but is highly misleading in time-course studies [7]. Compound aggregation can cause enzyme sequestration on the surface of the aggregate particles and is one of the main reasons for promiscuous enzyme inhibition [3].…”