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Importance of Product/ Reactant Equilibrium in the Kinetics of the Phosphoglucose Isomerization Reaction by Differential Stopped Flow Microcalorimetry

Published

Author(s)

M Stodeman, Frederick P. Schwarz

Abstract

The kinetics for the isomerization of fructose-6-phosphate to glucose-6-phosphate (F6P -> G6P) by baker's yeast phosphoglucose isomerase in terms of kcat and Km was determined from analysis of Differential Stopped Flow Microcalorimeter (DSFM) measurements using the integrated form of the Michaelis-Menten rate equation. Values for Km (F6P -> G6P) that were determined at 293.4 K, 298.4 K, 303.4 K, and 311.5 K, exhibited a linear dependence on the substrate concentration at each temperature because of the substantial substrate-product equilibrium. The minimum values for Km ranged from 2.62 ± 0.55 mM at 293.4 K to 7.8 ± 4.8 mM at 311.5 K, and were the same as the minimum values for the reverse reaction (G6P -> F6P) at 293.4 K and 298.4 K. Minimum values for kcat increased with temperature, from 2.78 ± 0.34 s-1 at 293.4 K to 11.4 ± 1.0 s-1 at 311.5 K and for the reverse reaction, G6P->F6P, from 0.852+0.086 s-1 at 293.4 K to 1.46 ± 0.06 s-1 at 298.4 K. The enzyme efficiency of 1.47 ± 0.90 X 106 s-1M-1 at 311.5 K is the same as the collision rate of 106 s-1M-1 for a diffusion-controlled process between molecules of the size of the PGI in solution. Equilibrium constants defined as [F6P]/[G6P] were determined from comparison of the values of kcat in both directions and were 0.307 ± 0.053 at 293.4 K and 0.395 ± 0.033 at 298.4 K. The heats of reaction were exothermic in the F6P->G6P direction and increased from -8.96 ± 0.26 kJ mol-1 at 311.5 K to -8.27 ± 0.40 kJ mol-1 at 293.4 K. At 293.4 K, the endothermic heat of reaction in the G6P -> F6P direction was 7.01 ± 0.32 kJ mol-1, a magnitude in fair agreement with the heat of reaction in the F6P -> G6P direction.
Citation
Analytical Biochemistry
Volume
329
Issue
2

Keywords

differential stopped flow microcalorimet, enzyme kinetics, fructose-6-phosphate glucose-6-phosphate, phosphoglucose isomerase, reaction enthalpies, thermodynamics

Citation

Stodeman, M. and Schwarz, F. (2004), Importance of Product/ Reactant Equilibrium in the Kinetics of the Phosphoglucose Isomerization Reaction by Differential Stopped Flow Microcalorimetry, Analytical Biochemistry (Accessed April 19, 2024)
Created June 14, 2004, Updated October 12, 2021