Role of Metal Ions in Catalysis by Enolase:  An Ordered Kinetic Mechanism for a Single Substrate Enzyme

Abstract

Spectroscopic and kinetic methods have been used to explore the roles of divalent metal ions in the enolase-catalyzed dehydration of 2-phosphoglycerate (2-PGA). Enolase requires 2 equiv of metal ion per active site for maximal activity. Previous crystallographic studies [Larsen, T. M., Wedekind, J. E., Rayment, I., and Reed, G. H. (1996) Biochemistry 35, 4349-4358] showed that both magnesium ions coordinated to the carboxylate group of the substrate/product-a scheme consistent with metal ion assistance in formation of the enolate intermediate. Electron paramagnetic resonance (EPR) data with 17O-labeled forms of phosphoenolpyruvate show that Mn(2+), bound at the lower affinity site, coordinates to one carboxylate oxygen and one phosphate oxygen of the substrate. These observations are fully consistent with the crystallographic data. Plots of activity versus log [metal ion] are bell-shaped, and the inhibitory phases of the profiles have been previously attributed to binding of metal ions at ancillary sites on the enzyme. However, the activation profiles and measurements of 2H kinetic isotope effects support an ordered kinetic mechanism wherein binding of 2-PGA precedes binding of the second metal ion, and release of the second metal ion occurs prior to departure of phosphoenolpyruvate. High concentrations of metal ion lead to inhibition in the ordered mechanism by interfering with product release. The 2H kinetic isotope effect is diminished in the inhibitory phases of the metal ion activation profiles in a manner that is consistent with the predominantly ordered mechanism. Zn(2+) gives lower maximal activity than Mg(2+), apparently due to slow release of Zn(2+) from the product complex. Addition of imidazole increases the maximal rate apparently by accelerating the release of Zn(2+) from the enzyme.

Stopped‐flow studies of the reaction of d‐tartronate semialdehyde‐2‐phosphate with human neuronal enolase and yeast enolase 1

“…This time would be within the dead time of the stopped-flow, as no lag phase has been observed by them or by us. Our data show no inhibition of the TSP reaction at Mg 2+ concentrations (30 mM) that are inhibitory to the steady-state physiological reaction, that is, there is no evidence for binding of inhibitory Mg 2+ , consistent with the interpretation of Poyner et al [18].…”

“…Originally, we wished to see if Mg 2+ concentrations above 1 mM inhibited the TSP reaction; in other words, is the inhibition of the physiological reaction due to slowing product (PEP) release [5,18], or, a possibility suggested by Faller et al [2], due to binding of a third, inhibitory Mg 2+ at the active site?…”

“…Since substrate and Mg 2+ binding are ordered and interdependent [5,18] that is equivalent to saying that more extensive or complete saturation by catalytic Mg 2+ binding produces the effect.…”

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

“…These results were later proved in Lebioda and Reed laboratories when the crystalline structures of yeast enolase and neuron-specific human enolase were established [2,45,46]. Selective mutagenesis of yeast enolase and its recent crystallographic studies revealed that the basic amino-acids Lys345, Lys396, His159, His373, and Arg374 in the catalytic center are important in the mechanism of the reversible transition of 2-PGA to PEP.…”

“…Magnesium ions play a double role in enolases. The so-called conformational ions ensure the proper spatial structure of the active center, whereas the catalytic ions are necessary for the reaction mechanism [45,46]. They ensure the insertion of the substrate into catalytic pocket and its transformation to the reaction product [50].…”