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

Abstract

a b s t r a c tWe determined the kinetics of the reaction of human neuronal enolase and yeast enolase 1 with the slowly-reacting chromophoric substrate D-tartronate semialdehyde phosphate (TSP), each in tris (tris (hydroxymethyl) aminomethane) and another buffer at several Mg 2+ concentrations, 50 or 100 lM, 1 mM and 30 mM. All data were biphasic, and could be satisfactorily fit, assuming either two successive first-order reactions or two independent first-order reactions. Higher Mg 2+ concentrations reduce the relative magnitude of the slower reaction. The results are interpreted in terms of a catalytically significant interaction between the two subunits of these enzymes.

A Unique Small Molecule Inhibitor of Enolase Clarifies Its Role in Fundamental Biological Processes

“…PhAH is thought to mimic the aci-carboxylate form of the intermediate carbanion in the reaction and is only applicable for crystallographic studies (e.g., ref ). Another two substrate analogues were developed, but these were only applied for direct spectrophotometric titration of the enolase active site and stopped-flow studies of enzyme kinetics ( d -tartronate semialdehyde phosphate and 3-aminoenolpyruvate phosphate). Moreover, these substrate analogues are not commercially available.…”

Thermal unfolding of apo- and holo-enolase from Saccharomyces cerevisiae: Different mechanisms, similar activation enthalpies

“…The second Mg 2+ atom forms a bridge with the oxygen atoms of Ser39 as well as with the oxygen atoms from the carboxylate and phosphate groups of the substrate/product [9][10][11][12][13][14][15]. Some reports indicate that Mg 2+ concentrations over 1 mM inhibit catalysis, probably due to a reduction in the rate of product release [16]. Structurally, enolase is a dimeric protein where each monomer has a large domain (C-terminal) with an ˛˛ˇˇ (˛ˇ) 6 barrel folding-type and a small domain (N-terminal) with 3 ˛-helices and 4 ˇ-sheets.…”