Reaction intermediate analogs for enolase

Abstract

A number of compounds that appear to be analogues of the aci form of the normal carbanion intermediate are good inhibitors of yeast enolase. These include (3-hydroxy-2-nitropropyl)phosphonate (I), the ionized (pK = 8.1) nitronate form of which in the presence of 5 mM Mg2+ has a Ki of 6 nM, (nitroethyl)phosphonate (III) (pK = 8.5; Ki of the nitronate in the presence of 5 mM Mg2+ = 1 microM), phosphonoacetohydroxamate (IV) (pK = 10.2; Ki with saturating Mg2+ for the ionized form = 15 pM), and (phosphonoethyl)nitrolate (VII) (Ki at 1 mM Mg2+ = 14 nM). The oxime of phosphonopyruvate (VI) has a pH-independent Ki of 75 microM. I, IV, VI, and VII are slow binding inhibitors. All of these compounds are trigonal at the position analogous to C-2 of 2-phosphonoglycerate and contain a phosphono group, but a negatively charged metal ligand at the position isosteric with the hydroxyl attached to C-3 of 2-phosphoglycerate (as in IV) appears to contribute more to binding than a nitro group isosteric with the carboxyl of 2-phosphoglycerate (I and III). These data support the carbanion mechanism for enolase and suggest that the 3-hydroxyl of 2-phosphoglycerate is directly coordinated to Mg2+ prior to being eliminated to give phosphoenolpyruvate.

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

“…Also, we carried out those measurements using solutions that also contained 0.15 M KCl, as this produces a small (15-20%) increase in the rate of the physiological reaction (unpublished observations). In addition, we carried out stopped-flow measurements in which the yeast enzyme in 30 mM Mg 2+ containing subsaturating levels (0.25 and 0.5 moles per mole of active site) of TSP or the very strongly bound competitive inhibitor phosphono-acetohydroxamate [14] were mixed with 30 mM Mg 2+ plus TSP (not shown). In no case did we observe single first-order kinetics; all data were fit, assuming either two successive first order or two parallel firstorder reactions and the rates obtained were consistent with those presented in Table 1.…”

The synthesis of 3‐phosphonoalanine, phosphonopyruvic acid and phosphonolactic acid

“…These authors followed Anderson et al (1984) in preparing phosphonopyruvate by esterifying phosphonoalanine, oxidizing the ester with a quinone, and hydrolysing. A simpler synthesis is that of Schwalbe and Freeman (1990) who hydrolysed thc triester by treatment with Me,SiBr; the triester was prepared (Coutrot et al, 1978) by treating (EtO),P(O) -CH2 -Cu with C1-CO-CO-OEt.…”

“…It was dissolved in 10 in1 water and passed through a column of 13 cm x 1 cm sulphonic resin (Dowex 50 X8, acid form) and washed through with water. The phosphonopyruvic acid was not retarded and w a s recovered (following Anderson et al, 1984) as the trilithium salt. The effluent was evaporated to dryness.…”

Cloning, overexpression and mechanistic studies of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus

“…The column was eluted with a linear gradient of this buffer (10-500 mM, 1 1 + 1 1). Fractions (20 ml) were collected and the A z S s was measured to detect the presence of the pyruvoyl group (Anderson et al, 1984). Fractions containing the desired compound were pooled and concentrated.…”

“…Thc eluate, containing disodium (hydroxyphosphiny1)pyruvate (Scheme 3, compound 4), was concentrated and dissolved in water (50 ml) for assay. Using the semicarbazide assay developed by Anderson et al (1984) for phosphonopyruvate, the yield of (hydroxyphosphiny1) …”