On the “phosphoryl-enzyme” of phosphoglycerate kinase

Johnson, Patricia E.; Abbott, Steven; Orr, George A.; Knowles, Jeremy R.

doi:10.1016/s0006-291x(75)80150-1

Cited by 7 publications

(7 citation statements)

References 11 publications

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“…These were shown, however, to be artifacts of contamination by traces of second substrates. Similar explanations of initially reported slow partial reactions have now been recognized in the cases of phosphoribosylpyrophosphate synthetase (Switzer and Simcox, 1974) and phosphoglycerate kinase (Johnson et al, 1975) demonstrating the caution which must be applied when very slow partial reactions are observed. Although the acyl donors can bind to free enzyme, no evidence for an acyl-enzyme could be isolated despite attempts to mimic substrate synergism with a variety of CoA or phosphate analogues.…”

Section: Discussionsupporting

confidence: 71%

Evidence against an acyl-enzyme intermediate in the reaction catalyzed by clostridial phosphotransacetylase

Henkin

Abeles²

1976

Biochemistry

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Clostridial phosphotransacetylase catalyzes acyl group transfer between coenzyme A (CoA) and inorganic phosphate and also the arsenolysis of acetyl-coenzyme A (AcCoA) to yield acetate and CoA-SH. The enzyme mobility on sodium dodecyl sulfate electrophoresis corresponds to a molecular weight of 70 000. Kinetics of both forward and reverse reactions are of the ternary type as previously reported and product inhibition data are consistent with a random binding scheme. One essential sulfhydryl group per 70 000 daltons was inactivated in a pseudo-first-order process by either N-ethylmaleimide or 5,5'-dithiobis (nitrobenzoic acid). Reduction of the rate of this inactivation by 50% in the presence of AcCoA or acetyl phosphate concentrations near their kinetic K values demonstrates binding of these acyl donors in simple enzyme-substrate complexes. Moreover, pulse-chase experiments show these binary complexes to be functional and also show that they do not dissociate rapidly compared with their rates of catalytic turnover. Incubation of the enzyme with 14C-labeled acyl donors failed to produce labeled protein after passage through Sephadex. This was true despite efforts to mimic "substrate synergism" with desulfo-CoA or to compensate for unfavorable equilibria by means of CoA traps. Very slow isotope exchange reactions of 32Pi into acetyl phosphate and [3H]CoA into AcCoA were at first observed. As in the cases of several other enzymes recently reexamined, these were shown on careful inspection to be artifacts of contamination by second substrates. Attempts to detect exchange reactions between acetyl phosphate and Pi, even in the presence of the CoA analogue, desulfo-CoA, were also unsuccessful. Therefore, no evidence for an acyl-enzyme could be detected. Furthermore, our data allow us to develop arguments which, we believe, indicate that an acyl-enzyme intermediate is extremely improbable in the reaction catalyzed by phosphotransacetylase.

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Section: Discussionsupporting

confidence: 71%

Evidence against an acyl-enzyme intermediate in the reaction catalyzed by clostridial phosphotransacetylase

Henkin

Abeles²

1976

Biochemistry

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“…It has previously been shown that the ki- ) and from yeast (Roustan et al, 1973) do catalyze the ATP-ADP exchange, but the reactions are very slow and are stimulated more than 103-fold by 1 mM 3-PGA. We have also demonstrated this exchange for the horse muscle enzyme (Johnson et al, 1975).…”

Section: Resultssupporting

confidence: 58%

“…Moreover, if the mechanism does involve a viable 1 Abbreviations used are: 3-PGA, 3-phospho-D-glycerate; BPGA, 1,3-bisphospho-D-glycerate; E ~P, phosphoryl-enzyme; Pj, inorganic phosphate; EDTA, (ethylenedinitrilo)tetraacetic acid; DEAE, diethylaminoethyl; NAD, nicotinamide adenine dinucleotide; NADH, reduced NAD; ATP, adenosine 5'-triphosphate; ADP, adenosine 5'-diphosphate; phosphoryl-enzyme, then a partial exchange reaction between 1,3-bisphosphoglycerate (BPGA) and [14C]3-PGA should exist in the absence of ADP, and the production of the phosphoryl-enzyme from BPGA should also be possible (see Scheme I). A preliminary report of some of this work has appeared (Johnson et al. 1975).…”

mentioning

confidence: 99%

The "phosphoryl-enzyme" from phosphoglycerate kinase. Appendix: Crystalline 3-phospho-D-glycerate kinase from horse muscle

Johnson¹,

Abbott²,

Orr³

et al. 1976

Biochemistry

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The "phosphoryl-enzyme" prepared from phosphoglycerate kinase and adenosine 5'-triphosphate in the presence of an adenosine 5'-diphosphate trap is shown to contain stoichiometric amounts of 3-phosphoglycerate. This "phosphoryl-enzyme" is chemically competent, but is probably just a tight complex between 1,3-bisphosphoglycerate and the enzyme. The two partial exchange reactions (between adenosine 5'-diphosphate, and adenosine 5'-triphosphate, and between 3-phosphoglycerate and 1,3-bisphosphoglycerate) can both be observed, but their rates are very much slower than the rate of overall catalysis. No substrate analogue was found that accelerated the partial exchange reactions. Catalysis of each of the two exchange reactions and of the kinase reaction coincides after isoelectric focusing of purified enzyme, but the amount of cosubstrate necessary to cause the observed partial exchange rates is so small that these reactions may well be artifactual. The balance of evidence does not support a ping-pong pathway via phosphoryl-enzyme, and the reaction may be a sequential one in which the phosphoryl group is transferred between substrates in a ternary complex. The results point to the dangers in the interpretation of experiments where very small amounts of contaminating cosubstrate can lead to large kinetic effects, and to the possibility of mistaken deductions about the identity of reaction intermediates.

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“…The C-terminal domain binds the nucleotide in a shallow hydrophobic cleft. The distance between these substrates as bound to the enzyme was modeled 6 as approximately 11 Å for the “open form” of PGK, which is too distant for direct phosphoryl transfer between the substrates. , In view of this bilobal shape of PGK, it has been proposed , that the two domains swing together about a hinge region, closing down on substrates and excluding water, and thereby effect catalysis. Such a domain movement to generate a “closed form” for the enzyme would support the sequential pathway for phosphoryl transfer demonstrated for PGK .…”

Section: Introductionmentioning

confidence: 99%

Highly Potent Bisphosphonate Ligands for Phosphoglycerate Kinase

Jakeman

Williamson

et al. 1998

J. Med. Chem.

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We have synthesized a series of novel analogs of 1, 3-bisphospho-D-glyceric acid, 1,3-BPG,3 and evaluated their binding to phosphoglycerate kinase, PGK (EC 2.7.2.3). Nonscissile methanephosphonic acids replace the two phosphate monoesters of 1, 3-BPG and lead to several stable, tight-binding mimics of this intermediate species in glycolysis. Multiple fluorine substitution for hydrogen in the alpha-methylene groups of the phosphonic acid 1, 3-BPG analogs markedly improves their binding to PGK as determined by NMR analysis. The best ligands bind some 50-100 times more strongly than does the substrate 3-phospho-D-glyceric acid and show a requirement for pKa3 to be generally below 6.0, while the presence of a beta-carbonyl group seems to be of secondary importance.

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On the “phosphoryl-enzyme” of phosphoglycerate kinase

Cited by 7 publications

References 11 publications

Evidence against an acyl-enzyme intermediate in the reaction catalyzed by clostridial phosphotransacetylase

Evidence against an acyl-enzyme intermediate in the reaction catalyzed by clostridial phosphotransacetylase

The "phosphoryl-enzyme" from phosphoglycerate kinase. Appendix: Crystalline 3-phospho-D-glycerate kinase from horse muscle

Highly Potent Bisphosphonate Ligands for Phosphoglycerate Kinase

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