Phylogeny and ontogeny of the phosphoglycerate mutases.--V. Inactivation of phosphoglycerate mutase isozymes by histidine-specific reagents

1. The three isozymes of glycerate-2,3-P2 dependent phosphoglycerate mutase present in tissues of mammals and reptiles were inactivated by both treatment with diethylpyrocarbonate and photooxidation with rose bengal. 2. Inactivation of type M isozyme purified from rabbit muscle was complete when two histidine residues per enzyme subunit were carboethoxylated. Hydroxylamine removed the carboethoxy groups, with partial recovery of the enzymatic activity. The cofactor protected the enzyme against inactivation. 3. The inactivation of rabbit muscle phosphoglycerate mutase by photooxidation with methylene blue and rose bengal was sharply pH dependent. The pH profile of enzyme inactivation followed the titration curve of histidine, suggesting that this amino acid was critical for enzyme activity. Glycerate-2,3-P2 did not protect phosphoglycerate mutase against photoinactivation.     

Multiple forms of human phosphoglycerate kinase.

Phosphoglycerate kinase was isolated by affinity chromatography from human skeletal muscle and erythrocytes. As in the tissue extracts, the purified enzyme showed in Cellogel electrophoresis one major and two minor bands with phosphoglycerate kinase activity. The multiple forms were separated by chromatography on CM-Sepharose. From the three separated forms, A, B, and C, the latter was not detectable in electrophoresis of tissue extracts or in the purified unresolved phosphoglycerate kinase. The faintest, most anodically migrating form observed in the tissue extracts could not be isolated in pure form by chromatography on CM-Sepharose. The electrophoretic mobility of the phosphoglycerate kinase forms depended strongly on the buffer systems used. The different forms had identical molecular weight, substrate affinity, and heat stability and were inhibited to the same extent by antibody. They could also not be separated by column affinity chromatography. Small differences were found in thiol group content and in the specific activity, the latter being a consequence of diminished free sulfhydryl residues. Exposure to either reductive or oxidative conditions changed the specific activity, but did not result in interconversion among the pure forms. The multiple forms probably arise as a result of epigenetic factors occurring after the primary polypeptide chain has been synthesized.     

Sequence and structure of yeast phosphoglycerate kinase.

The structure of yeast phosphoglycerate kinase has been determined with data obtained from amino acid sequence, nucleotide sequence, and X-ray crystallographic studies. The substrate binding sites, as deduced from electron density maps, are compatible with known substrate specificity and the stereochemical requirements for the enzymic reaction. A carboxyl-imidazole interaction appears to be involved in controlling the transition between the open and closed forms of the enzyme.     

Anion binding to yeast phosphoglycerate kinase.

The single thiol of yeast phosphoglycerate kinase was labelled with the chromophoric sulfhydryl reagent, 2-chloromercuri-4-nitrophenol. Sequential additions of individual anions to this modified enzyme brought about a decrease in absorbance at 410 nm that reflected the degree of saturation of the enzyme with anion. The binding curves were analyzed to determine the dissociation constants of a number of anions with charges varying from--1 to--4.1. A linear relationship was found between the charge of the anion and the negative logarithm of the dissociation constant for the labelled enzyme-anion complex. The highly charged anions, such as ATP, bound more tightly than did anions with less charge, such as Cl-. The average number of binding sites for those anions for which accurate results could be obtained was 1.06 mol per 47000 g of enzyme. Several lines of evidence suggested that titration of the active center was not being monitored. Anions bound to phosphoglycerate kinase decreased the rate of reaction between the enzyme thiol and 5,5'-dithiobis(2-nitrobenzoic acid). The relationship between the degree of saturation of the anion binding site and the reaction rate constant was used to calculate the dissociation constant between anion and enzyme. Dissociation constants determined in this manner were in good agreement with those determined by titration of the enzyme-mercurial complex.     

Stereospecificity of hydrogen transfer by phosphoglycerate dehydrogenase.

Phosphoglycerate dehydrogenase (EC 1.1.1.95) has been shown to be A site specific in its hydrogen transfer capacity unlike other dehydrogenases which use phosphorylated substrates. The experiments have been carried out using a coupled assay system with yeast alcohol dehydrogenase. The specific activity measurements of the reaction products indicate the possible influence of an isotope effect on this system.     

The two analogous phosphoglycerate mutases of Escherichia coli.

The glycolytic enzyme phosphoglycerate mutase exists in two evolutionarily unrelated forms. Vertebrates have only the 2,3-bisphosphoglycerate-dependent enzyme (dPGM), whilst higher plants have only the cofactor-independent enzyme (iPGM). Certain eubacteria possess genes encoding both enzymes, and their respective metabolic roles and activities are unclear. We have over-expressed, purified and characterised the two PGMs of Escherichia coli. Both are expressed at high levels, but dPGM has a 10-fold higher specific activity than iPGM. Differential inhibition by vanadate was observed. The presence of an integral manganese ion in iPGM was confirmed by EPR spectroscopy.     

Electrophoresis of phosphoglycerate kinase

A technique for the visualization of phosphoglycerate kinase on starch gel after electrophoresis is described. Three bands of activity were found in hemolysates prepared from normal red cells. When ATP, a substrate of the enzyme, was incorporated into the gel, only a single band was found. This suggested that ATP complexed with the enzyme and/or produced configurational changes. Incidentally, it was found that ATP markedly altered the electrophoretic mobility of hemoglobin. Red cells of 92 Caucasian males, 121 Caucasian females, 114 Negro males, 10 Negro females, 4 Oriental males, and 4 Oriental females were examined. No evidence of an electrophoretic polymorphism of this enzyme was found. Patterns of activity similar to those found in red cells were found in liver, heart, kidney, and skeletal muscle.This work was supported, in part, by Public Health Service Grant No. 07449 from the National Heart Institute, NIH. Presented, in part, at the annual meeting of the American Society of Human Genetics, Austin, Texas, October 12, 1968.     

Magnetic resonance studies on manganese-nucleotide complexes of phosphoglycerate kinase.

Measurements of the relaxation rate of water protons (PRR) have been used to study the interaction of yeast phosphoglycerate kinase with the manganous complexes of a number of nucleotides. The results indicate that phosphoglycerate kinase belongs to the same class of enzymes as creatine kinase, adenylate kinase, formyltetrahydrofolate synthetase, and arginine kinase, with maximal binding of metal ion to tne enzyme in the presence of the nucleotide substrate. However, an analysis of titration curves for a number of nucleoside diphosphates (ADP, IDP, GDP) showed that there is a substantial synergism in binding of the metal ion and nucleotide to the enzyme in the ternary complex. The metal-substrate binds to the enzyme approximately two orders of magnitude more tightly than the free nucleotide; Other evidence for an atypical binding scheme for Mn(II)-nucleoside diphosphates was obtained by electron paramagnetic resonance (EPR) studies; the EPR spectrum for the bound Mn(II) in the enzyme-MnADP complex differed substantially from those obtained for other kinases. An identical EPR spectrum is observed with the MnADP complex with the rabbit muscle enzyme as with the yeast enzyme. In contrast, the dissociation constant for the enzyme-MnATP complex is approximately fourfold lower than that for enzyme-ATP, and there are no substantial changes in the electron paramagnetic resonance spectrum of MnATP2- when the complex is bound to phosphoglycerate kinase. A small but significant change in the PRR of water is observed on addition of 3-phosphoglycerate (but not 2-phosphoglycerate) to the MnADP-enzyme complex. However, addition of 3-phosphoglycerate to enzyme-MnADP did not influence the EPR spectrum of the enzyme-bound Mn(II).