3‐Phosphoglycerate Kinase from Rabbit Sceletal Muscle and Yeast

Abstract

Phosphoglycerate kinase, isolated from rabbit muscle in highly purified crystalline form, was compared in terms of chemical, physical and kinetic properties to yeast phosphoglycerate kinase. A summary of the data is presented. Both proteins are composed of a single polypeptide chain, and in electrophoresis only one band was found. The N‐terminus is masked. The C‐terminal amino acid of the muscle enzyme is valine. Muscle and yeast enzyme have nearly identical molecular weights, Michaelis‐Menten constants, optimal pH values and Vmax. On the other hand, they differ significantly in their amino acid composition, especially in the content of sulfur and aromatic amino acids, in electrophoretic mobility and in heat stability. The muscle enzyme has an essential SH group. The single SH group of the yeast enzyme is apparently without importance for enzyme activity. Differences in the same direction as those observed with yeast and muscle phosphoglycerate kinase are discussed in connection with other glycolytic enzymes from the same organisms.

Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement

“…The rapid-equilibrium assumption is not valid for AK [25], hence expressions for the steady-state kinetic parameters in terms of the rate constants are : [77], 25 mC, pH 8.0 ; f k cat,r for the most purified human erythrocyte enzyme (4467i) was calculated from the data of Yoshida and Watanabe ( [75] ; specific activity 670 units/mg, pH 7.5, 25 mC) assuming that the M r of the enzyme is 49 600 ; g calculated from k cat,r assuming the activity in the direction of glycolysis is 2.5 times this value [74] ; h the concentration of PGK in human erythrocytes, e o , was estimated from the measured human erythrocyte activity (105 600 units/litre of erythrocytes, 37 mC, pH 7.6 ; [62] …”

Characterisation of yeast phosphoglycerate kinase modified by mutagenesis at residue 21

“…Remaining traces of nucleic acid (indicated by an A280/A260 lower than 1.5) were removed by passage over a Mono Q FPLC column in 20 mM Mes, pH 6.5. The protein concentration was determined using an absorption coefficient of e = 4.9 at 278 nm [16].For use in kinetic and crystallisation experiments the protein was further purified by FPLC on a Superose 12 column run in 20 mM Bistris, pH 6.5. The [R21M]PGK protein was separated from the 5% endogenous wild-type activity by binding to a Mono S FPLC column in 20 mM Bistris, pH 6.5, 0.01 M phenylmethylsulphony1 fluoride and eluted with an optimised gradient of 0 -30 mM NaC1.…”

Phosphoglycerate Kinase B from Ram Testis

“…That all mammalian phosphoglycerate kinases so far analysed contain 12 [5,31] or 13 [6,2,32,33] methionine residues for a molecular weight of 47 000 is remarkable, especially considering that there is only one triplet codon for methionine in the genetic code. Overall the mammalian phosphoglycerate kinase amino acid analyses are very similar and except for sulphurcontaining amino acids, so is yeast [2,34].…”

“…That all mammalian phosphoglycerate kinases so far analysed contain 12 [5,31] or 13 [6,2,32,33] methionine residues for a molecular weight of 47 000 is remarkable, especially considering that there is only one triplet codon for methionine in the genetic code. Overall the mammalian phosphoglycerate kinase amino acid analyses are very similar and except for sulphurcontaining amino acids, so is yeast [2,34]. The similarity of structure of the yeast and horse muscle enzymes from X-ray studies [35,36] further confirm that the basic structure and composition of phosphoglycerate kinase has been strongly conserved throughout evolution.…”

“…When compared to the analyses reported for rabbit [2] and erythrocyte [6] enzymes, it can be seen that there are very few differences in the composition despite the variety of sources from which the enzyme has been purified. This indicates a great degree of conservatism with respect to the composition of phosphoglycerate kinase throughout evolution.…”