Muscle Phosphoglycerate Mutase Research Articles

Amino acid sequences of active-site histidine peptides from rabbit muscle phosphoglycerate mutase

Monophosphogiyc~rate mutase (MPGM) catalyses the interconversion of 3-phosphoglycerate and 2-phosphoglycerate, in a reaction mechanism involving a pl~osphohistidine illter~nediate [ 1 ,I?]. A correlation of the 2.8 _& resolution electron-density map ffom X-ray crystallographic studies ofyeast MPGM with its amino acid sequence shows 2 histidines at the active site [3]. Structural studies of muscle MGP~I are much less complete, although the enzyme has been used extensively in kinetic work, e.g. [4,5]. The muscle and yeast enzymes have been shown to differ in several respects. The muscle enzyme is a dimer with identical subunits of mol. wt 28 000 [6], whereas the yeast enzyme is isolated as a tetramer composed of 4 identical subunits of mol. wt 27 000 [7]. The activity of the muscle enzyme is sensitive to modification of cysteinyl and arginyl residues [8,9], in contrast to the yeast enzyme that has no thiol [lo], and is only moderately sensitive to butanedione [l l]. In addition, a phosphohistidine peptide isolated from chicken breast muscle MGPM has been sequenced [ 121, and shows no homology with any of the 4 histidine sequences of the yeast enzyme (see table 2). We report here the purification and determination of the amino acid sequences of histidine-containing peptides from rabbit muscle MPGM to obtain an estimate of the extent of structural similarity at the active sites of the muscle and yeast enzymes.

Detection of an essential sulfhydryl group in phosphoglycerate mutase with an affinity-labeling reagent.

N-Bromoacetylethanolamine phosphate rapidly and irreversibly inactivates rabbit muscle phosphoglycerate mutase. At high molar ratios of reagent to enzyme, loss of activity (both mutase and phosphatase) approximates pseudo-first order kinetics. A rate-saturation effect is observed with half-maximal rate of inactivation occurring at 0.32 mM reagent, a value close to the Km for 3-phosphoglyceric acid. This datum and the dissociation constant of the 2,3-bisphosphoglycerate-enzyme complex, as determined from inactivation kinetics in the presence of the bisphosphate, suggest that the reagent reacts at the substrate binding site. Inactivation results from the covalent incorporation of about 0.8 mol of reagent/mol of catalytic subunit as determined with 14C-labeled reagent. Incorporation is negligible in the presence of substrate and is reduced 8-fold in the presence of 6 M urea. From amino acid analyses on acid hydrolysates of the inactivated enzyme, we have identified a sulfhydryl group as the site of alkylation. A peptide containing the essential sulfhydryl group has been isolated from a tryptic digest of the enzyme inactivated with labeled reagent; its amino acid composition is Trp1, Lys1,-Cys(Cm)1, Asp1, Ser1, Glu2, Gly1, Ala1, Leu1, Phe2.

Rates of phosphorylation and dephosphorylation of phosphoglycerate mutase and bisphosphoglycerate synthase.

Phosphoglycerate mutase and bisphosphoglycerate synthase (mutase) can both be phosphorylated by either glycerate-1,3-P2 or glycerate-2,3-P2 to form phosphohistidine enzymes. The present study uses a rapid quench procedure to determine if, for each enzyme, the formation of the phosphorylated enzyme and phosphate transfer from the enzyme can occur at rates consistent with the overall reactions. With bisphosphoglycerate synthase from horse red blood cells (glycerate-1,3-P2 leads to glycerate-2,3-P2) at pH 7.5, 25 degrees, phosphorylation of the enzyme appears rate-limiting, k = 13.5 s-1, compared with kcat = 12.5 s-1 for the overall synthase rate. Phosphoryl transfer from the enzyme to phosphoglycerate occurs at 38 s-1 at 4 degrees and was too fast to measure at 25 degrees. With chicken muscle phosphoglycerate mutase the half-times were too short to measure under optimal conditions. The rate of enzyme phosphorylation by glycerate-2,3-P2 at pH 5.5, 4 degrees, could account for the overall reaction rate of 170 s-1. The rate of phosphoryl transfer from the enzyme to glycerate-3-P was too rapid to measure under the same conditions. It is concluded that the phosphorylated enzymes have kinetic properties consistent with their participation as intermediates in the reactions catalyzed by these enzymes.

Inhibition of glycolytic enzymes by 2-phosphotartronate

2-Phosphotartronate has been synthesized by permanganate oxidation of glycerol 2-phosphate and has been tested as an inhibitor of five glycolytic enzymes that bind phosphoglycerate or phosphoglycollate. Competitive inhibition of rabbit muscle phosphoglycerate mutase, enolase and pyruvate kinase was observed. Triose phosphate isomerase and 3-phosphoglycerate kinase were not inhibited.

The effects of mercury and other resgents on Phosphoglycerate mutase−2,3−diphosphoglycerate Phosphatase from kidney, muscle and other tissues

Phosphoglycerate mutase (EC 2.7.5.3) was purified 2000-fold from pork kidney. The molecular weight estimated by Sephadex gel filtration was approx. 65000. This is in close agreement with other animal phosphoglyceromutases. The previously observed Hg 2+−induced reversible transformation of chicken and rabbit muscle phosphoglycerate mutase (EC 2.7.5.3) into 2,3-diphosphoglycerate mutase but does not occur with purified pork kidney phosphoglycerate mutase or with preparations of phosphoglycerate metase from beef liver, beef brain and human red cells. The uniqueness of the bahavvior of muscle phosphoglycerate mutase from a number of species is illustrated. On the other hand, the insensitivity of phosphoglycerate mutase from other tissues to Hg 2+ is typified by the kidney enzyme. Pyrophosphate and phosphoglycolate stimulated the 2,3-diphosphoglycerate phosphatase activity of purified preparations of phosphoglycerate mutase from pork kidney and chicken breast. The mutase activity was unaffected by these reagents.

The Purification and Properties of Phosphoglycerate Mutase from Chicken Breast Muscle

Abstract A very rapid procedure for the preparation of phosphoglycerate mutase from chicken breast muscle is presented. Commercially available frozen chicken breasts may be used. The enzyme is obtained in high yields, and the specific activity of the crystalline homogenous preparations is equal to that of the crystalline enzymes from yeast and rabbit muscle. The chicken breast enzyme requires 2,3-diphosphoglycerate for activity, and it is very similar in kinetic and molecular properties to the rabbit muscle phosphoglycerate mutase. Similarly, it also possesses 4 sulfhydryl groups per mole of enzyme. The molecular weight of the enzyme is 65,690. With the use of a gel filtration technique, it has been demonstrated that 2 moles of 2,3-diphosphoglycerate may be bound per mole of the enzyme. It appears that the binding of this phosphoglycerate may entail phosphoenzyme formation. Again, as with other pure phosphoglycerate mutases, the chicken breast muscle mutase shows some 2,3-phosphoglycerate phosphatase activity (about 1/40,000 the mutase activity).