Active site phosphohistidine peptides from red cell bisphosphoglycerate synthase and yeast phosphoglycerate mutase.

C.H Han,Z.B Rose

doi:10.1016/s0021-9258(19)86775-3

Abstract

Bisphosphoglycerate synthase (glycerate-1,3-P2 yields glycerate-2,3-P2) and phosphoglycerate mutase (glycerate-3-P formed from glycerate-2-P) are both phosphorylated by substrates at a histidine residue forming covalent intermediates which have been shown to function in the phosphoryl transfer reactions catalyzed by these enzymes (Rose, Z. B., and Dube, S. (1976) J. Biol. Chem. 251, 4817--4822). We have phosphorylated bisphosphoglycerate synthase from horse red blood cells with [U-32P]glycerate-2,3-P2, digested with trypsin, and purified the phosphopeptide. The amino acid sequence of the phosphohistidine peptide has been determined to be: His-Gly-Gln-Gly-Ala-Trp-Asn-Lys. In like manner, a phosphohistidyl peptide has now been purified from yeast phosphoglycerate mutase, for which the amino acid sequence is known (Winn, S. I., Watson, H. C., Fothergill, L. A., and Harkins, R. N. (1977) Biochem. Soc. Trans. 5, 657-659). The amino acid composition of the phosphopeptide indicates that histidine-8 was phosphorylated. The sequence of this peptide is closely homologous with the active site peptide from bisphosphoglycerate synthase. In yeast phosphoglycerate mutase, the denatured phosphoenzyme hydrolyzes with a single rate constant of 2.02 X 10(-4) s-1 at pH 3, 45 degrees C. The relevance of these observations to the enzymatic mechanism is discussed.

Highlights

Glycerate-2,3-Pz) and phosphoglycerate mutase are both phosphorylated by substrates at a histidine residue forming covalent intermediates which have been shown to function in the phosphoryl transfer reactions catalyzed by these enzymes
Bisphosphoglycerate synthase and phosphoglycerate mutase are both phosphorylated by substrates at a histidine residue forming covalent intermediates which have been shown to function in the phosphoryl transfer reactions catalyzed by these enzymes
Characterization of Histidyl Peptide from Bisphosphoglycerate Synthase-The two-step purification procedure allowed the isolation of phosphopeptide that subsequently met the criteria of purity

Summary

Introduction

Glycerate-2,3-Pz) and phosphoglycerate mutase (glycerate-3-P e glycerate-2-P) are both phosphorylated by substrates at a histidine residue forming covalent intermediates which have been shown to function in the phosphoryl transfer reactions catalyzed by these enzymes On histidine residues [6, 7] and in each case the phosphoryl group has kinetic properties compatible with participation in the phosphoryl transfer reactions catalyzed by the enzyme [4]. Both enzymes have subunits of M, = -30,000 yeast phosphoglycerate mutase is a tetramer [7,8,9] whereas muscle phosphoglycerate mutase [6, 8, 10] and red cell bisphosphoglycerate synthase are dimers [3].

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