Probing the role of arginines and histidines in the catalytic function and activation of yeast 3-phosphoglycerate kinase by site-directed mutagenesis

• Published in: The Journal of biological chemistry Vol. 265; no. 18; pp. 10659 - 10665
• Main Authors: Sherman, M.A. (Beckman Research Institute of the City of Hope, Duarte, CA), Szpikowska, B.K, Dean, S.A, Mathiowetz, A.M, McQueen, N.L, Mas, M.T
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25-06-1990
• Subjects: ACTIVE SITE; ACTIVE SITE AMINO ACIDS; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; ARGININA; ARGININE; Base Sequence; BINDING SITE; Binding Sites; Biological and medical sciences; Enzyme Activation; Enzymes and enzyme inhibitors; ENZYMIC ACTIVITY; Fundamental and applied biological sciences. Psychology; GENETIC ENGINEERING; GENIE GENETIQUE; HISTIDINA; HISTIDINE; INGENIERIA GENETICA; Kinetics; Molecular Sequence Data; Molecular Weight; MUTACION; MUTATION; phosphoglycerate kinase; Phosphoglycerate Kinase - genetics; Phosphoglycerate Kinase - metabolism; Protein Conformation; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; site-directed mutagenesis; TRANSFERASAS; TRANSFERASE; TRANSFERASES; Fungi; Regulation(control); Yeast; Enzymatic activity; Enzyme; Site directed mutagenesis; Ascomycetes; Binding site; Kinetics; Phosphoglycerate kinase; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)86997-6

A cluster of conserved histidines and arginines (His-62, His-167, Arg-21, Arg-38, and Arg-168) in 3-phosphoglycerate kinase
(PGK) has been implicated as possibly involved in the binding of 3-phosphoglycerate (3-PG) and/or stabilization of the negatively
charged transition state. The role of these residues in the catalytic function of yeast PGK and in the substrate- and sulfate-dependent
activation was investigated by site-directed mutagenesis. The following substitutions, R21A, R21Q, H62Q, H167S, and R168Q,
produced functional enzymes. In contrast, the R38A and R38Q mutations resulted in a complete loss of catalytic activity. These
results demonstrate that of the basic residues studied, only arginine 38 is essential for the catalytic function of PGK. A
moderate decrease in the catalytic efficiency as the result of the R21A, H167S, and R168Q mutations and an increased catalytic
efficiency of the H62Q mutant rule out a possible role of a positive charge at these positions in the mechanism of phosphoryl
transfer reaction. In contrast to the wild type PGK and the H62Q mutant, both of which are activated at low and inhibited
at high sulfate concentration, the H167S, R168Q, and R21A mutants exhibited a progressive inhibition with increased concentration
of sulfate. The activation observed at high concentration of either ATP or 3-PG as a variable substrate in the steady-state
kinetics of wild type PGK was abolished as the result of the latter three mutations. The results of this work support the
hypothesis that PGK has two binding sites for anionic ligands, the catalytic and regulatory sites for each substrate and the
activatory and inhibitory sites for sulfate, and suggest that arginine 21, arginine 168, and histidine 167 are located in
the activatory anion binding site, common for sulfate, 3-PG, and ATP. The increased Km values for both substrates and decreased
specific activities of the mutants suggest that this regulatory site is close to the catalytic site.