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Protein Engineering vol. 4 no. 8 pp. 935-940, 1991
© 1991 Oxford University Press

RESEARCH-ARTICLE

Site-directed mutations of arginine 65 at the periphery of the active site cleft of yeast 3-phosphoglycerate kinase enhance the catalytic activity and eliminate anion-dependent activation

Mark A. Sherman, Susan A. Dean, Alan M. Mathiowetz and Maria T. Mas1

Beckman Research Institute of the City of Hope, Physical Biochemistry Section, Division of Biology Duarte, CA 91010, USA

1To whom correspondence should be addressed

The function of arginine 65, a conserved residue located at the periphery of the active site cleft in yeast 3-phospho-glycerate kinase (PGK), has been investigated by site-directed mutagenesis. Mutant enzymes with glutamine, serine and alanine at position 65 all have very similar kinetic properties. The maximum velocities, determined in the absence of sulfate anion, are - 100% higher than the Vmax of wild-type PGK. The Km values are increased 2- to 3-fold for ATP and 5- to 6-fold for 3-phosphoglycerate (3PG). These results demonstrate that arginine 65 is not essential for catalysis. In contrast to wild-type enzyme, the mutants are not activated by sulfate ions. In addition, steady-state kinetic experiments indicate that the mutants are no longer activated by high concentrations of either 3PG or ATP. The dissociation constants for anions were determined by spectral titrations of the R65Q mutant labeled with a chromophoric probe. The Kd for 3PG is increased 6-fold, as compared to wild-type PGK, whereas the Kd for ATP is essentially unchanged. The KA for sulfate is decreased < 2-fold. The suppression of substrate- and sulfate-dependent activation suggests that arginine 65 participates in the regulatory mechanism responsible for activation of the enzyme.

Keywords: catalytic mechanism/enzyme activation/3-phospho-glycerate kinase/site-specific mutagenesis

Received March 18, 1991; revised September 5, 1991;
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