Site-directed mutagenesis of histidine-388 in the hinge region of yeast 3-phosphoglycerate kinase: effects on catalytic activity and activation by sulfate

Authors:
  • JBJerome M. Bailey
    Jerome M. Bailey
  • ZRZenaida E. Resplandor
    Zenaida E. Resplandor
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Abstract
It has been proposed that the catalytic mechanism of 3-phosphoglycerate kinase (PGK) and the regulation of its enzymatic activity by sulfate ions involve relatively large conformational changes. We have applied site-directed mutagenesis to assess the role of the interactions between glutamate-190 and histidine-388, located in the interdomain hinge region, in the substrate- and sulfate-induced conformational transitions. We have shown previously that substitutions of Glu-190 with either glutamine or aspartate resulted in a complete loss of sulfate activation and in decreased activities; corresponding to 26% and 36% of the activity of native PGK, respectively [Mas, M. T., Resplandor, Z. E., and Riggs, A. D. (1987) Biochemistry 26, 5369-5377]. In contrast, the Lys-388 and Ala-388 mutants retain the ability to undergo sulfate-induced activation and exhibit a larger decrease in activity (relative activities of 6% and 13%, respetively). The decrease of the enzymatic activities of these mutants and the relatively small changes of the Km values for the substrates imply that both residues participate in the catalytic mechanism by contributing to the conformational flexibility of the enzyme. Our results led to the following conclusions: (1) in contrast to glutamate-190, histidine-388 is not critical for the sulfate-induced activation; (2) the critical role of Glu-190 is not due to the specific interactions with histidine-388 but to new contacts with other amino acid residue(s) that occur as the result of sulfate-induced rearrangements of the structural elements of the hinge; (3) the substrate- and sulfate-induced conformational transitions seem to occur via different mechanisms; (4) a cumulative effect of multiple interactions of the side chains in the hinge region, rather than individual residues(s), is likely to be responsible for the efficient mechanism of ligand-induced domain movement.