Site-directed mutagenesis of histidine-388 in the hinge region of yeast 3-phosphoglycerate kinase: effects on catalytic activity and activation by sulfate.
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., & 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%, respectively). 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Mas
M T
MT
Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Bailey
J M
JM
Resplandor
Z E
ZE
D001483:Base Sequence; D004789:Enzyme Activation; D005796:Genes; D005800:Genes, Fungal; D006639:Histidine; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013431:Sulfates / Q000494:pharmacology*
D013431:Sulfates; C012036:sodium sulfate; D006639:Histidine; D010735:Phosphoglycerate Kinase