title: Substitution of a proline for alanine 183 in the hinge region of phosphoglycerate kinase: effects on catalysis, activation by sulfate, and thermal stability.
abstract: A "hinge-bending" domain movement has been postulated as an important part of the catalytic mechanism of phosphoglycerate kinase (PGK) (Banks et al., 1979). In order to test the role of the flexibility of a putative interdomain hinge in the substrate- and sulfate-induced conformational transitions, alanine-183 was replaced by proline using site-directed mutagenesis. The maximal velocity of the Ala 183----Pro mutant, measured at saturating concentrations of ATP and phosphoglycerate (5 mM and 10 mM, respectively) and in the absence of sulfate ions, is increased approximately 21% in comparison to the wild type PGK. The Km values for both substrates are essentially unchanged. The effect of sulfate on the specific activity of the Ala 183----Pro mutant and the wild type PGK was measured in the presence of 1 mM ATP and 2 mM 3-phosphoglycerate (3-PG). A maximum activation of 70% was observed at 20 mM sulfate for the mutant enzyme, as compared to 130% activation at 30 mM sulfate for the wild type PGK. These results demonstrate that the increased rigidity of the putative hinge, introduced by the Ala----Pro mutation, does not impair catalytic efficiency of phosphoglycerate kinase, while it appears to decrease the sulfate-dependent activation. The differential scanning calorimetry (DSC) studies demonstrate an increased susceptibility of the Ala 183----Pro mutant to thermal denaturation. In contrast to one asymmetric transition observed in the DSC scan for the wild type PGK, with Tm near 54 degrees C, two transitions are evident for the mutant enzyme with Tm values of about 45 and 54 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Division of Biology, Beckman Research Institute, City of Hope, Duarte, California 91010.
authors.forename: J M
authors.initials: JM
authors.lastname: Bailey
authors.forename: L N
authors.initials: LN
authors.lastname: Lin
authors.forename: J F
authors.initials: JF
authors.lastname: Brandts
authors.forename: M T
authors.initials: MT
authors.lastname: Mas
mesh_terms: D000409:Alanine / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D004789:Enzyme Activation; D006358:Hot Temperature; D009154:Mutation; D009841:Oligonucleotides; D010735:Phosphoglycerate Kinase / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D013431:Sulfates / Q000494:pharmacology; D015003:Yeasts / Q000201:enzymology
chemical_list: D009841:Oligonucleotides; D013431:Sulfates; C012036:sodium sulfate; D011392:Proline; D010735:Phosphoglycerate Kinase; D000409:Alanine