A variety of physico-chemical techniques have been used to probe the possible interactions between the characteristic structural domains of yeast phosphoglycerate kinase by comparison of the wild-type enzyme with the specific H388Q mutant in which a potential interaction between His388 and Glu190 in the crucial interdomain region is disrupted. Enzyme kinetic studies indicate that, despite being structurally remote from the active site, this mutation has significant effects on both the Vmax and Km values for various substrates. The single cysteine residue in the N domain of the protein is markedly more reactive in the mutant, and this enhanced accessibility is moderated by binding of substrates and various anions. Differences are also observed in the near-ultraviolet CD spectra of these proteins. The chemical and thermal stability of the mutant enzyme is reduced, as indicated from guanidinium chloride and differential-scanning calorimetry denaturation studies. Moreover, interdomain interactions seem to be altered in the mutant, resulting in the appearance of independent thermal transition for the two domains, in contrast to the single cooperative transition observed for the wild-type enzyme. The conformational and/or dynamic effects of the mutation on the H388Q enzyme are therefore various and not solely localised in the hinge region. Study holds ProTherm entries: 2748, 2749 Extra Details: additive : EDTA(0.25 mM), phosphoglycerate kinase; thermal stability; reactivity
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:19 p.m.
|Number of data points||5|
|Proteins||Phosphoglycerate kinase ; Phosphoglycerate kinase|
|Assays/Quantities/Protocols||Experimental Assay: m ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence SLSSKLSVQDLDLKDKRVFIRVDFNVPLDGKKITSNQRIVAALPTIKYVLEHHPRYVVLASHLGRPNGERNEKYSLAPVAKELQSLLGKDVTFLNDCVGPEVEAAVKASAPGSVILLENLRYHIEEEGSRKVDGQKVKASKEDVQKFRHELSSLADVYINDAFGTAHRAHSSMVGFDLPQRAAGFLLEKELKYFGKALENPTRPFLAILGGAKVADKIQLIDNLLDKVDSIIIGGGMAFTFKKVLENTEIGDSIFDKAVGPEIAKLMEKAKAKGVEVVLPVDFIIADAFSASANTKTVTDKEGIPAGWQGLDNGPESRKLFAATVAKATVILWNGPPGVFEFEKFAAGTKALLDEVVKSSAAGNTVIIGGGDTATVAKKYGVTDKISHVSTGGGASLELLEGKELPGVAFLSEKK|