A differential scanning calorimetric study of the effects of metal ions, substrate/product, substrate analogues and chaotropic anions on the thermal denaturation of yeast enolase 1.
The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding. Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing. The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation. 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism. More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion. We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.
Brewer
J M
JM
Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, GA 30602, USA.
Wampler
J E
JE
D000838:Anions; D002118:Calcium / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D004492:Edetic Acid / Q000737:chemistry; D004795:Enzyme Stability; D006877:Hydroxamic Acids / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008670:Metals / Q000737:chemistry / Q000494:pharmacology*; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D011189:Potassium Chloride / Q000737:chemistry; D011489:Protein Denaturation; D021122:Protein Subunits; D015203:Reproducibility of Results; D013379:Substrate Specificity; D015003:Yeasts / Q000201:enzymology*
D000838:Anions; D006877:Hydroxamic Acids; D008670:Metals; D021122:Protein Subunits; D011189:Potassium Chloride; C087412:phosphonoacetohydroxamate; D004492:Edetic Acid; D010751:Phosphopyruvate Hydratase; D008274:Magnesium; D010746:Phosphonoacetic Acid; D002118:Calcium