Introduction of internal cysteines as conformational probes in yeast phosphoglycerate kinase.
Several mutants of yeast phosphoglycerate kinase, each containing only one internal cysteine residue, were constructed from a single mutant devoid of cysteine. These cysteines were introduced as local conformational probes in selected buried positions. The enzyme activity, conformational characteristics and stability indicated that the mutations introduced only small perturbations in the molecule. The folding-unfolding process mediated by guanidine hydrochloride under equilibrium conditions was studied by following the variations in ellipticity and the reactivity of the cysteine residue towards 5,5'-dithiobis(nitrobenzoate). The process was found to be reversible except for mutant C97A, V49C, suggesting that this region located in helix I might be crucial in determining an intermediate on the folding pathway. The transitions obtained by the two signals did not coincide, indicating that the local structures, in several parts inside the molecule, are more sensitive to the denaturant than the overall conformation.
Ballery
N
N
Laboratoire d'Enzymologie physico-chimique et moléculaire, Université de Paris-Sud, Orsay, France.
Minard
P
P
Desmadril
M
M
Betton
J M
JM
Perahia
D
D
Mouawad
L
L
Hall
L
L
Yon
J M
JM
D002384:Catalysis; D002942:Circular Dichroism; D003545:Cysteine; D004355:Drug Stability; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation; D010735:Phosphoglycerate Kinase / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013438:Sulfhydryl Compounds; D013816:Thermodynamics
D006146:Guanidines; D013438:Sulfhydryl Compounds; D010735:Phosphoglycerate Kinase; D019791:Guanidine; D003545:Cysteine