Reaction of wild-type C365S, and C458S saccharomyces cerevisiae phosphoenolpyruvate carboxykinases with fluorescent iodoacetamide derivatives.
The reactivities of Cys365 and Cys458 of ATP-dependent Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase against a range of sulfhydryl reagents have been investigated. The effect of pH on the second order reaction constants of N-(1-pyrenyl)maleimide with mutant C458S and C365S PEP carboxykinases allowed the determination of pKa values of 9.4 and 9.1 for Cys365 and Cys458, respectively. The analysis of the inactivation rates of C458S and C365S mutant enzymes by several sulfhydryl reagents of different hydrophobicity showed that the microenvironment of these residues is rather polar. Anisotrophy measurements and acrylamide quenching experiments carried out with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled mutant enzymes indicated a higher rotational freedom and solvent exposure for the probe linked to Cys458 than to Cys365. These findings point to differences in the protein microenvironments around Cys365 and Cys458 in S. cerevisiae PEP carboxykinase. A comparison of the results obtained with published data for GTP-dependent PEP carboxykinases, suggest significant differences for the protein region around the reactive cysteinyl residues in these enzymes.
Krautwurst
H
H
Departamento de Ciecias Qúimicas, Facultad de Quimica y Biología, Universidad de Santiago de Chile, Chile.
Berti
M
M
Encinas
M V
MV
Frey
P A
PA
D000595:Amino Acid Sequence; D003545:Cysteine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008301:Maleimides; D009282:Naphthalenesulfonates; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008301:Maleimides; D009282:Naphthalenesulfonates; D011994:Recombinant Proteins; C048876:1,5-AEDANS; C003944:N-(3-pyrene)maleimide; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D003545:Cysteine