Reduction in water activity greatly retards the phosphoryl transfer from ATP to enzyme protein in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase.
Cys-674 of the sarcoplasmic reticulum Ca2+-ATPase was labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity. The ATP-induced drop in the fluorescence of the label, which was shown in our previous studies to reflect the conformational change upon formation of the calcium.enzyme.ATP complex, was followed by the stopped-flow method. The subsequent phosphoenzyme formation was followed by the rapid quenching method. Effects of a partial substitution of organic solvents for water in the medium on the conformational change and phosphoenzyme formation were investigated in the presence of 100 microM CaCl2 at pH 7.5, 0 degrees C. The rate of the conformational change increased with increasing ATP concentration (0.1 100 microM) and was unaffected by 30% (v/v) dimethyl sulfoxide. In contrast, the rate of phosphoenzyme formation decreased sharply with increasing concentration of dimethyl sulfoxide (20-40% (v/v)), even when phosphoenzyme formation was saturated with ATP. N,N-Dimethylformamide and glycerol had essentially the same effects as dimethyl sulfoxide. These results show that the reduction in water activity does not affect the rate of the conformational change upon formation of the calcium.enzyme.ATP complex, but greatly retards the subsequent phosphoryl transfer from ATP to the enzyme protein. This strongly suggests that in this early stage of the catalytic cycle water plays a critical role in ensuring the rapid turnover of the enzyme.
Suzuki
H
H
Department of Biochemistry, Asahikawa Medical College, Asahikawa 078, Japan.
Kanazawa
T
T
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002122:Calcium Chloride / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D003545:Cysteine; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004126:Dimethylformamide / Q000494:pharmacology; D005456:Fluorescent Dyes; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology*; D010750:Phosphoproteins / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014867:Water / Q000494:pharmacology*
D005456:Fluorescent Dyes; D010750:Phosphoproteins; D012997:Solvents; D014867:Water; D004126:Dimethylformamide; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D003545:Cysteine; D002122:Calcium Chloride; D005990:Glycerol; D004121:Dimethyl Sulfoxide