Specificity of the sarcoplasmic reticulum calcium ATPase at the hydrolysis step.
The coupling of Ca2+ transport to ATP hydrolysis by the SR ATPase requires that the enzyme operate with considerable specificity, which is different at different steps. The limits of specificity of the calcium-free phosphorylated enzyme for transfer of its phosphoryl group to water have been examined. The rate of transfer of the phosphoryl group to the simple nucleophile methanol was compared to its transfer to water by following the formation of methyl phosphate from inorganic phosphate. The reverse reaction, hydrolysis of methyl phosphate, was compared to phosphate-water oxygen exchange. The reactions involving methanol as nucleophile or leaving group are at least 2-3 orders of magnitude slower than those involving water. This result indicates that the transition state for this reaction involves strong and specific interactions of the H2O molecule with the enzyme. These interactions may also involve the bound Mg2+ ion. The results also suggest that the difference in specificity between Ca2+ free and Ca2+ bound states of the enzyme involves significant differences in the structure of the catalytic site.
Chipman
D M
DM
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Jencks
W P
WP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D000432:Methanol / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013379:Substrate Specificity; D014867:Water / Q000378:metabolism
D014867:Water; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D000432:Methanol