Uncoupling of the calcium transporting ATPase of sarcoplasmic reticulum by removal of the catalytic metal ion will be characterized further. The metal ion specificity and rate and equilibrium constants for development and disappearance of the uncoupling will be determined. The steps involving the binding and release of calcuim at the low affinity site will be measured directly to determine the mechanism and thermodynamics of these steps. Structure- reactivity correlations for nucleophilic reagents with phosphorylated pyridines will be done in order to determine if there is a metaphosphate intermediate in catalysis. Although an enormous amount of work has been carried out on chemical reactions that generate carbocations a limited amount of work has probed these indirectly by trapping reactions. This will be done. The substitution reactions at carbon will be investigated as will the ionization of thiamine. A mechanistic understanding of vectorial processes in biology is being established with this work. Knowledge of the basis the efficiency of catalytic processes in living systems is of great importance for fundamental work and for applications. The most rational approach to genetic engineering requires information at the level of mechanistic detail that this work provides
