The mechanisms by which enzymes catalyze reactions are only beginning to be elucidated. An approach of significant potential is provided by the transition state analog theory which predicts that an enzyme binds the transition state of the reaction which it catalyzes much more tightly than the substrate. In theory one can synthesize a stable analog which will complex with the enzyme, and allow the enzyme- substrate interactions to be ascertained near the point of maximum energy for the substrate in the reaction pathway. A comparison of these enzyme-transition state interactions with those for enzyme-substrate (Michaelis complex) or acyl-enzyme should elucidate the changes necessarily responsible for catalysis. We propose to synthesize specific transition state analogs for several enzymes, and to study their interactions with the active site groups. The methods of study include pH-inhibition profiles, spectrophotometric complex determinations and electron spin resonance of the enzyme analog adducts. It is proposed that oxovanadyl ion (VO2 plus) will be an effective active site probe for enzyme transition state interactions of alkaline and acid phosphatase. Specific boronic acids will be synthesized as potential analogs for papain (sulfhydryl) and subtilisin (serine) esterases. In a second study the reactions of 3 enzyme systems of physiological importance bound to solid supports will be studied. Urokinase, alpha- galactosidase A and alpha-galactosidase B will be separately entrapped or immobilized on solid particles, simple assay methods developed, and their reaction characteristics and stability investigated. Potential for enzyme "replacement therapy" is proposed.