Determination of the rate constant of enzyme modification by measuring the substrate reaction in the presence of the modifier.
On the basis of the equations derived previously [Tsou, C. L. (1965) Sheng Wu Hua Hsueh Yu Sheng Wu Wu Li Hsueh Pao 5, 398-408, 409-417] for the substrate reaction during the course of enzyme modification, the kinetic behavior of the system chymotrypsin-substrate-modifier has been studied. The kinetics of benzoyltyrosine ester hydrolysis during the course of irreversible inhibition of the enzyme has been found to be in satisfactory agreement with equations obtained previously. The apparent rate constant between the enzyme and an irreversible inhibitor can be easily obtained in one single experiment by following the course of substrate hydrolysis in the presence of the inhibitor. The results are also in accord with the assumption that diisopropyl fluorophosphate can be classified as an irreversible competitive inhibitor. For both phenylmethanesulfonyl fluoride and L-1-[(p-toluene-sulfonyl)amino]-2-phenylethyl chloromethyl ketone, the inhibition has been found to be in agreement with the kinetics of the complexing type; i.e., a noncovalent enzyme-inhibitor complex is formed before irreversible enzyme modification. Both the equilibrium constants for the complex formation and the first-order rate constants for the irreversible modification step have been determined also by following the course of substrate hydrolysis in the presence of the irreversible inhibitor.
D001665:Binding Sites; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D013329:Structure-Activity Relationship
D002918:Chymotrypsin