The activation parameters of acylation of subtilisin with alkyl and p-nitrophenyl esters of N-acylamino acid enantiomers were determined. It was found that (1) the activation entropy is much higher with the nitrophenyl esters than with the corresponding methyl esters, (2) the difference in rate constants between enantiomers is 10(4)--10(5) with methyl esters whereas it is only of the order of 10 with nitrophenyl esters. The results indicate that the catalytic mechanism is simpler for nitrophenyl esters than for alkyl esters. The simple mechanism requires only general base catalysis, and thus permits more freedom of motion in the transition state, whereas the complex mechanism involves both general base and general acid catalysis. Furthermore, the strikingly low enantiomeric specificity with nitrophenyl esters indicates that not only binding but also the catalytic mechanism is an important factor in determining the stereospecificity of an enzyme. The activation parameters for enantiomeric nitrophenyl ester reactions suggest that structurally related substrates can be transformed by the enzyme in different conformations which may be energetically similar or not. The energetically different conformations may account for the activation enthalpy-entropy compensation.
|Number of pages||6|
|Journal||European Journal of Biochemistry|
|Publication status||Published - Dec 17 1979|
ASJC Scopus subject areas