Probing an Acyl Enzyme of Selenosubtilisin by Raman Spectroscopy

摘要

Replacement of the catalytieally essential serine in subtilisin by selenocysteine dramatically alters the properties of the protease. In addition to affording it novel peroxidase activity, the single atom mutation (from oxygen to selenium) converts the enzyme into an acyl transferase, favoring aminolysis over hydrolysis of the acyl enzyme intermediate formed in reactions with activated esters and imidazolides.3 The latter properties (and the lack of amidase activity) make selenosubtilisin a potentialcandidate for the development of a peptide ligase. We have initiated spectroscopic investigations of enzyme-bound seleno esters to establish the origins of the altered hydrolytic chemistry. This report describes the characterization of a typical intermediate by Raman Spectroscopy and shows that selenosubtilisin polarizes the carbonyl of the acyl enzyme to a much lesser extent than the native enzyme. These results have important implications for the design of the more active variants.