Covalent modification of subtilisin Bacillus lentus cysteine mutants with enantiomerically pure chiral auxiliaries causes remarkable changes in activity

Methanethiosulfonate reagents may be used to introduce virtually unlimited structural modifications in enzymes via reaction with the thiol group of cy steine. The covalent coupling of enantiomerically pure (R) and (S) chiral a uxiliary methanethiosulfonate ligands to cysteine mutants of subtilisin Bac illus lentus induces spectacular changes in catalytic activity between dias tercomeric enzymes. Amidase and esterase kinetic assays using a low substra te approximation were used to establish k(cat)/K-M values for the chemicall y modified mutants, and up to 3-fold differences in activity were found bet ween diastereomeric enzymes. Changing the length of the carbon chain linkin g the phenyl or benzyl oxazolidinone ligand to the mutant N62C by a methyle ne unit reverses which diastereomeric enzyme is more active. Similarly, cha nging from a phenyl to benzyl oxazolidinone ligand at S166C reverses which diastereomeric enzyme is more active. Chiral modifications at S166C and L21 7C give CMMs having both high esterase k(cat)/K-M's and high esterase to am idase ratios with large differences between diastereomeric enzymes. (C) 200 0 Elsevier Science Ltd. All rights reserved.