Site-selective glycosylation of subtilisin Bacillus lentus causes dramaticincreases in esterase activity

Using site directed mutagenesis combined with chemical modification, we hav e developed a general and versatile method for the glycosylation of protein s which is virtually unlimited in the scope of proteins and glycans that ma y be conjugated and in which the site of glycosylation and the nature of th e introduced glycan can be carefully controlled. We have demonstrated the a pplicability of this method through the synthesis of a library of 48 glycos ylated forms of the serine protease subtilisin Bacillus lentus (SBL) as sin gle, pure species. As part of our ongoing program to tailor the activity of SBL for use in peptide synthesis, we have screened these enzymes for activ ity against the esterase substrate succinyl-Ala-Ala-Pro-Phe-S-benzyl. Grati fyingly, 22 enzymes displayed greater than wild type (WT) activity. Glycosy lation at positions 62, in the S-2 pocket, resulted in five glycosylated fo rms of SBL that were 1.3- to 1.9-fold more active than WT. At position 217, in the S-1' pocket, all glycosylations increased k(cat)/K-M up to a remark able 8.4-fold greater than WT for the glucosylated enzyme L217C-S-beta-Glc( Ac)(3). Furthermore, the ratio of amidase to esterase activity, (k(cat)/K-M )(esterase)/(k(cat)/K-M)(amidase) (E/A), is increased relative to wild type for all 48 glycosylated forms of SBL. Again, the most dramatic changes are observed at positions 62 and 217 and L217C-S-beta-Glc(Ac)(3) has an E/A th at is 17.2-fold greater than WT. The tailored specificity and high activity of this glycoform can be rationalized by molecular modeling analysis, whic h suggests that the carbohydrate moiety occupies the S-1' leaving group poc ket and enhances the rate of deacylation of the acyl-enzyme intermediate. T hese glycosylated enzymes are ideal candidates for use as catalysts in pept ide synthesis as they have greatly increased (k(cat)/K-M)(esterase) and sev erely reduced (k(cat)/K-M)(amidase) and will favor the formation of the ami de bond over hydrolysis. (C) 2000 Published by Elsevier Science Ltd. All ri ghts reserved.