Site-directed mutagenesis combined with chemical modification as a strategy for altering the specificity of the S1 and S1' pockets of subtilisin Bacillus lentus.
By combining site-directed mutagenesis with chemical modification, we have altered the S1 and S1' pocket specificity of subtilisin Bacillus lentus (SBL) through the incorporation of unnatural amino acid moieties, in the following manner: WT --> Cysmutant + H3CSO2SR --> Cys-SR, where R may be infinitely variable. A paradigm between extent of activity changes and surface exposure of the modified residue has emerged. Modification of M222C, a buried residue in the S1' pocket of SBL, caused dramatic changes in kcat/KM, of an up to 122-fold decrease, while modification of S166C, which is located at the bottom of the S1 pocket and is partially surface exposed, effected more modest activity changes. Introduction of a positive charge at S166C does not alter kcat/KM, whereas the introduction of a negative charge results in lowered activity, possibly due to electrostatic interference with oxyanion stabilization. Activity is virtually unaltered upon modification of S156C, which is located toward the bottom of the S1 pocket and surface exposed and whose side chain is solvated. An unexpected structure-activity relationship was revealed for S166C-SR enzymes in that the pattern of activity changes observed with increasing steric size of R was not monotonic. Molecular modeling analysis was used to analyze this unprecedented structure-activity relationship and revealed that the position of the beta-carbon of Cys166 modulates binding of the P1 residue of the AAPF product inhibitor.
DeSantis
G
G
Department of Chemistry, University of Toronto, Ontario, Canada.
Berglund
P
P
Stabile
M R
MR
Gold
M
M
Jones
J B
JB
D019943:Amino Acid Substitution / Q000235:genetics; D001407:Bacillus / Q000201:enzymology*; D018360:Crystallography, X-Ray; D007202:Indicators and Reagents; D007700:Kinetics; D013058:Mass Spectrometry; D008698:Mesylates; D008715:Methionine / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics*; D013438:Sulfhydryl Compounds; D017186:Titrimetry
D007202:Indicators and Reagents; D008698:Mesylates; D013438:Sulfhydryl Compounds; C074502:methanethiosulfonate; D012694:Serine; D008715:Methionine; D013381:Subtilisins