title: Benzophenone boronic acid photoaffinity labeling of subtilisin CMMs to probe altered specificity.
abstract: A transition state analogue inhibitor, boronic acid benzophenone (BBP) photoprobe, was used to study the differences in the topology of the S1 pocket of chemically modified mutant enzymes (CMMs). The BBP proved to be an effective competitive inhibitor and a revealing active site directed photoprobe of the CMMs of the serine protease subtilisin Bacillus lentus (SBL) which were chemically modified with the hydrophobic, negatively charged and positively charged moieties at the S1 pocket S166C residue. As expected, in all cases BBP bound best to WT-SBL. BBP binding to S166C-SCH2C6H5 and S166C-CH2-c-C6H11, with their large hydrophobic side chains, was reduced by 86-fold and 9-fold, respectively, compared to WT. Relative to WT, BBP binding to the charged CMMs, S166C-S-CH2CH2SO3- or S166C-S-CH2CH2NH3+, was reduced 170-fold and 4-fold respectively. Photolysis of the WT-SBL-BBP enzyme inhibitor (EI) complex, inactivated the enzyme and effected the formation of a covalent crosslink between WT and BBP. The crosslink was identified at Gly127 by peptide mapping analysis and Edman sequencing. Gly127 is located in the S1 hydrophobic pocket of SBL and its modification thus established binding of the benzophenone moiety in S1. Photolysis of the EI complex of S166C-SCH2C6H5, S166C-S-CH2CH2SO3-, or S166C-S-CH2CH2NH3+ and BBP under the same conditions did not inactivate these enzymes, nor effect the formation of a crosslink. These results corroborated the kinetic evidence that the active site topology of these CMMs is dramatically altered from that of WT. In contrast, while photolysis of the S166C-CH2-c-C6H11-BBP EI complex only inactivated 50% of the enzyme after 12 h, it still effected the formation of a covalent crosslink between the CMM and BBP, again at Gly127. However, this photolytic reaction was less efficient than with WT, demonstrating that the S1 pocket of S166C-CH2-c-C6H11 is significantly restricted compared to WT, but not as completely as for the other CMMs.
authors.affiliation: Department of Chemistry, University of Toronto, Ontario, Canada.
authors.forename: G
authors.initials: G
authors.lastname: DeSantis
authors.forename: C
authors.initials: C
authors.lastname: Paech
authors.forename: J B
authors.initials: JB
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry; D001577:Benzophenones / Q000737:chemistry* / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D001897:Boronic Acids / Q000737:chemistry*; D003432:Cross-Linking Reagents; D005818:Genetic Engineering; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010782:Photolysis / Q000187:drug effects; D017319:Photosensitizing Agents / Q000737:chemistry / Q000494:pharmacology; D015202:Protein Engineering; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry; D020860:Subtilisin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: C408472:4-boronic acid benzophenone; D001426:Bacterial Proteins; D001577:Benzophenones; D001897:Boronic Acids; D003432:Cross-Linking Reagents; D017319:Photosensitizing Agents; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; D020860:Subtilisin