Benzophenone boronic acid photoaffinity labeling of subtilisin CMMs to probe altered specificity

A transition state analogue inhibitor, boronic acid benzophenone (BBP) phot oprobe, was used to study the differences in the topology of the S-1 pocket of chemically modified mutant enzymes (CMMs). The BBP proved to be an effe ctive 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 posit ively 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 th e charged CMMs, S166C-S-CH2CH2SO3- or S166C-S-CH2CH2NH3+, was reduced 170-f old and 4-fold respectively. Photolysis of the WT-SBL-BBP enzyme-inhibitor (EI) complex, inactivated the enzyme and effected the formation of a covale nt crosslink between WT and BBP. The crosslink was identified at Gly127 by peptide mapping analysis and Edman sequencing. Gly127 is located in the S-1 hydrophobic pocket of SBL and its modification thus established binding of the benzophenone moiety in S-1. Photolysis of the EI complex of S166C-SCH2 C6H5. S166C-S-CH2CH2SO3-, or S166C-S-CH2CH2NH3+: and BBP under the same con ditions did not inactivate these enzymes, nor effect the formation of a cro sslink. These results corroborated the kinetic evidence that the active sit e topology of these CMMs is dramatically altered from that of WT. In contra st, while photolysis of the S166C-CH2-c-C6H11-BBP EI complex only inactivat ed 50% of the enzyme after 12 h, it still effected the formation of a coval ent crosslink between the CMM and BBP, again at Gly127. However, this photo lytic reaction was less efficient than with WT, demonstrating that the S-1 pocket of S166C-CH2-c-C6H11 is significantly restricted compared to WT, but not as completely as for the other CMMs. (C) 2000 Elsevier Science Ltd. Al l rights reserved.