PMID- 10732973
OWN - NLM
STAT- MEDLINE
DCOM- 20000601
LR  - 20190831
IS  - 0968-0896 (Print)
IS  - 0968-0896 (Linking)
VI  - 8
IP  - 3
DP  - 2000 Mar
TI  - Benzophenone boronic acid photoaffinity labeling of subtilisin CMMs to probe 
      altered specificity.
PG  - 563-70
AB  - 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.
FAU - DeSantis, G
AU  - DeSantis G
AD  - Department of Chemistry, University of Toronto, Ontario, Canada.
FAU - Paech, C
AU  - Paech C
FAU - Jones, J B
AU  - Jones JB
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Bioorg Med Chem
JT  - Bioorganic & medicinal chemistry
JID - 9413298
RN  - 0 (4-boronic acid benzophenone)
RN  - 0 (Bacterial Proteins)
RN  - 0 (Benzophenones)
RN  - 0 (Boronic Acids)
RN  - 0 (Cross-Linking Reagents)
RN  - 0 (Photosensitizing Agents)
RN  - 0 (Serine Proteinase Inhibitors)
RN  - EC 3.4.21.- (Serine Endopeptidases)
RN  - EC 3.4.21.62 (Subtilisin)
SB  - IM
MH  - Amino Acid Sequence
MH  - Bacillus/*enzymology
MH  - Bacterial Proteins/chemistry
MH  - Benzophenones/*chemistry/pharmacology
MH  - Binding Sites
MH  - Binding, Competitive
MH  - Boronic Acids/*chemistry
MH  - Cross-Linking Reagents
MH  - Genetic Engineering
MH  - Kinetics
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Photolysis/drug effects
MH  - Photosensitizing Agents/chemistry/pharmacology
MH  - Protein Engineering
MH  - Serine Endopeptidases/chemistry/genetics/metabolism
MH  - Serine Proteinase Inhibitors/chemistry
MH  - Subtilisin/antagonists & inhibitors/*chemistry/genetics/metabolism
MH  - Time Factors
EDAT- 2000/03/25 09:00
MHDA- 2000/06/03 09:00
CRDT- 2000/03/25 09:00
PHST- 2000/03/25 09:00 [pubmed]
PHST- 2000/06/03 09:00 [medline]
PHST- 2000/03/25 09:00 [entrez]
AID - S0968-0896(99)00320-X [pii]
AID - 10.1016/s0968-0896(99)00320-x [doi]
PST - ppublish
SO  - Bioorg Med Chem. 2000 Mar;8(3):563-70. doi: 10.1016/s0968-0896(99)00320-x.