Robillard, G.

Shulman, R.G.

English

The proton nuclear magnetic resonance signal of the His57-Asp102 hydrogen bonded proton in the charge relay system of chymotrypsinogen A and chymotrypsin Aδ has been monitored to determine the influence of substrate analogues and competitive inhibitors on the electronic state of the active site regions. Borate ion, benzene boronic acid and 2-phenylethylboronic acid, when bound to chymotrypsin at pH 9.5 shift the resonance position of the His-Asp hydrogen bonded proton to -15.9, -16.3 and -17.2 parts per million, respectively. These positions are intermediate between the low pH position in the free enzyme of -18.0 parts per million and the high pH position of -14.9 parts per million. The presence of these analogues prevents the His-Asp proton resonance from titrating in the region of pH 6 to 9.5. Similar low field shifts are observed for the hydrogen bonded proton resonance of subtilisin BPN’ when complexed with these boronic acids. The results support the chemical and crystallographic data which show that negatively charged tetrahedral adducts of the boronic acid substrate analogues are formed at the active sites of these enzymes. When combined with similar nuclear magnetic resonance data for the binding of N-acetyl-L-tryptophan to chymotrypsin Aδ, they suggest that a direct interaction occurs between the active site histidine and the atom occupying the leaving group position of the substrate, presumably a hydrogen bond. The His-Asp proton resonance was also monitored in complexes of chymotrypsin Aδ with bovine pancreatic trypsin inhibitor over the pH range 4 to 9. In the complex the low field proton resonance had a field position of -14.9 parts per million over the pH range 4 to 9 indicating that His57 is in the neutral form, similar to the active enzyme at high pH

Dissertations of the University of Groningen

High Resolution Nuclear Magnetic Resonance Studies of the Active Site of Chymotrypsin. II. Polarization of Histidine 57 by Substrate Analogues and Competitive Inhibitors

https://pure.rug.nl/ws/files/14658988/1974JMolBiolRobillard2.pdf

High Resolution Nuclear Magnetic Resonance Studies of the Active Site of Chymotrypsin. II. Polarization of Histidine 57 by Substrate Analogues and Competitive Inhibitors

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Dissertations of the University of Groningen