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Biochemistry. 1997 Apr 15;36(15):4576-84.

A new concept for the mechanism of action of chymotrypsin: the role of the low-barrier hydrogen bond.

Author information

1
Institute for Enzyme Research, Department of Biochemistry, University of Wisconsin-Madison 53705, USA.

Abstract

The basicities of the diad H57-D102 at N(epsilon)2 in the tetrahedral complexes of chymotrypsin with the peptidyl trifluoromethyl ketones (TFK) N-acetyl-L-Leu-DL-Phe-CF3 and N-acetyl-DL-Phe-CF3 have been studied by 1H-NMR. The protons bridging His 57 and Asp 102 in these complexes are engaged in low-barrier hydrogen bonds (LBHBs). In 1H-NMR spectra at pH 7.0, these protons appear at delta 18.9 and 18.6 ppm, and the pK(a)s of the diads are 12.0 +/- 0.2 and 10.8 +/- 0.1, respectively. The difference indicates that removal of leucine from the second aminoacyl site S2 of chymotrypsin weakens the LBHB and decreases the basicity of the H57-D102 diad relative to the case in which S2 is occupied by leucine. Consideration of the available structural data on chymotrypsin and other serine proteases, together with the high pK(a)s of the hemiketals formed with TFKs, suggests that LBHB formation in catalysis arises through a substrate-induced conformational transition leading to steric compression between His 57 and Asp 102. Because the N-O distance in the LBHB is shorter than the Van der Waals contact distance, the LBHB is proposed to stabilize the tetrahedral intermediate through relief of steric strain between these residues. In this mechanism, substrate-induced steric compression within the diad increases the basicity of N(epsilon)2 in His 57, making it a more effective base for abstracting a proton from Ser 195 in the formation of the tetrahedral intermediate. The values of pK(a) for N(epsilon)2 in TFK adducts lie between those of Ser 195 (pK(a) approximately 14) and the leaving group in tetrahedral adducts (pK(a) approximately 9), making N(epsilon)2 of the H57-D102 diad strong enough as a base to abstract the proton from Ser 195 in tetrahedral adduct formation but not so strong that its conjugate acid cannot protonate the leaving group. According to this theory, the "normal" pK(a) of His 57 in free chymotrypsin arises from the use of part of the stabilization energy provided by the LBHB to drive the conformational compression required for its formation. In catalysis, the energy for conformational compression is supplied by the binding of remote portions of the substrate, including the side chains of P1 and P2.

PMID:
9109667
DOI:
10.1021/bi962013o
[Indexed for MEDLINE]

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