A NEW CONCEPT FOR THE MECHANISM OF ACTION OF CHYMOTRYPSIN - THE ROLE OF THE LOW-BARRIER HYDROGEN-BOND

The basicities of the diad H57-D102 at N-epsilon 2 in the tetrahedral complexes of chymotrypsin with the peptidyl trifluoromethyl ketones (T FK) N-acetyl-L-Leu-DL-Phe-CF3 and N-acetyl-DL-Phe-CF3 have been studie d by H-1-NMR. The protons bridging His 57 and Asp 102 in these complex es are engaged in low-barrier hydrogen bonds (LBHBs). In H-1-NMR spect ra 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 amino acyl site S-2 Of chymotrypsin weakens the LBHB and decreases the basic ity of the H57-D102 diad relative to the case in which S-2 is occupied by leucine. Consideration of the available structural data on chymotr ypsin and other serine proteases, together with the high pK(a)s of the hemiketals formed with TFKs, suggests that LBHB formation in catalysi s arises through a substrate-induced conformational transition leading to steric compression between His 57 and Asp 102. Because the N-O dis tance 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, su bstrate-induced steric compression within the diad increases the basic ity of N-epsilon 2 in His 57, making it a more effective base for abst racting a proton from Ser 195 in the formation of the tetrahedral inte rmediate. The values of pK(a) for N-epsilon 2 in TFK adducts lie betwe en those of Ser 195 (pK(a) approximate to 14) and the leaving group in tetrahedral adducts (pK(a) approximate to 9), making N-epsilon 2 of t he H57-D102 diad strong enough as a base to abstract the proton from S er 195 in tetrahedral adduct formation but not so strong that its conj ugate acid cannot protonate the leaving group. According to this theor y, 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 catalys is, the energy for conformational compression is supplied by the bindi ng of remote portions of the substrate, including the side chains of P -1 and P-2.