INVESTIGATION OF HYDROGEN-BONDS IN THE CATALYTIC TRIAD OF TRYPSIN USING H-1-NMR, C-13-NMR, AND N-15-NMR SPECTROSCOPY

C-13-NMR spectra of polymer-modified trypsin and several of its comple xes with inhibitors simulating its movement along the axis of the enzy matic reaction over a wide range of pH (1-10) were acquired and interp reted. A strong (quasisymmetrical) hydrogen bond was formed between th e carboxylate and imidazolyl groups. The data explain contradictions i n the literature on experimental location of the protonation center. A ll hydrogen bonds in the catalytic triad are cooperatively strengthene d on simulating an electrophilic attack on the Ser hydroxyl group. The data suggest that the smooth movement of the proton in the field of t he three main sites without discrete steps of proton transfer is a cha racteristic feature of the mechanism of acid-base catalysis during enz ymatic hydrolysis of amide substrates. Resulting from the cooperative interaction between hydrogen bonds in the catalytic triad, the effecti ve basicity of the catalytic site of the nitrogen atom N-epsilon 2 (Im ) is a function of the reaction coordinate. The transition state is st abilized by a system of quasisymmetrical hydrogen bonds. The model exp lains the effective function of the enzyme in alkaline media (pH(opt) 8.0) and anomalous pK(a) for fragments of the catalytic traid: pK(a) ( Asp) 1.3:pK(a)(His)7.5.