IDENTIFICATION OF SERINE AND HISTIDINE ADDUCTS IN COMPLEXES OF TRYPSIN AND TRYPSINOGEN WITH PEPTIDE AND NONPEPTIDE BORONIC ACID INHIBITORS BY H-1-NMR SPECTROSCOPY

We have previously shown, in N-15 NMR studies of the enzyme's active site histidine residue, that boronic acid inhibitors can form two distinct types of complexes with alpha-lytic protease. Inhibitors that are structural analogs of good alpha-lytic protease substrates form transition-state-like tetrahedral complexes with the active site serine whereas those that are not form complexes in which N(epsilon2) of the active site histidine is covalently bonded to the boron of the inhibitor. This study also demonstrated that the serine and histidine adduct complexes exhibit quite distinctive and characteristic low-field H-1 NMR spectra [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. A. (1988) Biochemistry 27,7689-7697]. Here we have used low-field H-1 NMR diagnostically for a series of boronic acid inhibitor complexes of trypsin and trypsinogen. The results show that H-D-Val-Leu-boroArg and Ac-Gly-boroArg, analogs of good trypsin substrates, form transition-state-like serine adducts with trypsin, whereas the nonsubstrate analog inhibitors boric acid, methane boronic acid, butane boronic acid, and triethanolamine borate all form histidine adducts, thereby paralleling the previous results obtained with alpha-lytic protease. However, with trypsinogen, Ac-Gly-boroArg forms predominantly a histidine adduct while H-D-Val-Leu-boroArg forms both histidine and serine adducts, with the histidine adduct predominating below pH 8.0 and the serine adduct predominating above pH 8.0. The addition of exogenous Ile-Val, the dipeptide formed at the amino terminus of trypsin upon activation of trypsinogen to trypsin, induces both the Ac-Gly-boroArg and the H-D-Val-Leu-boroArg trypsinogen complexes to switch from histidine and pH-dependent multiple mode adducts to pH-independent, well-defined serine adducts. All the nonsubstrate analogs listed above that form histidine adducts with trypsin also form histidine adducts with trypsinogen. The significance of these findings for understanding the mechanisms of catalysis, inhibitor binding, and zymogen activation is discussed.