The effect of the hydrogen bond network in the S1-pocket on catalytic activity of serine protease, Achromobacter protease I (API)

Crystal structural analyses of the API-TLCK complex revealed that the E-amino group (NZ) of the lysyl part of TLCK forms hydrogen bonds with OD1 of Asp(225) which is a substrate specificity determinant of API, OG of Ser(214), O Of Ser(214), OG1 Of Thr(189), and O of Thr(189). The beta-carboxyl oxygen of Asp(225) forms hydrogen bonds with the NE1 of Trp(182). From these observations, it is thought that besides Asp(225), Thr(189), Ser(214), and Trp(182) may also contribute to the steric specificity for lysine and high proteolytic activity of API. The side-chain hydroxyl groups of Thr(189) and Ser(214) were removed to elucidate the role of these hydrogen bonds in the S-1-pocket. The k(cat)/K-m, of T189V, S214A, and T189V.S214A were decreased to 1/4, 1/3, and 1/46, respectively, of the value for native API. The decreased activities were mainly due to the increase of K-m. The CD and fluorescence spectra of the three mutants were similar to those of wild-type API. With regards to the kinetic parameters (Ki and k,) of mutants for the reaction involving TLCK and DFP, k, decreased by increase of K, only. These results suggest that the decreased catalytic activity of these mutants is caused by the partial loss of the hydrogen bond network in the S-1-pocket. On the other hand, the similarity of enzymatic properties between W182F and the native enzyme suggests that the hydrogen bond between OD2 of Asp(225) and NE1 of Trp(182), not directly related to the reaction of Asp(225) with the substrate.