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KMID : 0545119980080020158
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Journal of Microbiology and Biotechnology
1998 Volume.8 No. 2 p.158 ~ p.164
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The Effect of the Hydrogen Bond Network in the S1-pocket on Catalytic Activity of Serine Protease, Achromobacter protease 1 (API)
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Lim, Seon Gil
Byun, Myung Woo/Choi, Cheong
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Abstract
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Crystal structural analyses of the API-TLCK complex revealed that the ε-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 β-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 T1 89V·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 fluoroscence spectra of the three mutants were similar to those of wild-type API. With regards to the kinetic parameters (K_1 and k_2) of mutants for the reaction involving TLCK and DFP, k_2 decreased by increase of K_1 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 is not directly related to the reaction of Asp^225 with the substrate.
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