tailieunhanh - Báo cáo khóa học: The structure–function relationship in the clostripain family of peptidases

In this study we investigate the active-site structure and the catalytic mechanism of clostripain by using a combination of three separate techniques: affinity labelling, site-directed mutagenesis and molecular modelling. A benzamidinyl-diazo dichlorotriazine dye (BDD) was shown to act as an efficient active site-directed affinity label for Clostridium histolyticumclostripain. The enzyme, upon incubation with BDD in pH , exhibits a time-dependent loss of activity. | Eur. J. Biochem. 271 983-992 2004 FEBS 2004 doi The structure-function relationship in the clostripain family of peptidases Nikolaos E. Labrou1 and Daniel J. Rigden2 1Laboratory of Enzyme Technology Department of Agricultural Biotechnology Agricultural University of Athens Greece 2School of Biological Sciences University of Liverpool UK In this study we investigate the active-site structure and the catalytic mechanism of clostripain by using a combination of three separate techniques affinity labelling site-directed mutagenesis and molecular modelling. A benzamidinyl-diazo dichlorotriazine dye BDD was shown to act as an efficient active site-directed affinity label for Clostridium histolyticum clostripain. The enzyme upon incubation with BDD in M Hepes NaOH buffer pH exhibits a timedependent loss of activity. The rate of inactivation exhibits a nonlinear dependence on the BDD concentration which can be described by reversible binding of dye to the enzyme prior to the irreversible reaction. The dissociation constant of the reversible formation of an enzyme-BDD complex is Kd M and the maximal rate constant of inactivation is k3 . Effective protection against inactivation by BDD is provided by the substrate N-benzoyl-L-arginine ethyl ester BAEE . Cleavage of BDD-modified enzyme with trypsin and subsequent separation of peptides by reverse-phase HPLC gave only one modified peptide. Amino acid sequencing of the modified tryptic peptide revealed the target site of BDD reaction to be His176. Site-directed mutagenesis was used to study further the functional role of His176. The mutant His176Ala enzyme exhibited zero activity against BAEE. Together with previous data these results confirm that a catalytic dyad of His176 and Cys231 is responsible for cysteine peptidase activity in the C11 peptidase family. A molecular model of the catalytic domain of clostripain was constructed using a manually extended fold .