tailieunhanh - Báo cáo Y học: The role of the second binding loop of the cysteine protease inhibitor, cystatin A (stefin A), in stabilizing complexes with target proteases is exerted predominantly by Leu73

The aimof this workwas to elucidate the roles of individual residues within the flexible second binding loop of human cystatin A in the inhibition of cysteine proteases. Four recombinant variants of the inhibitor, each with a single mutation, L73G, P74G, Q76G or N77G, in the most exposed part of this loopwere generated by PCR-based site-directed mutagenesis. The binding of these variants to papain, cathepsin L, and cathepsin B was characterized by equilibrium and kinetic methods. | Eur. J. Biochem. 269 5649-5658 2002 FEBS 2002 doi The role of the second binding loop of the cysteine protease inhibitor cystatin A stefin A in stabilizing complexes with target proteases is exerted predominantly by Leu73 Alona Pavlova Sergio Estrada and Ingemar Bjork Department of Veterinary Medical Chemistry Swedish University of Agricultural Sciences Uppsala Biomedical Centre Sweden The aim of this work was to elucidate the roles of individual residues within the flexible second binding loop of human cystatin A in the inhibition of cysteine proteases. Four recombinant variants of the inhibitor each with a single mutation L73G P74G Q76G or N77G in the most exposed part of this loop were generated by PCR-based site-directed mutagenesis. The binding of these variants to papain cathepsin L and cathepsin B was characterized by equilibrium and kinetic methods. Mutation of Leu73 decreased the affinity for papain cathepsin L and cathepsin B by w 300-fold 10-fold and w 4000-fold respectively. Mutation of Pro74 decreased the affinity for cathepsin B by w 10-fold but minimally affected the affinity for the other two enzymes. Mutation of Gln76 and Asn77 did not alter the affinity of cystatin A for any of the proteases studied. The decreased affinities were caused exclusively by increased dissociation rate constants. These results show that the second binding loop of cystatin A plays a major role in stabilizing the complexes with proteases by retarding their dissociation. In contrast with cystatin B only one aminoacid residue of the loop Leu73 is of principal importance for this effect Pro74 assisting to a minor extent only in the case of cathepsin B binding. The contribution of the second binding loop of cystatin A to protease binding varies with the protease being largest w 45 of the total binding energy for inhibition of cathepsin B. Keywords cathepsins cystatin cysteine proteases papain second binding loop. Cystatins are effective protein