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Department of Biochemistry, Eotvos Lorand University, and 2Biotechnology Research Group of the Hungarian Academy of Sciences, ´ ´ ´ ´ Pazmany setany 1/C, Budapest, Hungary The molecular mechanism of the autolysis of rat a-chymotrypsin B was investigated. In addition to the two already known autolytic sites, Tyr146 and Asn147, a new site formed by Phe114 was identified. The former two sites and the latter one are located in the autolysis and the interdomain loops, respectivel | Eur. J. Biochem. 268 6238-6246 2001 FEBS 2001 Structural determinants of the half-life and cleavage site preference in the autolytic inactivation of chymotrypsin Arpad Bodi1 2 Gyula Kaslik1 Istvan Venekei1 and László Graf1 2 1 Department of Biochemistry Eotvos Lorand University and 2Biotechnology Research Group of the Hungarian Academy of Sciences Pazmany setany 1 C Budapest Hungary The molecular mechanism of the autolysis of rat a-chymotrypsin B was investigated. In addition to the two already known autolytic sites Tyr146 and Asn147 a new site formed by Phe114 was identified. The former two sites and the latter one are located in the autolysis and the interdomain loops respectively. By eliminating these sites by site-directed mutagenesis their involvement in the autolysis and autolytic inactivation processes was studied. Mutants Phe114 lle and Tyr146 His Asn147 Ser that had the same enzymatic activity and molecular stability as the wild-type enzyme displayed altered routes of autolytic degradation. The Phe114 lle mutant also exhibited a significantly slower autolytic inactivation its half-life was 27-fold longer in the absence and sixfold longer in the presence of Ca21 ions that obeyed a first order kinetics instead of the second order displayed by wild-type chymotrypsin inactivation. The comparison of autolysis and autolytic inactivation data showed that a the preferential cleavage of sites followed the order of Tyr146-Asn147 Phe114 other sites b the cleavage rates at sites Phe114 and Tyr146-Asn147 were independent from each other and c the hydrolysis of the Phe114-Ser115 bond was the rate determining step in autolytic inactivation. Thus it is the cleavage of the interdomain loop and not of the autolysis or other loops that determines the half-life of chymotrypsin activity. Keywords autolysis inactivation chymotrypsin cleavage site preference proteolytic half-life. A number of physiological studies on humans rats and pigs show that chymotrypsin and trypsin .
