tailieunhanh - Báo cáo Y học: Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis

Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the )1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower kcat and a slightly higher Km than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with 2% residual activity | Eur. J. Biochem. 269 893-901 2002 FEBS 2002 Expression and characterization of active site mutants of hevamine a chitinase from the rubber tree Hevea brasiliensis Evert Bokma1 Henriette J. Rozeboom2 Mark Sibbald1 Bauke W. Dijkstra2 and Jaap J. Beintema1 Departments of 1 Biochemistry and 2Biophysical Chemistry Rijksuniversiteit Groningen the Netherlands Hevamine is a chitinase from the rubber tree Hevea brasil-iensis. Its active site contains Asp125 Glu127 and Tyr183 which interact with the -1 sugar residue of the substrate. To investigate their role in catalysis we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purilcation they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower kcat and a slightly higher Km than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with w 2 residual activity. In contrast the Asp125Asn mutant retained substantial activity with an approximately twofold lower kcat and an approximately twofold higher Km than the wild-type enzyme. More interestingly it showed activity to higher pH values than the other variants. The X-ray structure of the Asp125Ala Glu127Ala double mutant soaked with chito-tetraose shows that compared with wild-type hevamine the carbonyl oxygen atom of the N-acetyl group of the -1 sugar residue has rotated away from the C1 atom of that residue. The combined structural and kinetic data show that Asp125 and Tyr183 contribute to catalysis by positioning the carbonyl oxygen of the N-acetyl group near to the C1 atom. This allows the stabilization of a positively charged transient intermediate in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis. Keywords chitinase site-directed mutagenesis substrate-assisted catalysis X-ray structure. Chitin b- 1 4 -linked poly N-acetylglucosamine is one of the