tailieunhanh - Báo cáo khóa học: Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase

Glycoside hydrolysis by retaining family 18 chitinases involves a catalytic acid (Glu) which is part of a conserved DXDXEsequence motif that spans strand four of a (ba)8 barrel (TIMbarrel) are unusual in that thepositive charge emergingon theanomeric carbon after departure of the leaving group is stabilized by the substrate itself (the N-acetyl group of the distorted )1 sugar), rather than by a carboxylate group on the enzyme. | Eur. J. Biochem. 271 253-262 2004 FEBS 2003 doi Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase Bjernar Synstad1 Sigrid Gaseidnes1 Daan M. F. van Aalten2 Gert Vriend3 Jens E. Nielsen and Vincent G. H. Eijsink1 1 Department of Chemistry and Biotechnology Agricultural University of Norway As Norway 2Division of Molecular Microbiology and Biological Chemistry Wellcome Trust Biocentre University of Dundee UK CMBl University of Nijmegen the Netherlands 4Howard Hughes Medical Institute Department of Chemistry and Biochemistry University of California San Diego La Jolla CA USA Glycoside hydrolysis by retaining family 18 chitinases involves a catalytic acid Glu which is part of a conserved DXDXE sequenee moiil that spans trrand four of a ba 8 barrel TIM barrel structure. These glycoside hydrolases are unusual in that the positive charge emerging on the anomeric carbon after departure of the leaving group is stabilized by the substrate itself the N-acetyl group of the distorted -1 sugar rather than by a carboxylate group on the enzyme. We have studied seven conserved residues in the catalytic center of chitinase B from Serratia marcescens. Putative roles for these residues are proposed on the basis of the observed mutational effects the pH-dependency of these effects pKa calculations and available structural information. The results indicate that the pKa of the catalytic acid Glu144 is cycled during catalysis as a conseeuence of substrate-binding and release and possibly by a back and forth movement of Asp142 between Asp140 and Glu144. Rotation of Asp142 towards Glu144 also contributes to an essential distortion of the N-acetyl group of the -1 sugar. Two other conserved residues Tyr10 and Ser93 are important because they stabilize the charge on Asp140 while Asp142 points towards Glu144. Asp215 lying opposite Glu144 on the other side of the scissile glycosidic bond .