Effects of introduced aspartic and glutamic acid residues on the Pi substrate specificity, pH dependence and stability of carboxypeptidase Y |
| |
Authors: | Stennicke Henning R; Mortensen Uffe H; Christensen Ulla; Remington SJames; Breddam Klaus |
| |
Affiliation: | Carlsberg Laboratory, Department of Chemistry Gamle Carlsberg Vej 10, 2500 Copenhagen
1University of Copenhagen, Department of Chemistry Universitetsparken 5, 2100 Copenhagen, Denmark
2University of Oregon, Institute of Molecular Biology Eugene, Oregon 97403, USA |
| |
Abstract: | Carboxypeptidase Y is a serine carboxypeptidase isolated fromSaccharomyces cerevisiae with a preference for Cterminal hydrophobicamino acid residues. In order to alter the inherent substratespecificity of CPD-Y into one for basic amino acid residuesin P'1, we have introduced Asp and/or Glu residues at a numberof selected positions within the Si binding site. Hie effectsof these substitutions on the substrate specificity, pH dependenceand protein stability have been evaluated. The results presentedhere demonstrate that it is possible to obtain significant changesin the substrate preference by introducing charged amino acidsinto the framework provided by an enzyme with a quite differentspecificity. The introduced acidic amino acid residues providea marked pH dependence of the (kcat/Km)FA-A-R-OH/(kcatm)FA-A-R-OHratio. The change in stability upon introduction of Asp/Gluresidues can be correlated to the difference in the mean buriedsurfac surface area between the substituted and the substitutingamino acid. Thus, the effects of acidic amino acid residueson the protein stability depend upon whether the introducedamino acid protrudes from the solvent accessible surface asdefined by the surrounding residues in the wild type enzymeor is submerged below. |
| |
Keywords: | carboxypeptidase Y/ pH dependence/ protein stability/ site-directed mutagenesis/ substrate specificity |
本文献已被 Oxford 等数据库收录! |
|