Structure Report for: 1CPY

ESTHER: Sorensen_1995_J.Am.Chem.Soc_117_5944
PubMedSearch: Sorensen 1995 J.Am.Chem.Soc 117 5944
Gene_locus related to this paper: yeast-cbpy1

Abstract

In (serine)carboxypeptidase Y, the flexible side chain of Met 398 forms one side of the Si' binding pocket and the beta -and gamma-carbon atoms of Thr60 form the opposite side. Met398 has been substituted with the residues Gly,Ala,Val,lie,Leu,Phe,and Tyr while Thr60 has been substituted with the residues Ala,Val,Leu,Met,Phe,and Tyr by site-directed mutagenesis,and the resulting enzymes have been characterized with respect to their Pi' substrate preferences using thes ubstrate series FA-Phe-Xaa-OH (Xaa=Gly,Ala,Val,orLeu) and FA-Ala-Yaa-OH (Yaa=Leu,Gin,Glu,Lys,or Arg). The results show that Met398 is much more important for transition state stabilization than Thr60 although itappears that the selected non bulky amino acid residue(Thr) at position 60 is important for high Kcat values. The results further suggest that bulky amino acid side chains at position 398 are able to adjust the size of the Si' pocket such that favorable interactions with the substrate can be obtained with even small Pi' side chains,e.g., Gly. Accordingly,the hydrolysis of substrates with bulky/hydrophobic Pi' side chains is less dependent on the nature of the amino acid residue at position 398 than that of a substrate with a non bulky Pi' sid echain.The three-dimensional structure oft hemutant enzymeE65A+E145A has been determined, and it provides support for the high mobility of the Met398 side chain. In transpeptidation reactions the substitutions at position 398 also influence the interactions between the binding pocket and the amino acid leaving group as well as the added nucleophile competing with water in the deacylation reaction.Much higher aminolysis was obtained with some of the mutant enzymes, presumably due to a changed accessibility of water to the acyl-enzyme intermediate while the nucleophile/leaving group isbound at the Si' binding site.

ESTHER: Endrizzi_1994_Biochemistry_33_11106
PubMedSearch: Endrizzi 1994 Biochemistry 33 11106
PubMedID: 7727362
Gene_locus related to this paper: yeast-cbpy1

Abstract

The structure of monomeric serine carboxypeptidase from Saccharomyces cerevisiae (CPD-Y), deglycosylated by an efficient new procedure, has been determined by multiple isomorphous replacement and crystallographic refinement. The model contains 3333 non-hydrogen atoms, all 421 amino acids, 3 of 4 carbohydrate residues, 5 disulfide bridges, and 38 water molecules. The standard crystallographic R-factor is 0.162 for 10,909 reflections observed between 20.0- and 2.8-A resolution. The model has rms deviations from ideality of 0.016 A for bond lengths and 2.7 degrees for bond angles and from restrained thermal parameters of 7.9 A2. CPD-Y, which exhibits a preference for hydrophobic peptides, is distantly related to dimeric wheat serine carboxypeptidase II (CPD-WII), which has a preference for basic peptides. Comparison of the two structures suggests that substitution of hydrophobic residues in CPD-Y for negatively charged residues in CPD-WII in the binding site is largely responsible for this difference. Catalytic residues are in essentially identical configurations in the two molecules, including strained main-chain conformational angles for three active site residues (Ser 146, Gly 52, and Gly 53) and an unusual hydrogen bond between the carboxyl groups of Glu 145 and Glu 65. The binding of an inhibitor, benzylsuccinic acid, suggests that the C-terminal carboxylate binding site for peptide substrates is Asn 51, Gly 52, Glu 145, and His 397 and that the "oxyanion hole" consists of the amides of Gly 53 and Tyr 147. A surprising result of the study is that the domains consisting of residues 180-317, which form a largely alpha-helical insertion into the highly conserved cores surrounding the active site, are quite different structurally in the two molecules. It is suggested that these domains have evolved much more rapidly than other parts of the molecule and are involved in substrate recognition.