A structural comparison of three isoforms of anionic trypsin from chum salmon (Oncorhynchus keta).
Three anionic salmon trypsin isoforms (CST-1, CST-2 and CST-3) were isolated from the pyloric caeca of chum salmon (Oncorhynchus keta). The order of catalytic efficiency (K(m)/k(cat)) of the isoforms during BAPA hydrolysis was CST-2 > CST-1 > CST-3. In order to find a structural rationalization for the observed difference in catalytic efficiency, the X-ray crystallographic structures of the three isoforms were compared in detail. Some structural differences were observed in the C-terminal alpha-helix, interdomain loop and active-site region. From the results of the detailed comparison, it appears that the structural flexibility of the C-terminal alpha-helix, which interacts with the N-terminal domain, and the substrate-binding pocket in CST-3 are lower than those in CST-1 and CST-2. In addition, the conformation of the catalytic triad (His57, Asp102 and Ser195) differs among the three isoforms. The imidazole N atom of His57 in CST-1 and CST-2 forms a hydrogen bond to the hydroxyl O atom of Ser195, but the distance between the imidazole N atom of His57 and the hydroxyl O atom of Ser195 in CST-3 is too great (3.8 A) for the formation of a hydrogen bond. Thus, the nucleophilicity of the hydroxyl group of Ser195 in CST-3 is weaker than that in CST-1 or CST-2. Furthermore, the electrostatic potential of the substrate-binding pocket in CST-2 is markedly lower than those in CST-1 and CST-3 owing to the negative charges of Asp150, Asp153 and Glu221B that arise from the long-range effect. These results may explain the higher catalytic efficiency of CST-2 compared with CST-1 and CST-3.
Toyota
Eiko
E
Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Japan. toyota@hoku-iryo-u.ac.jp
Iyaguchi
Daisuke
D
Sekizaki
Haruo
H
Tateyama
Midori
M
Ng
Kenneth K S
KK
D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D029941:Fish Proteins / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D017685:Oncorhynchus keta / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
D000838:Anions; D029941:Fish Proteins; D007527:Isoenzymes; D014357:Trypsin