Amphipathic property of free thiol group contributes to an increase in the catalytic efficiency of carboxypeptidase Y.
Cys341 of carboxypeptidase Y, which constitutes one side of the solvent-accessible surface of the S1 binding pocket, was replaced with Gly, Ser, Asp, Val, Phe or His by site-directed mutagenesis. Kinetic analysis, using Cbz-dipeptide substrates, revealed that polar amino acids at the 341 position increased K(m) whereas hydrophobic amino acids in this position tended to decrease K(m). This suggests the involvement of Cys341 in the formation of the Michaelis complex in which Cys341 favors the formation of hydrophobic interactions with the P1 side chain of the substrate as well as with residues comprising the surface of the S1 binding pocket. Furthermore, C341G and C341S mutants had significantly higher k(cat) values with substrates containing the hydrophobic P1 side chain than C341V or C341F. This indicates that the nonhydrophobic property conferred by Gly or Ser gives flexibility or instability to the S1 pocket, which contributes to the increased k(cat) values of C341G or C341S. The results suggest that Cys341 may interact with His397 during catalysis. Therefore, we propose a dual role for Cys341: (a) its hydrophobicity allows it to participate in the formation of the Michaelis complex with hydrophobic substrates, where it maintains an unfavorable steric constraint in the S1 subsite; (b) its interaction with the imidazole ring of His397 contributes to the rate enhancement by stabilizing the tetrahedral intermediate in the transition state.
Mima
Joji
J
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. mima@kais.kyoto-u.ac.jp
Jung
Giman
G
Onizuka
Takuo
T
Ueno
Hiroshi
H
Hayashi
Rikimaru
R
D002268:Carboxypeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D043402:Cathepsin A; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014633:Valine / Q000235:genetics
D013438:Sulfhydryl Compounds; D010649:Phenylalanine; D006639:Histidine; D002268:Carboxypeptidases; D043402:Cathepsin A; D014633:Valine