In rat dipeptidyl peptidase III, His⁵⁶⁸ is essential for catalysis, and Glu⁵⁰⁷ or Glu⁵¹² stabilizes the coordination bond between His⁴⁵⁵ or His⁴⁵⁰ and zinc ion.
Dipeptidyl peptidase (DPP) III is a zinc-dependent exopeptidase that has a unique motif, "HELLGH," as the zinc-binding site. In the present study, a three-dimensional (3D) model of rat DPP III was generated with the X-ray crystal structure of human DPP III (PDB: 3FVY [Dobrovetsky E. et al. (2009) SGC]) as a template. The replacement of the seven charged amino acid residues with a hydrophobic amino acid around the zinc ion did not cause any significant changes in K(m) values or in the substrate specificity. However, the k(cat) values of H568R and H568Y were remarkably reduced, by factors of 50 and 400, respectively. The His⁵⁶⁸ residue of rat DPP III is essential for enzyme catalysis. The k(cat) values of the mutants E507A and E512A were 2.38 and 3.88 s⁻¹ toward Arg-Arg-NA, and 0.097 and 0.59 s⁻¹ toward Phe-Arg-NA, respectively. These values were markedly lower than those of the wild-type DPP III. Furthermore, the zinc contents of E507A and E512A were 0.29 and 0.08 atom per mol of protein, respectively, and those mutations caused remarkable increases in the dissociation constants of the zinc ions from DPP III by factors of 5 x 10³ to 2 x 10⁴. The 3D model of the catalytic domain of rat DPP III showed that the carboxyl oxygen atoms of Glu⁵⁰⁷ and Glu⁵¹² form the hydrogen bonds to the nitrogen atoms of His⁴⁵⁵ and His⁴⁵⁰. All of these results showed that Glu⁵⁰⁷ or Glu⁵¹² stabilizes the coordination bond between the zinc ion and His⁴⁵⁵ or His⁴⁵⁰.
Fukasawa
Kayoko M
KM
Department of Hard Tissue Research, Graduate School of Oral Medicine, Matsumoto Dental University, School of Dentistry, Shiojiri, Nagano 399-0781, Japan. kmf@po.mdu.ac.jp
Hirose
Junzo
J
Hata
Toshiyuki
T
Ono
Yukio
Y
D000818:Animals; D002384:Catalysis; D018360:Crystallography, X-Ray; D004151:Dipeptides / Q000737:chemistry* / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry* / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D004151:Dipeptides; C048251:phenylalanylarginine; C076685:arginylarginine; D018698:Glutamic Acid; D006639:Histidine; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III; D015032:Zinc