Importance of Glu-125 in the catalytic activity of human renal dipeptidase.
The carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and dansyl-ethylenediamine have shown to inhibit human renal dipeptidase (hrDP) irreversibly in a time-dependent manner. Cilastatin, a competitive inhibitor of the enzyme, partially protected the enzyme from inactivation. To identify the site(s) modified by EEDQ and dansylethylenediamine, the amino-acid sequence of tryptic fragments of modified enzyme were analyzed extensively. A comparison of the determined amino-acid sequences with the predicted primary structure of hrDP revealed that Glu-125 within the Glu115-Arg138 fragment was modified. In consequence, the role of Glu-125 in catalytic activity was investigated by site-directed mutagenesis. Glu-125 was replaced by a glutamine, aspartic acid or cysteine residue. cDNAs for wild-type or mutated enzymes were expressed in CHO cells, and the resulting proteins were purified to apparent homogeneity. The mutated enzyme, Gln-125-hrDP exhibited specific activity of 28.5 U/mg, corresponding to 11.4% of the wild-type. In contrast, Asp-125-hrDP and Cys-125hrDP were found to be inactive (< or = 0.1% of wild-type enzyme). These results suggest that the polarity and/or length of the side chain of Glu-125 residue are important for the enzyme activity.
Adachi
H
H
Suntory Institute for Biomedical Research, Osaka, Japan.
Katayama
T
T
Nakazato
H
H
Tsujimoto
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D015377:Cilastatin / Q000494:pharmacology; D003619:Dansyl Compounds / Q000494:pharmacology; D004150:Dipeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D005029:Ethylenediamines / Q000494:pharmacology; D005971:Glutamates; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011804:Quinolines / Q000494:pharmacology; D014357:Trypsin
D003619:Dansyl Compounds; D005029:Ethylenediamines; D005971:Glutamates; D011804:Quinolines; D015377:Cilastatin; C032377:dansylethylenediamine; D018698:Glutamic Acid; C000470:EEDQ; D004150:Dipeptidases; C038937:dipeptidase; D014357:Trypsin