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.