Alteration of rat dipeptidyl peptidase III by site-directed mutagenesis: cysteine(176) is a regulatory residue for the enzyme activity.
To comprehend the importance of cysteine residues for the regulation of enzyme activity, we replaced each of seven cysteine residues in rat dipeptidyl peptidase III cDNA with alanine, glycine, or glutamic acid residue using site-directed mutagenesis. Each mutated cDNA was expressed in E. coli (BL21), and each expressed DPP III was purified to apparent homogeneity on SDS-polyacrylamide gel electrophoresis. Six of the mutant proteins had similar activity to that of the wild-type enzyme, whereas the activity of the Cys(176) --> Ala mutant enzyme was only 25-35% of that of the wild-type enzyme activity. This mutant enzyme was resistant against both PCMB and NEM. Furthermore, both Cys(176) --> Gly and Cys(176) --> Glu mutated enzymes showed no DPP III activity. These seven mutated enzymes contained significant amounts of zinc, as determined by atomic absorption spectrometry. The results indicate that Cys(176) is essential for the regulation of DPP III activity.
Li
Y H
YH
Department of Medical Biochemistry, Shiga University of Medical Science, Seta Otsu, 520-2192, Japan.
Maeda
T
T
Yamane
T
T
Ohkubo
I
I
D019943:Amino Acid Substitution; D000818:Animals; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015032:Zinc / Q000378:metabolism
D010447:Peptide Hydrolases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III; D015032:Zinc; D003545:Cysteine