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 enz yme activity, me replaced each of seven cysteine residues in rat dipeptidyl peptidase III cDNA with alanine, glycine, or glutamic acid residue using s ite-directed mutagenesis. Each mutated cDNA was expressed in E. coli (BL21) , and each expressed DPP III was purified to apparent homogeneity on SDS-po lyacrylamide gel electrophoresis. Six of the mutant proteins had similar ac tivity 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 act ivity. This mutant enzyme was resistant against both PCMB and NEM. Furtherm ore, both Cys(176) --> Gly and Cys(176) --> Glu mutated enzymes showed no D PP LII activity. These seven mutated enzymes contained significant amounts of zinc, as determined by atomic absorption spectrometry. The results indic ate that Cys(176) is essential for the regulation of DPP III activity. (C) 2000 Academic Press.