The HELLGH motif of rat liver dipeptidyl peptidase III is involved in zinccoordination and the catalytic activity of the enzyme

The role of the HELLGH (residues 450-455) motif in the sequence of rat dipe ptidyl peptidase III (EC 3.4.14.4) was investigated by replacing Glu(451) w ith an alanine or an aspartic acid residue and by replacing His(450) and Hi s(455) with a tyrosine residue by site-directed mutagenesis. Mutated cDNAs were expressed three or four times in Escherichia coli, and the resulting p roteins were purified to apparent homogeneity. None of the expressed mutate d proteins exhibited DPP III activity. The mutants of Glu(451) contained 1 mol of zinc per mole of protein, but mutants His(450) and His(455) did not contain significant amounts of zinc as determined by atomic absorption spec trometry. The Leu(453)-deleted enzyme (having the zinc aminopeptidase motif HExxH-18-E) had almost the same order of binding affinity (for Arg-Arg-2-n aphthylamide) as the wild-type enzyme, but the specificity constant was abo ut 10%. These results provide evidence that the suitable number of amino ac ids included between Glu(451) and His(455) is three residues for the enzyme activity and confirm that residues His(450), His(455), Glu(451) are involv ed in zinc coordination and catalytic activity.