The HELLGH motif of rat liver dipeptidyl peptidase III is involved in zinc coordination and the catalytic activity of the enzyme.
The role of the HELLGH (residues 450-455) motif in the sequence of rat dipeptidyl peptidase III (EC 3.4.14.4) was investigated by replacing Glu451 with an alanine or an aspartic acid residue and by replacing His450 and His455 with a tyrosine residue by site-directed mutagenesis. Mutated cDNAs were expressed three or four times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. None of the expressed mutated proteins exhibited DPP III activity. The mutants of Glu451 contained 1 mol of zinc per mole of protein, but mutants His450 and His455 did not contain significant amounts of zinc as determined by atomic absorption spectrometry. The Leu453-deleted enzyme (having the zinc aminopeptidase motif HExxH-18-E) had almost the same order of binding affinity (for Arg-Arg-2-naphthylamide) as the wild-type enzyme, but the specificity constant was about 10%. These results provide evidence that the suitable number of amino acids included between Glu451 and His455 is three residues for the enzyme activity and confirm that residues His450, His455, and Glu451 are involved in zinc coordination and catalytic activity.
Fukasawa
K
K
Department of Oral Biochemistry, School of Dentistry, Matsumoto Dental University, Shiojiri, Nagano 399-0781, Japan. kmf@po.mdu.ac.jp
Fukasawa
K M
KM
Iwamoto
H
H
Hirose
J
J
Harada
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D018076:DNA, Complementary / Q000302:isolation & purification; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D007930:Leucine / Q000235:genetics; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006639:Histidine; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III; D007930:Leucine; D015032:Zinc