Directed mutagenesis of pig renal membrane dipeptidase. His219 is critical but the DHXXH motif is not essential for zinc binding or catalytic activity.
Pig renal membrane dipeptidase cDNA has been expressed in COS-1 cells. Directed mutagenesis was used to investigate the roles of some conserved histidyl and aspartyl residues. Mutation of His219 to Arg, Lys or Leu results in complete abolition of enzyme activity, although the mutants are expressed at the cell-surface. Residues in a proposed motif (DHXDH; residues 269-273) for zinc binding have been mutated individually. Each retained activity comparable to that of the wild-type, excluding an essential role for components of this motif. The zinc-binding ligands in membrane dipeptidase therefore represent a novel domain for a metallopeptidase with His219 being one candidate.
Keynan
S
S
Department of Biochemistry and Molecular Biology, University of Leeds, UK.
Hooper
N M
NM
Turner
A J
AJ
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D004252:DNA Mutational Analysis; D004150:Dipeptidases / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013552:Swine; D015032:Zinc / Q000378:metabolism*
D006639:Histidine; D004150:Dipeptidases; C038937:dipeptidase; D015032:Zinc