Inactivation of carboxypeptidase Y by mutational removal of the putative essential histidyl residue.
Carboxypeptidase Y is a serine carboxypeptidase assumed to contain a catalytic triad similar to the serine endopeptidases. On the basis of the homology between various serine carboxypeptidases His-397 is suspected to be part of the catalytic triad. To test this it was exchanged with Ala and Arg by site-directed mutagenesis of the cloned PRC1 gene. The catalytic efficiency of the mutant enzymes were reduced by a factor of 2 X 10(4) and 7 X 10(2), respectively, confirming the key role of His-397 in catalysis. Treatment of Ala-397-CPD-Y with Hg++ or CNBr, hence modifying Cys-341 located in the vicinity of the active site abolished the residual activity of the enzyme, indicating an additional involvement of this residue in catalysis.
Bech
L M
LM
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby.
Breddam
K
K
D000595:Amino Acid Sequence; D001665:Binding Sites; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D012689:Sequence Homology, Nucleic Acid
D006639:Histidine; D002268:Carboxypeptidases; C022633:serine carboxypeptidase