Essential roles of zinc ligation and enzyme dimerization for catalysis in the aminoacylase-1/M20 family.
Members of the aminoacylase-1 (Acy1)/M20 family of aminoacylases and exopeptidases exist as either monomers or homodimers. They contain a zinc-binding domain and a second domain mediating dimerization in the latter case. The roles that both domains play in catalysis have been investigated for human Acy1 (hAcy1) by x-ray crystallography and by site-directed mutagenesis. Structure comparison of the dinuclear zinc center in a mutant of hAcy1 reported here with dizinc centers in related enzymes points to a difference in zinc ligation in the Acy1/M20 family. Mutational analysis supports catalytic roles of zinc ions, a vicinal glutamate, and a histidine from the dimerization domain. By complementing different active site mutants of hAcy1, we show that catalysis occurs at the dimer interface. Reinterpretation of the structure of a monomeric homolog, peptidase V, reveals that a domain insertion mimics dimerization. We conclude that monomeric and dimeric Acy1/M20 family members share a unique active site architecture involving both enzyme domains. The study may provide means to improve homologous carboxypeptidase G2 toward application in antibody-directed enzyme prodrug therapy.
Lindner
Holger A
HA
Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec H4P 2R2, Canada.
Lunin
Vladimir V
VV
Alary
Alain
A
Hecker
Regina
R
Cygler
Miroslaw
M
Ménard
Robert
R
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D004795:Enzyme Stability; D018698:Glutamic Acid; D006639:Histidine; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D015032:Zinc / Q000378:metabolism*
D018698:Glutamic Acid; D006639:Histidine; D000581:Amidohydrolases; C019672:aminoacylase I; D015032:Zinc