The roles of Glu93 and Tyr149 in astacin-like zinc peptidases.
The catalytic zinc of astacin, a prototype of the astacin family and the metzincin superfamily of metalloproteinases is coordinated by three histidines, a glutamate bound water and a tyrosine. In order to assess the roles of active site key residues, two mutants, Glu93Ala-astacin and Tyr149Phe-astacin, were expressed in Escherichia coli, affinity-purified and renatured. While the Glu93Ala mutant was inactive, the Tyr149Phe mutant retained about 2. 5% residual activity toward Dns-Pro-Lys-Arg*Ala-Pro-Trp-Val, based on the k(cat)/K(m) value for recombinant wild-type astacin. These results support a model in which Glu93 is the general base in substrate hydrolysis, whereas Tyr149 contributes to transition state binding.
Yiallouros
I
I
Institute of Zoophysiology, University of Münster, Hindenburgplatz 55, D-48143, Münster, Germany. yiallou@uni-muenster.de
Grosse Berkhoff
E
E
Stöcker
W
W
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D004926:Escherichia coli; D018698:Glutamic Acid; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D018698:Glutamic Acid; D014443:Tyrosine; D008666:Metalloendopeptidases; C071288:astacin; D015032:Zinc