Mutational analysis of active site residues of human adenosine deaminase.
Adenosine deaminase was overexpressed in a baculovirus system. The pure recombinant and native enzymes were identical in size, Zn2+ content, and activity. Five amino acids, in proximity to the active site, were replaced by mutagenesis. The altered enzymes were purified to homogeneity and compared to wild-type adenosine deaminase with respect to zinc content, enzymatic activity, and kinetic parameters. All but one of the alterations produced significant activity perturbations. Replacement of Cys262 produced a protein that retained at least 30-40% of wild-type activity. In contrast, replacements of His17, His214, His238, and Glu217 resulted in dramatic losses of enzyme activity. None of these mutants exhibited large variations in Km. The proteins produced from alterations of amino acids implicated in metal coordination were slightly activated by inclusion of Zn2+ throughout purification. These experiments confirm that in the active enzyme Zn2+ plays a critical role in catalysis, that a histidine or glutamate residue plays a mechanistic role in the hydrolytic deamination step, and that cysteine is not involved in the catalytic mechanism of adenosine deaminase. These data support the roles for these amino acid residues suggested from the x-ray structure of murine adenosine deaminase (Wilson, D. K., Rudolf, F. B., and Quicho, F. A. (1991) Science 252, 1278-1284).
Bhaumik
D
D
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
Medin
J
J
Gathy
K
K
Coleman
M S
MS
D000243:Adenosine Deaminase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins; D015032:Zinc / Q000032:analysis
D011994:Recombinant Proteins; D004247:DNA; D000243:Adenosine Deaminase; D015032:Zinc