title: Three-dimensional structure and catalytic mechanism of cytosine deaminase.
abstract: Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a K(i) of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pK(a) of 6.0, and Zn-CDA has a kinetic pK(a) of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on k(cat) and k(cat)/K(m), consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.
authors.affiliation: Department of Chemistry, Texas A&M University, College Station, Texas 77842-3012, United States.
authors.forename: Richard S
authors.initials: RS
authors.lastname: Hall
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Fedorov
authors.forename: Chengfu
authors.initials: C
authors.lastname: Xu
authors.forename: Elena V
authors.initials: EV
authors.lastname: Fedorov
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Frank M
authors.initials: FM
authors.lastname: Raushel
mesh_terms: D002384:Catalysis; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism; D043525:Cytosine Deaminase / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D015032:Zinc / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D018698:Glutamic Acid; D003596:Cytosine; D043525:Cytosine Deaminase; C468827:codA protein, E coli; D015032:Zinc