Richard Hall - Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase

Version 1

      Publication Details (including relevant citation   information):

      Richard S. Hall, Alexander A.   Fedorov, Chengfu   Xu, Elena V.   Fedorov, Steven C.   Almo*,   and Frank M. Raushel*

     

      Department of Chemistry, P.O. Box 30012,   Texas A&M University,   College Station, Texas 77842-3012, United States  

        Albert Einstein College     of Medicine, 1300 Morris Park Avenue, Bronx, New York     10461, United States  

      Biochemistry, Article ASAP

      DOI: 10.1021/bi200483k

      Publication Date (Web): May 5, 2011

      Copyright © 2011 American Chemical Society

      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 Ki 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   pKa of 6.0, and Zn-CDA has a kinetic pKa 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 kcat and kcat/Km,   consistent with the formation of a low-barrier hydrogen bond   during the conversion of cytosine to uracil. A chemical   mechanismfor substrate deamination by CDA was proposed.

      Address (URL): http://pubs.acs.org/doi/abs/10.1021/bi200483k