THE STRUCTURE OF THE CYTIDINE DEAMINASE PRODUCT COMPLEX PROVIDES EVIDENCE FOR EFFICIENT PROTON-TRANSFER AND GROUND-STATE DESTABILIZATION

Crystal structures of the cytidine deaminase-uridine product complex p repared either by cocrystallizing enzyme with uridine or by diffusing cytidine into ligand-free crystals show that the product binds as a 4- ketopyrimidine. They reveal four additional features of the catalytic process. (1) A water molecule bound to a site previously observed to b ind the incoming 4-NH2 group represents the site for the leaving ammon ia molecule. The conserved Pro 128 accommodates both moieties by orien ting the carbonyl group of the previous residue. (2) The Glu 104 carbo xylate group rotates from its hydrogen bond to the O4 hydroxyl group i n transition-state analog complexes, forming a new hydrogen bond to th e leaving group moiety. Thus, after stabilizing the hydroxyl group in the transition state, Glu 104 transfers a proton from that group to th e leaving amino group, promoting enol-to-keto isomerization of the pro duct. (3) Difference Fourier comparisons with transition-state complex es indicate that the pyrimidine ring rotates toward the zinc by simila r to 10 degrees. The active site thus ''pulls'' the ring and 4-NH2 gro up in opposite directions during catalysis. To preserve coplanarity of the 4-keto group with the pyrimidine ring, the Nl-Cl' glycosidic bond bends by similar to 19 degrees out of the ring plane. This distortion may ''spring-load'' the product complex and promote dissociation. Fai lure to recognize a similar distortion could explain an earlier crysta llographic interpretation of the adenosine deaminase-inosine complex [ Wilson, D. K., & Quiocho, F. A. (1994) Nat. Struct. Biol. 1, 691-694]. (4) The Zn-S(gamma)132 bond, which lengthens in transition-state comp lexes, shortens as the O4 atom returns to a state of lower negative ch arge in the planar product, consistent with our previous proposal that this bond buffers the zinc bond valence, compensating buildup of nega tive charge on the oxygen nucleophile during catalysis.