Role of glutamate-67 in the catalytic mechanism of human cytidine deaminase.

Abstract

Human cytidine deaminase (CDA, EC 3.5.4.5) is a tetramer of identical subunits (16KDa/subunit) each containing a single zinc atom1 and catalyzes the deamination of cytidine (CR) and deoxycytidine (CdR) to their corresponding uracil compounds. Several cytosine-based drugs, such as cytosine arabinoside and 5-Aza-CdR,2 which are anti-tumor agents used in the chemotherapy, are also deaminated by CDA. Thereby they loose their pharmacological properties. The deamination mechanism of CDA is similar to that of adenosine deaminase (ADA)3,4 and consists of an enzyme-assisted direct water attack on the nucleoside C-4 and C-6 carbon atoms, respectively. In both enzymes the hydroxyl group thus formed interacts with an active-site zinc atom and a negatively charged carboxylate group. The amino acid sequences of ADA and CDA are unrelated, except for a short sequence within the active site: in Escherichia coli these sequences are TVHAE and TVHAGE for CDA and ADA, respectively5 and the H residue is involved in zinc coordination in both deaminases. The E residue participates to the catalytic mechanism of CDA,3 whereas in the crystal structure of ADA4 the glutamic residue is shown to share a proton with N-l of the purine ring, while an aspartic residue (D 295) plays the same role as the glutamate in CDA, suggesting the importance of the presence of a carboxyl group in the catalytic site of nucleotide deaminases. In fact, in both the human and the Bacillus subtilis CDA the E residue is conserved, whereas the H residue is replaced by a C residue. In the present paper we demonstrate the importance of the Glu-67 carboxylic group by studying the effect on enzyme activity of reacting wild-type CDA with the specific carboxyl reagent N-ethoxy- carbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ), and by analyzing two mutant enzymes in which Glu-67 has been replaced with Asp and Gln (E67D and E67Q), respectively.