Cytidine deaminase structure

A cell extract was subjected to ammonium sulfate fractionation and the dialyzed protein was then poured through the affinity column which held the cytidine deaminase molecules because of their affinity for the cytidine structures that were bound to the agarose. 

Fig. 7 Structure of cytidine deaminase with zebularine 3,4-hydrate bound.33 The low-barrier hydrogen bond between Glul04 and the Zn OH would help stabilize the tetrahedral intermediate, which would have an NH2 in place of the H at C4 of the ring in this structure.

It should therefore be possible to design chemical structures, modeled after known or putative reaction intermediates that resemble postulated transition states. They may exhibit high affinities for the reactive sites of enzymes and therefore function as effective, but reversible, inhibitors. A successful example is the potent and specific cytidine deaminase inhibitor 3,4,5,6-tetrahydrouridine, which effectively blocks the conversion of cytidine to uridine. It was similarly demonstrated that 1,6-dihydro-6-hydroxymethylpurine effectively blocked the deamination of adenine to hypoxanthine by adenine deaminase (Eq. 2.13). 

E. coli cytidine deaminase (CDA) is an enzyme that catalyzes hydrolytic deamination of cytidine to uridine (or from 2 -deoxycytidine to 2 -deoxyuridine) (Scheme 8). This enzyme is considered to be a member of the MMP family, although the Zn -binding site contains two Cys and one His residues. On the basis of the X-ray crystal structure of a transition-state analogue complex,a mechanism was proposed as shown in Scheme 8, in which Glul04 serves multiple roles the deprotonation of the Zn " -bound water and simultaneous protonation to N3 of the substrate (Scheme 8a), stabilization of the first tetrahedral transition state (Scheme 8b), and protonation of the leaving amino group at the 4-position of the substrate in the second tetrahedral intermediate (Scheme 8c).Upon elimination of NH3, an E-P complex is formed (Scheme 8d) and the enzyme goes into the next turnover. The deamination mechanism of E. coli... 

It was snbseqnently discovered that the first enzyme in the pathway for isoleucine synthesis, which is threonine deaminase, was inhibited by isoleucine in an extract of E. coli. No other amino acid caused inhibition of the enzyme. Threonine deaminase is, in fact, the rate-limiting enzyme in the pathway for isoleucine synthesis, so that this was interpreted as a feedback control mechanism (Fignre 3.13(a)). Similarly it was shown that the hrst enzyme in the pathway for cytidine triphosphate synthesis, which is aspartate transcarbamoylase, was inhibited by cytidine triphosphate (Fignre 3.13(b)). Since the chemical structures of isoleucine and threonine, or cytidine triphosphate and aspartate, are completely different, the qnestion arose, how does isolencine or cytidine triphosphate inhibit its respective enzyme The answer was provided in 1963, by Monod, Changenx Jacob. 

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