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Adenosine deaminase catalyzes conversion of adenosine and water into inosine and ammonia. Its zinc-containing active site activates water and stabilizes the reacting configuration; proton transfers assist addition and leaving-group departure. Mutation kinetics constrain catalytic roles but do not uniquely locate every proton.

Hypoxanthine is the 6-oxopurine base of inosine.

Inosine is the ribonucleoside of hypoxanthine. Hydrolytic deamination of adenosine replaces its exocyclic amino group with a carbonyl oxygen while retaining its sugar attachment.

The reactive fragment is the C6 amidine-like portion of the purine ring; the N9–ribose attachment is retained. Write it locally as $N1=C6(\mathrm{N}{\mathrm{H}}_2)-$, with the rest of the fused ring understood. One productive acid catalysis route protonates N1, making C6 more electrophilic. water then adds at C6 while the C6=N1 pi electrons move onto N1. Subsequent proton transfers generate a carbinolamine and then a protonated amino leaving group:These are local bonding fragments: C6 retains its bond to the adjoining ring carbon at every step. In the addition step, an oxygen lone pair forms C6–O and the C6=N1 pi bond opens. During collapse, an oxygen lone pair reforms C6=O and the C6–N6 bond breaks toward the departing nitrogen. Solvent mediates each proton transfer; no direct unsupported transfer across a long distance is required.

water supplies the new C6 oxygen, and ammonia is expelled after protonation of the leaving group. Final deprotonation gives the lactam form of inosine. In acidic bulk solution the released ammonia is largely trapped as ammonium. The catalytic proton transfers regenerate acid around the substitution cycle; ammonium trapping separately affects the net proton balance. This is hydrolytic deamination, not cleavage of the glycosidic bond.

The oxygen installed at C6 is derived from solvent water. An attacking zinc-bound water or hydroxide therefore provides the straightforward explanation of the oxygen-18 label. The result supports hydrolytic addition rather than direct oxygen transfer from a pre-existing protein carboxyl group as the sole oxygen source.

It does not by itself establish the identity of the base, the timing of proton transfers, or exclusion of a transient covalent enzyme intermediate: water could introduce the labeled oxygen during breakdown of such an intermediate. Product oxygen exchange must also be excluded. Incubate preformed unlabeled inosine in labeled water with and without enzyme, and use early-time quenched reactions to compare incorporation with product formation. These controls test whether labeling reports the formation step rather than later exchange.

A chemically coherent mechanism combines zinc-activated water addition with a proton relay involving His238 and Glu217. In a simple route consistent with the displayed contacts, neutral His238 accepts a proton from zinc-bound water while protonated Glu217 donates to substrate N1. These transfers can be coupled to the bond-forming event rather than requiring accumulation of a free, doubly activated substrate. Zinc-bound hydroxide attacks C6; the C6=N1 pi electrons move onto N1, yielding the tetrahedral C6 intermediate bearing OH and NH${}_2$ while N1 bears H.

Next, the exocyclic amino group receives a proton, through His238, Glu217 and/or intervening water as permitted by the changing geometry. The oxygen is deprotonated as it forms the C6 carbonyl, and the C6–N6 bond cleaves to release ammonia. These transfers can be coupled in the collapse step, avoiding expulsion of $\mathrm{N}{\mathrm{H}}_2^{-}$. Zinc stabilizes the reacting oxygen and the organized intermediate; the ring regains conjugation in the inosine product. Product dissociation, fresh water binding and proton exchange with solvent reset the catalytic groups.

The core result is zinc-assisted addition–elimination, with acid/base proton transfers and water supplying C6 oxygen. The contributions are metal ion catalysis, general base catalysis, general acid catalysis, electrostatic catalysis and proximity and orientation catalysis. The proposed route does not require covalent catalysis by a protein side chain: a substrate–water C–O bond is not an enzyme–substrate covalent linkage. Asp296 can retain a structural hydrogen-bonding role without acting as an indispensable acid/base.

The data establish the importance of His238 and Glu217 but do not uniquely assign the entire proton relay. In particular, an alternative has Glu217 first deprotonate water and then deliver that proton to N1, with His238 assisting hydroxide stabilization and later amino-group protonation. The primary observation that His238 substitution need not abolish analogue hydration cautions against calling its initial-base role proven.^[ref] pH-rate profiles, solvent isotope effects and time-resolved intermediate experiments would discriminate these refinements. The diagram is a proposed catalytic route, not a structure directly observed by the crystal experiment.