Zinc-bound hydroxyl

This is of relevance to the mechanism of carbonic anhydrase. This enzyme, which catalyzes the hydration of C02, has at its active site a Zn2+ ion ligated to the imidazole rings of three of its histidines. The classic mechanism for the reaction is that the fourth ligand is a water molecule which ionizes with a pKa of 7.37 The reactive species is considered to be the zinc-bound hydroxyl. Chemical studies show that zinc-bound hydroxyls are no exception to the rule of high reactivity. The H20 in structure 2.31 ionizes with a pKa of 8.7 and catalyzes the hydration of carbon dioxide and acetaldehyde.38... 

In order to obtain a proper molecular description of LFERs, it is essential to represent each reactant, product or intermediate by a parabolic free-energy function.22 In the case of CA III, we describe the proton transfer from residue 64 (Lys or His) to the zinc bound hydroxyl via a bridging water molecule (and alternatively two water molecules), by considering the three states ... 

The mechanism of action of mononuclear zinc enzymes depends on the Zn +-OH2 centre, which can participate in the catalytic cycle in three distinct ways (Figure 12.2) — either by ionisation, to give zinc-bound hydroxyl ion (in carbonic anhydrase), polarisation by a general base (in carboxypeptidase), or displacement of... 

The zinc-bound hydroxyl is hydrogen bonded through a water chain to His 64. In the absence of the CO2 substrate an additional water molecule will be hydrogen bonded to the hydroxyl and this must be displaced by the incoming CO2. 

With the pK value of water lowered to near physiological pH values, an appreciable amount of zinc-bound hydroxyl ion will be formed by dissociation of a proton from the zinc-bound water. The hydroxyl ion is the nucleophile that attacks the carbonyl carbon of CO2 to form the bicarbonate ion. Thus, the enzyme generates a reactive substrate by binding water to Zn ", thereby facilitating its dissociation to form the reactive substrate. 

Fig. 21. Indirect carboxylate-zinc interactions through bridging hydroxyl groups (Fig. 20) orient the nucleophilic lone electron pair (stippled dumbbell) of zinc-bound solvent. This reduces the conformational disorder about the Zn -O axis, and thereby reduces the entropic barrier to catalysis (Merz, 1990).

The active site contains two Zn2+ ions and one Mg2+ ion which are held by imidazole and carboxylate groups. The inorganic phosphate in an enzyme-product complex is bound to both zinc ions (Fig. 12-23). The Ser 102 side chain is above one Zn. In the enzyme-P intermediate it would be linked to the phospho group as an ester which would then be hydrolyzed, reversibly, by a water molecule bound to Zn.712 713a This water presumably dissociates to Zn+-OH and its bound hydroxyl ion carries out the displacement. This reaction may be preceded by a proton transfer to an oxygen atom of the phospho group.714... 

Crystallographic studies suggest that this is the ionization of the zinc-bound water molecule. The nicotinamide ring of the NAD+ is bound close to the zinc ion at the bottom of the pocket. The 2 -hydroxyl of the ribose ring is located between the hydroxyl of Ser-48 and the imidazole ring of His-51, within hydrogenbonding distance of both. 

The positive charge on the zinc ion withdraws electrons from the oxygen of the bound water, weakens the bonds to its hydrogen atoms, and promotes the dissociation of a proton to form an enzyme-bound hydroxyl. 

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