Molecular Biology: Protein Structure and Function - Stepanov V.M. 2005

Enzymes
Alcohol dehydrogenase

No matter how vast the capabilities of protein functional group assemblies may be, in A number of cases they prove insufficient, for instance, in catalyzing certain redox reactions. Such difficulties, however, can be overcome by incorporating non-protein ligands—Metal Ions or Coenzymes—into the enzyme's MECHANISM OF ACTION. A prime example of this mechanism is the action of Liver alcohol dehydrogenase.

This enzyme catalyzes The transfer of a hydride ion H- from the CH₂OH group of primary alcohols with various structures to the nicotinamide moiety of the coenzyme, nicotinamide adenine dinucleotide (NAD). Schematically, this reaction can be represented as follows:

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As is typical, the same enzyme is also capable of catalyzing the reverse reaction: the reduction of an aldehyde to an alcohol.

The well-characterized horse liver alcohol dehydrogenase is built from two identical subunits, each containing 374 amino acid residues in a single polypeptide chain and forming two domains. The catalytic domain, which houses all Components of the enzyme's active center—including the residues that bind yet another non-protein Ligand, the catalytically active zinc ion—comprises the N-terminal (residues 1-175) and C-terminal (residues 319-374) fragments of the polypeptide chain.

The coenzyme (nucleotide)-binding domain is formed by the central fragment (residues 176-318). In its secondary and tertiary Structure, it is remarkably similar to the nucleotide-binding domains of Other Enzymes, although their primary structures show no significant correspondence.

It is hypothesized that the characteristic Spatial Structure of the nucleotide-binding domain emerged at a very early stage of evolution and was repeatedly utilized as a ready-made functional module that could combine with various catalytic centers to form different enzymes containing NAD as a coenzyme. Clearly, this evolutionary pathway for enzymes is far more efficient than creating a functionally identical structure de novo every time.

The contact between subunits is driven primarily by the interaction of their nucleotide-binding domains, which form the structural "core" of the dimer. As is often the case in The formation of quaternary structure, the ß-pleated sheet of one subunit "docks" with the corresponding STRUCTURE OF THE other, creating an extensive shared ß-pleated layer. This interaction is reinforced by an extensive network of hydrophobic contacts involving about 30 amino acid residues, making the dissociation of the dimer extremely difficult even in 8 M urea.

Coenzyme binding. The adenosine diphosphoribose moiety of NAD forms numerous non-covalent contacts with the enzyme. The adenine ring resides in a hydrophobic pocket, the 2'-hydroxyl of the adenosine ribose forms a Hydrogen bond with the carboxyl group of Asp-223, and the pyrophosphate group forms a salt bridge with the guanidine group of Arg-47, which belongs to the catalytic domain. The "terminal" ribose residue, i.e., the one closest to the nicotinamide ring, forms Hydrogen Bonds with the carbonyl groups of Gly-293 and Ile-269. The multiplicity of non-covalent bonds ensures the precise positioning of the coenzyme within the Active Site of alcohol dehydrogenase.

Substrate binding. The substrate-binding site is located in a deep "pocket"—a channel situated between the catalytic and nucleotide-binding domains of one subunit, though it also incorporates individual amino acid residues from the second subunit. This channel is essentially lined with hydrophobic side chains, enabling hydrophobic interactions with the alkyl radical R of the alcohol. Interestingly, the formation of this channel is completed only after NAD binding and the subsequent conformational change in the enzyme. Liver alcohol dehydrogenase can bind A wide variety of alcohols, ranging from ethanol to benzyl alcohol derivatives C₆H₅CH₂OH, ω-hydroxy acids, and even steroidal alcohols. It is only essential that they contain a hydrophobic moiety in their structure. Among the residues involved in binding benzyl alcohol are Leu-116, Leu-57, Val-294, and Phe-93. Upon sorption, the substrate displaces several Water molecules from the binding site and to some extent tightens the adjacent region of the enzyme into a more compact structure. It is quite possible that this subtle structural shift induced by the bound substrate plays a crucial role in catalysis.

Components of the catalytic center. The catalytic center of alcohol dehydrogenase contains a zinc ion coordinated by four ligands arranged at the vertices of a distorted tetrahedron. Three of these belong to the protein: the thiol groups of residues Cys-16 and Cys-174, and the imidazole ring of His-67. The fourth coordination site is occupied by the oxygen atom of the substrate, or by a water molecule in its absence. The hydroxyl group of the Ser-48 residue is capable of forming a hydrogen bond with the oxygen atom of the ligand—whether water or alcohol.

Probable METABOLISM/10.html">Mechanism of enzyme Action. It is believed that the alcohol, whose hydrophobic chain is bound within the enzyme's hydrophobic channel, is oriented such that its oxygen atom establishes direct contact with the zinc ion, displacing the water molecule previously occupying the fourth coordination position (Fig. 10.10). This leads to the abstraction of a proton from the hydroxyl group, with the alcoholate anion R—CH₂O^- forming a bond with the zinc ion:

Clearly, the existence of such a structure, which is extremely sensitive to Hydrolysis, is possible only within the catalytic center, where the reaction takes place shielded from contact with water.

Fig. 10.10. Arrangement of the substrate in the active center of alcohol dehydrogenase.

Upon substrate (alcohol) binding, its hydroxyl group moves into proximity with the zinc ion and loses a proton, turning into an alcoholate. A system of hydrogen bonds involving Ser-48 and His-51 participates in proton abstraction and alcoholate stabilization.

This once again illustrates a hallmark feature of Enzymatic Catalysis: shifting the reaction environment to a favorable milieu.

The alcoholate anion is further stabilized by a shift in electron density from the alcoholate oxygen atom toward the C—O bond, which ultimately transforms into a double bond. Meanwhile, the hydride ion is transferred from the carbon atom, presumably along a structurally predetermined pathway, to position 4 of the coenzyme's nicotinamide ring, thereby converting NAD⁺ into its reduced form, NADH:

In the reverse reaction, the hydride ion is transferred from NADH to the carbon atom of the aldehyde group, whose double bond becomes polarized due to the electron-withdrawing effect of the enzyme-bound zinc ion on the oxygen atom. This polarization enhances the partial positive charge on the carbonyl carbon atom, facilitating the nucleophilic attack and addition of the hydride ion.

The examined example demonstrates how the functional repertoire of Enzymes can be expanded by incorporating ligands—specifically, a metal ion and a cofactor—into The structure of the active center.