Fundamentals of Biochemistry - A. A. Anisimov 1986

Enzymes
Structure and Functions of Individual Coenzymes and Prosthetic Groups

A wide variety of organic and inorganic substances (Metal Ions) can function as the non-protein parts of Enzymes. All of this diversity is generally divided into conventional groups. There are several different Classification principles for non-protein moieties. Based on their chemical nature, they can be tentatively subdivided into 4 groups: 1) nucleotide-type Structure, 2) Vitamins AND THEIR derivatives, 3) metals and metal-containing non-protein moieties, and 4) other non-protein components. The non-protein components of enzymes have a relatively low molecular weight and, unlike apoenzymes, are thermostable.

3.4.1. Non-protein moieties of nucleotide-type structure.

Nicotinamide Coenzymes. Nicotinamide coenzymes include NAD⁺ (nicotinamide adenine dinucleotide) and NADP⁺ (nicotinamide adenine dinucleotide phosphate). The NAD⁺ and NADP⁺ molecules consist of two heterocycles—pyridine and purine—linked by a chain of two ribose monosaccharide residues and a pyrophosphoric acid residue.

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These coenzymes contain Vitamin PP, which is why they can also be classified as vitamin-derived coenzymes. NADP⁺ differs from NAD⁺ by containing an additional orthophosphate residue attached to the C—2 position of the adenosine ribose moiety. NAD⁺ and NADP⁺ are typical coenzymes because they are loosely bound to the protein and shuttle between different enzymes during the cycle of biochemical reactions.

Fig. 3.4. Binding of NAD⁺ and L-lactate (highlighted by a wavy line) in the Active Site of Lactate dehydrogenase

Nicotinamide coenzymes function as part of numerous dehydrogenases. The coenzyme is bound to the protein via electrostatic bonds and hydrophobic interactions (Fig. 3.4). NAD⁺- and NADP⁺-dependent dehydrogenases catalyze The transfer of a hydride ion (H^-) from the substrate to the nicotinamide moiety of the coenzyme molecule, releasing a proton into the medium:

Reduced NADH and NADPH carry no charge on the nitrogen atom of the pyridine ring and have the hydrogen acquired from the substrate attached to position 4 of the pyridine ring. The NADH and NADPH formed As a result of hydride ion transfer lose their affinity for the apoenzyme and dissociate from the dehydrogenase. These reduced coenzymes are subsequently oxidized by transferring electrons and protons to an acceptor associated with a second enzyme. Thus, NAD⁺ and NADH, as well as NADP⁺ and NADPH, function cyclically and participate in dismutations, i.e., the reduction of one metabolite by another.

In living organisms, the majority of NAD is found in the oxidized form, whereas NADP is predominantly in the reduced form, which is related to the distinct roles of these coenzymes in METABOLISM. Redox enzymes involved in Cellular energy supply are NAD⁺-dependent, whereas enzymes catalyzing reductive synthesis reactions utilize NADPH. NADPH is required for the reductive Biosynthesis of most cellular components; in plants, it also participates in the Synthesis of glucose from CO₂ driven by light energy. NADH plays a lesser role in biosynthetic processes.

The conversion of NAD⁺ and NADP⁺ to their reduced states is accompanied by a shift in their UV absorption spectrum. While the oxidized forms of the coenzymes exhibit a single narrow absorption band with a maximum at 260 nm, the reduced forms develop an additional peak with a maximum at 340 nm. This property of nicotinamide coenzymes forms The basis of a widely used spectrophotometric assay for determining The activity of NAD⁺- and NADP⁺-dependent dehydrogenases.

NAD⁺- and NADP⁺-dependent dehydrogenases are found in All living organisms; such universal distribution highlights The Unity of fundamental Metabolic Pathways in living nature. NAD⁺- and NADP⁺-dependent dehydrogenases catalyze reversible dehydrogenation reactions of alcohols, hydroxy acids, and Amino Acids. Well-studied dehydrogenases to date include LDH (lactate dehydrogenase), MDH (malate dehydrogenase), ADH (Alcohol dehydrogenase), and glyceraldehyde-3-phosphate dehydrogenase.

All of the aforementioned dehydrogenases share a similar protein fragment consisting of six parallel ß-strands and several a-helical regions. It is precisely this fragment that binds the coenzyme and is referred to as the NAD⁺-binding domain. Dehydrogenases are characterized by a quaternary structure; specifically, MDH and ADH exist as dimers, whereas LDH is a tetramer.

It has now been demonstrated that NAD⁺ also Functions as a coenzyme in reactions that do not utilize its redox properties—for instance, it is required for the activity of DNA ligase from E. coli, which catalyzes The formation of a phosphodiester bond.

Flavin prosthetic groups. This group comprises FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide). Both of these compounds can also be simultaneously considered as derivatives of vitamin B₂ (riboflavin).

FMN is not a typical nucleotide because it contains the sugar alcohol ribitol instead of ribose, which does not form a glycosidic bond. Its nitrogenous base is demethylisoalloxazine, which is not a derivative of purine or pyrimidine. Both flavin NUCLEOTIDES are tightly bound to the protein moieties of enzymes, sometimes even covalently, which is why they are classified as prosthetic groups.

FMN and FAD function as components of various redox enzymes (Flavoproteins). Currently, about 80 flavin enzymes are known, the majority of which contain FAD as their non-protein moiety. The active site of flavins is the isoalloxazine ring, which is capable of accepting two hydrogen atoms in its oxidized form.

Oxidized flavoproteins exhibit three absorption maxima: at 280, 350–380, and 450 nm. Upon reduction, the visible absorption band at 450 nm—which is responsible for the yellow color of flavoproteins—almost completely disappears, and there is also a partial decrease in absorption at 280 and 350–380 nm.

Flavoproteins catalyze a diverse range of redox reactions: The oxidation of hemiacetals to lactones, alcohols to aldehydes, amines to Imines, saturated carbonyl compounds to a,ß-unsaturated ones, and NADH and NADPH to NAD⁺ and NADP⁺ (FAD and FMN are stronger oxidizing agents than nicotinamide coenzymes). An example of such reactions is the glucose oxidase-catalyzed oxidation of glucose to gluconic acid.

Glucose oxidase, like some other flavoproteins, is autooxidizable, meaning it can transfer hydrogen cleaved from the substrate directly to molecular oxygen, bypassing the Electron Transport Chain to form hydrogen peroxide; such Enzymes are classified as oxidases.

The resulting H₂O₂ is either used as an oxidizing agent by several peroxidases or degraded by catalase.

Other flavin enzymes are represented by dehydrogenases, which transfer electrons and protons from oxidizable substrates to intermediate carriers (see Section 7.3.1). α,β-Dehydrogenation involving flavin enzymes occurs, for example, during the oxidation of succinic acid to fumaric acid in the tricarboxylic acid (TCA) cycle and is catalyzed by succinate dehydrogenase (SDH). SDH abstracts hydrogen directly from the oxidizable substrate, acting as a primary dehydrogenase. Among flavoproteins, There are also secondary dehydrogenases that accept hydrogen from NADH and NADPH generated by the action of primary NAD⁺ and NADP⁺-dependent dehydrogenases. Flavoproteins may contain a metal (metalloflavoproteins), a heme, or iron-sulfur centers. In such complex flavoproteins, a single protein harbors an entire miniature electron transport chain. Metalloflavoproteins include, in particular, aldehyde oxidase and xanthine oxidase. The latter catalyzes the oxidation of not only hypoxanthine but also aldehydes.

Xanthine oxidase is a dimer (M ≈ 275,000) containing two FAD molecules, two Mo atoms, and eight Fe atoms. Xanthine oxidase belongs to iron-sulfur Proteins. Aldehyde oxidase catalyzes only the oxidation of aldehydes and has a structure very similar to that of xanthine oxidase. In iron-sulfur proteins, Fe atoms are linked to the SH groups of Cys residues. Flavin-containing iron-sulfur proteins also include succinate dehydrogenase, nitrate reductase, and nitrite reductase.

Nucleoside Triphosphates and NDSs. ATP and other nucleoside triphosphates (GTP, UTP, CTP) serve as coenzymes for phosphotransferases, which catalyze the transfer of a phosphate group from nucleoside triphosphates to Other Compounds, thereby activating them (see Section 3.10). In some cases, activation is achieved by transferring a pyrophosphate, adenosine, or adenosine monophosphate moiety of the ATP molecule. For instance, the transfer of pyrophosphate—two phosphate groups from ATP—occurs during the formation of Thiamine diphosphate (TDP), a phosphorylated form of vitamin B₁ (see Section 10.3); the transfer of adenosine monophosphate (AMP) takes place during Amino Acid Activation in Protein Biosynthesis (see Section 5.3.2); and the transfer of an adenosyl moiety (adenine-ribose) is directed to R—S—CH₃ type compounds, notably The amino acid Methionine. The latter reaction leads to the Synthesis of the active form of methionine, S-adenosylmethionine, in which the methyl group exhibits enhanced reactivity.

Nucleoside diphosphate sugars (NDSs) act as coenzymes for Glycosyltransferases in Reactions Involving the transfer of monosaccharide residues during The biosynthesis of oligo- and Polysaccharides (see Section 6.6.3).

The Acetylation coenzyme is coenzyme A (CoA).

The nucleotide moiety in CoA serves as a "handle" by which the coenzyme attaches to the protein, while the other part of the molecule, which is 1.9 nm long, terminates in the functionally essential SH group.

CoA is most typically involved in the activation and transfer reactions of acetyl groups; however, it can also mediate the transfer of other acyl groups, which is why it is additionally referred to as the acyl coenzyme. Acyl groups attach to the sulfur atom within CoA via an energy-rich thioester bond, thereby activating the acid residue. The synthesis of acetyl-coenzyme A (acetyl-CoA) from acetate and CoA occurs in The Liver and Nervous Tissue of mammals at the expense of ATP energy, with the participation of the enzyme acetate thiokinase.

CoA is a component of the Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase systems, which catalyze The oxidative decarboxylation of the corresponding keto acids. As a coenzyme, CoA also functions within fatty acid thiokinase, an enzyme that carries out their activation prior to β-oxidation (see Section 5.8.2). Acetyl-CoA, in turn, serves as a coenzyme for acetyltransferases, which catalyze biological acetylation reactions—the attachment of acetyl groups (CH₃CO—) to other molecules. Acetyl-CoA participates in such biochemical processes as the synthesis of Fatty acids and Steroids from CARBOHYDRATES, and mediates acetylation during the synthesis of acetylcholine and acetylaminosugars.

3.4.2. Metals and Metal-Containing Non-Protein Moieties. Enzymes that function as complexes of varying stability with a metal are called metalloenzymes. Based on the strength of the metal-protein bond, they are conventionally divided into true metalloenzymes and metal-activated enzymes. True metalloenzymes contain a tightly bound metal that does not dissociate from the apoenzyme during purification, and they are characterized by a fairly strict Specificity toward the metal. In metal-activated enzymes, the metal is loosely bound to the protein and readily dissociates partially or completely during purification; specificity toward the metal is generally poorly pronounced.

Currently, about a hundred enzymes are known that should be classified as true metalloenzymes. Most frequently, the composition of these enzymes, like other Metalloproteins, includes Zn, Cu, Fe, and Mo. Metal ions located in the active sites of enzymatic molecules can participate in the catalytic act, serve as a bridging link between the enzyme and the substrate, or take part in the Formation of the enzyme-coenzyme complex. If a metal ion is located outside the active site, it maintains the Tertiary and Quaternary structures of the enzyme.

Metal ions in true metalloenzymes are bound to specific protein groups (ligands) via coordination bonds, forming complexes. Stable complexes with nitrogen-containing ligands are formed by Cu²⁺, Co²⁺, Ni²⁺, and Fe²⁺ ions. In addition to nitrogen atoms, transition metal ions also bind well to sulfur (Zn²⁺, Cu²⁺, Fe²⁺, etc.). Ca and Mg ions in proteins bind predominantly to ligands such as phosphate and carboxylate ions. It is hypothesized that specific sites for the binding of alkali metals K⁺ and Na⁺ also exist in protein molecules, although within The Cell they are largely present in a free state.

Below are Examples of true metalloenzymes, along with some information on their structure and the function performed by the metal. Ascorbate oxidase and polyphenol oxidase (tyrosinase) are widely distributed in plants. The former catalyzes the oxidation of ascorbic acid by atmospheric O₂ to dehydroascorbic acid (see Section 10.3), while the latter oxidizes phenols to Quinones. These enzymes have a relative molecular mass of approximately 120,000, are copper-containing, and their molecules are built from multiple subunits.

Sometimes, alongside Cu²⁺, enzymes incorporate other ions as well. For example, eukaryotic cytoplasmic superoxide dismutase (see Section 7.5) is a dimer, each subunit of which contains tightly bound Cu²⁺ and Zn²⁺.

In oxidation-reduction enzymes, metals usually take a direct part in the catalytic act.

Iron-containing enzymes are primarily the so-called iron-Porphyrins, i.e., proteins in which iron is part of the heme group (heme iron). Such enzymes include catalase, peroxidase, cytochrome oxidase, etc.

Zinc-containing enzymes include Carbonic anhydrase, alcohol dehydrogenase, Carboxypeptidase A, etc. Carbonic anhydrase catalyzes the reversible Cleavage of carbonic acid: H₂CO₃ ⇄ H₂O + CO₂. The enzyme is widespread in plants and animals. In the tissue capillaries of animals, the reaction is shifted toward the formation of carbonic acid, which facilitates its removal via the bloodstream, whereas in the lung capillaries, the reaction proceeds more actively in the opposite direction, thereby accelerating the elimination of CO₂ through the Lungs.

The oxidation of alcohols proceeds with the participation of alcohol dehydrogenase;

This Zn²⁺-dependent enzyme is active in the human liver, where it oxidizes ethanol to acetaldehyde, a product toxic to humans. A complex protein containing a single Zn²⁺ ion is pancreatic carboxypeptidase A. Zn²⁺ ions are also present in DNA polymerase I from E. coli. Zinc ions in Zn²⁺-dependent enzymes can frequently be replaced by Mn²⁺ or Co²⁺ without any significant loss of catalytic activity.

Several selenium-containing enzymes involved in oxidation-reduction reactions are known. This is presumably one of the reasons why the element Se, which is extremely toxic at certain concentrations, is nevertheless an essential dietary component whose absence can lead to cell death, as demonstrated in experiments with laboratory animals.

3.4.3. Other Non-Protein Moieties of Enzymes. Lipoic Acid. As a coenzyme, lipoic acid participates in the Oxidative Decarboxylation of α-keto acids. Lipoic acid is covalently linked via an amide bond to the ε-NH₂ group of a Lysine residue within the apoenzyme.

Oxidative decarboxylation of α-keto acids proceeds According to the following scheme:

Glutathione. This tripeptide serves as a coenzyme for A number of isomerases acting on unsaturated compounds and performs this function in intramolecular hydrogen transfer (dismutation). For instance, glutathione participates in the isomerization of maleic acid derivatives into fumaric acid derivatives, i.e., it carries outcis-trans isomerization. Glutathione is also a coenzyme in the oxidation of formaldehyde to formate. This tripeptide is a component of DDT dehydrochlorinase, an enzyme that splits off HCl from the insecticide and participates in its detoxification. High activity of this enzyme is characteristic of DDT-resistant insects.

The Role of vitamins as the non-protein moieties of enzymes is discussed in Chapter 10.