BIOCHEMISTRY: A TEXTBOOK FOR MEDICAL UNIVERSITIES - E. S. Severin - 2004

SECTION 2. ENZYMOLOGY

III. Cofactors and Coenzymes

Most Enzymes require low-molecular-weight non-protein Organic compounds (Coenzymes) and/or Metal Ions (Cofactors) to exhibit catalytic activity.

The term "coenzyme" was introduced in the early 20th century to designate a fraction of certain enzymes that readily dissociated from the protein molecule and passed through a semipermeable membrane during dialysis. It was later discovered that most enzymes consist of a thermolabile protein moiety and a thermostable non-protein factor, known as the coenzyme. The protein component is called the "apoenzyme," which lacks catalytic activity in the absence of the coenzyme. The coenzyme combined with the protein molecule (apoenzyme) forms the holoenzyme molecule, which possesses catalytic activity.

A. Cofactors

Over 25% of all enzymes require metal ions for full catalytic activity. Let us examine The Role of cofactors in Enzymatic Catalysis.

1. The role of metals in substrate binding at the Active Site of an enzyme

Metal ions function as stabilizers of the substrate molecule, the enzyme's active site, and the conformation of the protein molecule, specifically its Tertiary and Quaternary structures.

Metal ions as stabilizers of the substrate molecule

For certain enzymes, the substrate is a complex of the transformed substance with a metal ion. For example, in most Kinases, one of the substrates is not an ATP molecule, but rather the Mg²⁺-ATP complex. In this case, the Mg²⁺ ion does not interact directly with the enzyme; instead, it participates in stabilizing the ATP molecule and neutralizing the negative charge of the substrate, thereby facilitating its binding to the active site of the enzyme (see scheme).

Class="center">Scheme

The role of the cofactor in the interaction between the enzyme and the substrate can be schematically represented as the E-S-Me complex, where E is the enzyme, S is the substrate, and Me is the metal ion.

An example is the arrangement of substrates in the active site of hexokinase (Fig. 2-3).

Hexokinase catalyzes The transfer of the terminal y-phosphate residue from an ATP molecule to glucose, yielding glucose-6-phosphate:

Fig. 2-3. Involvement of magnesium ions in substrate binding at the active site of hexokinase. The active site of hexokinase contains binding domains for the glucose molecule and the Mg²⁺-ATP complex. The enzymatic reaction results in the transfer of the terminal y-phosphate group of the ATP molecule to glucose, forming glucose-6-phosphate.

The Mg²⁺ ion participates in binding and correctly orienting the ATP molecule within the enzyme's active site, weakening the phosphoester bond and facilitating the transfer of the phosphate group to glucose.

Metal ions as stabilizers of the enzyme's active site

In some cases, metal ions serve as a "bridge" between the enzyme and the substrate. They function as active site stabilizers, facilitating substrate binding and the chemical reaction itself. In certain instances, metal ions may promote coenzyme binding. The Functions listed above are performed by metals such as Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, and Mo²⁺. In the absence of the metal, these enzymes are inactive. Such enzymes are termed "metalloenzymes." The interaction process involving the enzyme, substrate, and metal can be schematically represented as follows:

E-Me-S

Metalloenzymes include, for example, the enzyme Pyruvate kinase (Fig. 2-4), which catalyzes the reaction:

Fig. 2-4. Involvement of magnesium ions in substrate binding at the active site of pyruvate kinase. The active site of pyruvate kinase has binding sites for phosphoenolpyruvate and ADP. Mg²⁺ participates in stabilizing the active site, which facilitates the binding of phosphoenolpyruvate. The enzymatic reaction yields pyruvate and ATP.

2. The role of metals in stabilizing the tertiary and Quaternary Structure of enzymes

Metal ions ensure the preservation of the secondary, tertiary, and quaternary structure of enzyme molecules. While such enzymes remain capable of chemical catalysis in the absence of metal ions, they are structurally unstable. Their activity declines—and may even vanish completely—under minor fluctuations in pH, Temperature, or other subtle environmental conditions. Thus, metal ions act as stabilizers of the optimal conformation of the protein molecule.

Occasionally, alkaline-earth or alkali metal ions participate in stabilizing secondary and tertiary structures. For instance, K⁺ ions are required to maintain the tertiary conformation of pyruvate kinase.

Stabilizing The quaternary structure of Alcohol dehydrogenase, which catalyzes The oxidation of ethanol, requires zinc ions. Alcohol dehydrogenase consists of 4 subunits with a Molecular Weight of 151 kD and contains 4 Zn²⁺ atoms. Removing Zn²⁺ leads to a loss of enzymatic activity due to dissociation into 4 inactive subunits, each with a molecular weight of 36 kD (Fig. 2-5).

Fig. 2-5. The role of zinc ions in stabilizing the quaternary structure of alcohol dehydrogenase.

3. The role of metals in enzymatic catalysis

Metal ions play an equally vital role in facilitating enzymatic catalysis.

Participation in electrophilic catalysis

This function is most commonly performed by transition metal ions with variable valence, possessing a vacant d-orbital that allows them to act as electrophiles. These predominantly include metals such as Zn²⁺, Fe²⁺, Mn²⁺, and Cu²⁺. Alkaline-earth and alkali metal ions, such as Na⁺ and K⁺, lack this property. A classic example is the function of Carbonic anhydrase. Carbonic anhydrase is a zinc-containing enzyme that catalyzes The formation of carbonic acid:

CO₂ + H₂O <-> H₂CO₃.

Through an electrophilic attack, the Zn²⁺ ion assists in generating H⁺ and OH^- ions from a Water molecule:

The proton and hydroxyl group sequentially attach to carbon dioxide to yield carbonic acid (see Scheme A).

During electrophilic catalysis, metal ions frequently participate in stabilizing reaction intermediates.

Participation in redox reactions

Transition metal ions with variable valence can also take part in electron transfer. For example, in Cytochromes (heme-containing Proteins), the iron ion is capable of accepting and donating a single electron:

This property enables cytochromes to participate in oxidation-reduction reactions.

Another example of metal ions participating in redox reactions is the action of dopamine beta-hydroxylase, an enzyme that catalyzes the formation of norepinephrine in the presence of Vitamin C (see Scheme B).

The copper ion is responsible for the Redox Properties of dopamine beta-hydroxylase (Fig. 2-6).

Fig. 2-6. The role of the copper ion in activating an oxygen molecule during the functioning of dopamine beta-hydroxylase. 1 - reduction of Cu²⁺ within the active site of dopamine beta-hydroxylase to Cu⁺ by ascorbic acid; 2 - interaction of Cu⁺ with oxygen to form a peroxide intermediate; 3 - transfer of the hydroxyl group to the dopamine molecule, yielding norepinephrine.

The Cu²⁺-containing enzyme does not react with oxygen molecules. Upon the reduction of Cu²⁺ to Cu⁺ by ascorbic acid, a copper ion is formed that is capable of interacting with oxygen to yield a peroxide compound. Subsequently, the hydroxyl group is transferred to a dopamine molecule to produce norepinephrine.

4. The Role of Metals in the Introduction/15.html">Regulation of enzyme Activity

Metal ions sometimes act as regulatory molecules. For instance, Ca²⁺ ions serve as activators of protein kinase C, an enzyme that catalyzes protein phosphorylation reactions (see Section 5). Ca²⁺ ions also alter The activity of A number of calcium-calmodulin-dependent enzymes (see Subsection V).

B. Coenzymes

As previously mentioned, most enzymes require a coenzyme to exhibit catalytic activity. Exceptions include hydrolytic enzymes (such as proteases, lipases, and Ribonuclease), which perform their functions in the absence of a coenzyme.

Localized within the catalytic region of the active site, the coenzyme takes direct part in the chemical reaction, acting as an acceptor and donor of chemical groups, atoms, or electrons. A coenzyme can be linked to the protein moiety of the molecule by covalent and non-covalent bonds. In the former case, it is termed a prosthetic group (e.g., FAD, FMN, biotin, Lipoic Acid). At the same time, instances are known where a coenzyme binds to the enzyme via non-covalent bonds so tightly that it does not dissociate from the protein molecule, as is the case with Thiamine diphosphate.

In the latter case, the coenzyme interacts with the enzyme only for the duration of the chemical reaction and can be regarded as a second substrate. Examples include NАD⁺ and NАDР⁺.

The apoenzyme ensures reaction Specificity and dictates the type of chemical transformation the substrate undergoes. The same coenzyme, when interacting with different apoenzymes, can participate in various Chemical transformations of the substrate. For example, depending on which apoenzyme it interacts with, Pyridoxal phosphate participates in AMINO ACID Transamination or decarboxylation reactions.

The Chemical Nature of Coenzymes and Their functions in enzymatic reactions are extremely diverse. Traditionally, vitamin derivatives are classified as coenzymes, although alongside them, There is a significant class of non-protein compounds that also participate in the manifestation of the catalytic functions of enzymes.

Coenzymes include the following compounds:

✵ vitamin derivatives;

✵ Hemes, which are part of cytochromes, catalase, peroxidase, guanylyl cyclase, and NO synthase, and serve as the prosthetic group of these enzymes;

✵ NUCLEOTIDES—Donors and acceptors of the phosphoric acid residue;

✵ ubiquinone, or coenzyme Q, which participates in electron and proton transport within the ETC;

✵ phosphoadenosylphosphosulfate, which participates in Sulfate transfer;

✵ S-adenosylmethionine (SAM)—a methyl group donor;

✵ Glutathione, which participates in oxidation-reduction reactions.

The STRUCTURE AND FUNCTIONS of these coenzymes are examined in detail in the relevant sections of the textbook.

C. Multi-Substrate Reactions

Most enzymes catalyze reactions involving more than one substrate. If a coenzyme is not a prosthetic group, it can also be regarded as yet another substrate. Consequently, an enzymatic reaction may involve several participants: the enzyme itself, multiple substrates, and a coenzyme.

In such cases, The Mechanism of the enzymatic reaction generally follows one of two pathways: the ping-pong mechanism (double-displacement mechanism) or the sequential mechanism. Let us examine both mechanisms.

1. Ping-Pong Mechanism

Schematically, the ping-pong mechanism can be represented as follows:

Substrate A interacts with the enzyme (E) and is converted into a product (P₁). As a result of this transformation, the enzyme remains not in its native form, but in a modified state (E') due to the Modification of the coenzyme. Next, substrate B binds to the active site of E' and undergoes conversion into a product (P₂) with the simultaneous release of the native form of the enzyme (E).

A classic example of the ping-pong mechanism is the Transamination reaction involving aminotransferase enzymes (with Pyridoxal phosphate as a coenzyme). Aminotransferases, discovered by the Soviet scientist A. E. Braunshtein, catalyze the reversible transfer of an amino group from an amino acid to a keto acid. The ping-pong mechanism of this reaction is schematically illustrated in Fig. 2-7.

Fig. 2-7. Events within the active site of an aminotransferase as an example of the ping-pong mechanism. The enzyme-bound coenzyme pyridoxal phosphate (PLP) accepts an α-amino group from the first amino acid (AA₁), which is thereby converted into α-keto acid 1 (KA₁) and released from the enzyme's active site. Subsequently, α-keto acid 2 (KA₂) binds to the active site, accepts the amino group from the coenzyme, and is converted into the corresponding α-amino acid (AA₂).

Another example of the ping-pong mechanism is found in dehydrogenation Reactions Involving the coenzymes FAD (flavin adenine dinucleotide) or FMN (flavin mononucleotide), which are tightly bound to the enzyme and therefore cannot act as a second substrate.

The schematic structures of these coenzymes and their corresponding chemical formulas are presented in Fig. 2-8.

Fig. 2-8. Structure (A) and chemical constitution (B) of the FMN and FAD coenzymes.

FMN and FAD participate in oxidation-reduction reactions by accepting 2 e^- and 2 H+ at the isoalloxazine ring (see the scheme below).

The Scheme for the dehydrogenation reaction (serving as an example of the ping-pong mechanism involving FMN and FAD coenzymes) can be represented as follows:

where AH₂ is the hydrogen donor, i.e., the oxidized substrate 1; A is the oxidized form of substrate 1; B is the hydrogen acceptor, i.e., substrate 2; BH₂ is the reduced form of substrate 2; and E(FAD) and E(FADH₂) are the oxidized and reduced forms of the FAD coenzyme associated with enzyme E.

A prominent example of an FAD-dependent reaction is the succinate dehydrogenase reaction. In this pathway, ubiquinone—one of the Electron Transport Chain intermediates—serves as the second substrate (see scheme).

2. Sequential Mechanism

In a sequential mechanism, the enzyme-catalyzed reaction requires the simultaneous interaction of two substrates. Under these conditions, substrate binding can occur via two distinct pathways:

Ordered mechanism of substrate binding to the enzyme's active site:

Substrate A binds to the enzyme's active site first, which facilitates the subsequent binding of substrate B. Following chemical modification, the release of reaction products also proceeds in a strictly defined order.

Random mechanism of substrate binding to the enzyme's active site:

There is no preferential order for the binding of substrates A and B to the active site (each substrate possesses its own distinct binding pocket within the active site). Likewise, there is no strict pattern for the release of reaction products.

An example of a sequential ordered mechanism is the dehydrogenation reaction involving NAD⁺ and NADP⁺ coenzymes.

The schematic structures and chemical formulas of these coenzymes are shown in Fig. 2-9.

Fig. 2-9. Structure (A) and chemical constitution (B) of the NAD⁺ and NADP⁺ coenzymes.

Both coenzymes function as carriers transferring two electrons and one proton from a donor to an acceptor, while the other proton is released into the medium (see Scheme A).

The donor and acceptor do not necessarily participate in the same metabolic pathway. In other words, the reduced form of these nucleotides acts as a common electron pool generated by oxidation reactions and can be utilized in various reduction reactions. Such reactions are referred to as coupled reactions (see Scheme B).

where AH₂ is the hydrogen donor, the reduced form of substrate 1; A is the oxidized form of substrate 1; B is the hydrogen acceptor, the second substrate; BH₂ is the reduced form of substrate 2; NAD⁺ and NADH are the oxidized and reduced forms of the coenzyme; E₁ and E₂ are enzymes.

The two enzymatic Reactions Catalyzed by enzymes E₁ and E₂ are coupled with each other through the coenzyme NAD⁺, which serves as a substrate in each case. For the first enzyme, the oxidized form of NAD acts as a substrate, with the hydrogen donor serving as the second substrate—an example of sequential reactions—yielding the reduced form of NAD, whereas for enzyme E₂ the reverse is true.

As an example, we can consider the following coupled reactions (see scheme), where E₁ is glyceraldehyde-3-phosphate dehydrogenase and E₂ is Lactate dehydrogenase.

scheme