BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 7. ENZYMOLOGY

7.3. Cofactors and Coenzymes

7.3.1. Cofactors

Over 25 % of all Enzymes require Metal Ions to exhibit full catalytic activity. Let us examine The Role of Cofactors in Enzymatic Catalysis.

Metal ions function as stabilizers of the substrate molecule, the Active Site of the enzyme, and the conformation of the protein molecule itself, specifically its Tertiary and Quaternary structures.

Metal ions as stabilizers of the substrate molecule. For certain enzymes, the true substrate is a complex of the converted substance with a metal ion. For instance, in most Kinases, one of the substrates is not the ATP molecule itself, but the Mg²+-ATP complex. In this case, the Mg²⁺ ion does not interact directly with the enzyme, but is

rather involved in stabilizing the ATP molecule and neutralizing the negative charge of the substrate, which facilitates its binding to the active site of the enzyme:

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Schematically, the role of a cofactor in enzyme-substrate interaction can be depicted as an 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. 7.3). Hexokinase catalyzes The transfer of the terminal γ-phosphate group from an ATP molecule to glucose, yielding glucose-6-phosphate:

The Mg²⁺ ion participates in the binding and "correct" orientation of the ATP molecule within the active site of the enzyme, weakening the phosphoester bond and facilitating the transfer of the phosphate group to glucose.

Metal ions as stabilizers of the active site.

Sometimes metal ions serve as a "bridge" between the enzyme and the substrate. They act as active site stabilizers by facilitating substrate binding and the progression of the chemical reaction. In certain cases, a metal ion can also promote coenzyme binding. These Functions are performed by metals such as Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, and Mo²⁺. In the absence of the metal, these enzymes are inactive and are referred to as "metalloenzymes". Schematically, the interaction among the enzyme, substrate, and metal can be represented as follows: E-Me-S. Pyruvate kinase is an example of a metalloenzyme (Fig. 7.4), catalyzing the reaction:

Fig. 7.3. The role of magnesium ions in substrate binding at the active site of hexokinase.

The active site of hexokinase contains binding pockets for the glucose molecule and the Mg²⁺-ATP complex. The enzymatic reaction results in the transfer of the terminal γ-phosphate group of the ATP molecule to glucose, forming glucose-6-phosphate

Fig. 7.4. The role of magnesium ions in substrate binding at the active site of pyruvate kinase:

the active site of pyruvate kinase possesses 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

Metal ions ensure the Maintenance of the tertiary and Quaternary Structure of the enzyme molecule. Such enzymes are still capable of chemical catalysis in the absence of metal ions, but they are unstable. Their activity decreases or even ceases entirely upon minor shifts in pH, Temperature, or other slight environmental changes. Consequently, metal ions function as stabilizers of the optimal conformation of the protein molecule.

In some cases, alkali and alkaline-earth metal ions take part in stabilizing the tertiary structure. For instance, maintaining the tertiary conformation of pyruvate kinase requires K⁺ ions. Zinc ions are essential for stabilizing The quaternary structure of Alcohol dehydrogenase, which catalyzes The oxidation of ethanol. Alcohol dehydrogenase consists of four subunits with a molecular mass of 151 kDa and contains four Zn²⁺ atoms. Removal of Zn²⁺ leads to a loss of enzymatic activity due to dissociation into four inactive subunits, each with a molecular mass of 36 kDa (Fig. 7.5).

Fig. 7.5. Stabilization of the quaternary structure of alcohol dehydrogenase by zinc ions

The role of metals in enzymatic catalysis. The Role of Metal ions in executing enzymatic catalysis is equally crucial. During electrophilic catalysis, they frequently participate in stabilizing intermediate compounds. This function is most commonly fulfilled by variable-valence metal ions that possess a vacant d-orbital and act as electrophiles—primarily metals such as Zn²⁺, Fe²⁺, Mn²⁺, and Cu²⁺. Alkali metal ions like Na⁺ and K⁺ lack this capability. A prime example is the functioning of Carbonic anhydrase, a zinc-containing enzyme that catalyzes The formation of carbonic acid:

As a result of the hydrophilic attack of the zinc enzyme, the Zn²₊ ion participates in the formation of H⁺ and OH⁺ ions from a Water molecule:

The proton and the hydroxyl group sequentially attach to carbon dioxide to form carbonic acid:

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

Due to this property, cytochromes take part in oxidation-reduction reactions.

Another example of the Involvement of metal ions in redox reactions is the action of the enzyme dopamine beta-hydroxylase, which catalyzes the synthesis of noradrenaline in the presence of Vitamin C. The Redox Properties of dopamine beta-hydroxylase are mediated by a copper ion (Fig. 7.6). The fragment containing the Cu²+ ion does not react directly with the oxygen molecule. Upon the reduction of Cu²⁺ to Cu⁺ by ascorbic acid, a copper ion is formed that is capable of interacting with oxygen to produce a peroxide intermediate. Subsequently, the hydroxyl group is transferred to the dopamine molecule, yielding noradrenaline.

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. Ca²⁺ ions also modulate The activity of several calcium-calmodulin-dependent enzymes.

Fig. 7.6. Involvement of the copper ion in the activation of the oxygen molecule during the functioning of dopamine beta-hydroxylase:

1 - reduction of Cu²⁺, located in the active site of dopamine beta-hydroxylase, to Cu⁺ by ascorbic acid; 2 - interaction of Cu⁺ with oxygen to form a peroxide compound; 3 - transfer of the hydroxyl group to the dopamine molecule with the formation of noradrenaline