BIOCHEMISTRY - Textbook - L. I. Ostapchenko - 2012

Chapter 7. ENZYMOLOGY

7.3. Cofactors and Coenzymes

7.3.3. Multi-substrate Reactions

Most Enzymes catalyze reactions involving more than one substrate. If a coenzyme is not a prosthetic group, it can also be considered as an additional 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 proceeds via one of two pathways: the "ping-pong" mechanism (double-displacement mechanism) or the sequential mechanism. Let us examine both of them. Schematically, the "ping-pong" mechanism can be represented as follows:

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

A prime example of the "ping-pong" mechanism is Transamination reactions involving aminotransferases (with Pyridoxal phosphate as the 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 such a reaction is schematically illustrated in Fig. 7.7.

Fig. 7.7. Events in the active center of an aminotransferase as an example of the "ping-pong" mechanism:

the coenzyme pyridoxal phosphate (PLP), bound to the enzyme, accepts the 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 center.

Then, α-keto acid 2 (KA₂) binds to the active center of the enzyme, accepts the amino group from the coenzyme, and is converted into the α-amino acid (AA₂).

Another example of the "ping-pong" mechanism is dehydrogenation reactions involving FAD (flavin adenine dinucleotide) or FMN (flavin mononucleotide) Coenzymes, which are tightly bound to the enzyme and, accordingly, cannot be considered as a second substrate. The schematic structures of these Coenzymes and Their corresponding chemical formulas are shown in Fig. 7.8.

Fig. 7.8. 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⁺ into the isoalloxazine ring.

The scheme of the dehydrogenation reaction (as an example of the "ping-pong" mechanism involving FAD and FMN coenzymes) can be depicted as follows:

where AH₂ is the hydrogen donor that oxidizes 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 (FAD), (FADH₂) are the oxidized and reduced forms of the FAD coenzyme, which is part of the enzyme E.

An example of an FAD-dependent reaction is the succinate dehydrogenase reaction. In this reaction, the second substrate is ubiquinone, one of the intermediates in the Respiratory Chain:

In the case of a sequential mechanism, the enzymatic reaction requires the simultaneous interaction of two substrates. Under such conditions, substrate binding can occur via two different pathways:

✵ the ordered mechanism of substrate interaction with the active center of the enzyme:

Substrate A binds to the enzyme's active center first, facilitating the subsequent binding of substrate B. Following chemical modification, a specific order of product release is also observed;

✵ mechanism of random substrate interaction with the Active Site of an enzyme:

There is no preferential interaction of substrates A and B with the active site of the enzyme (each substrate has its own binding site within the active center). Furthermore, there is no strict pattern in the release of reaction products.

An example of an ordered sequential mechanism is the dehydrogenation reaction involving the coenzymes NAD⁺ and NADP⁺. The Structure and Chemical formulas of these coenzymes are shown schematically in Fig. 7.9. Both coenzymes function as intermediaries in The transfer of two electrons and one proton from a donor to an acceptor, while the second proton is released into the medium:

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 pool of electrons generated by oxidation reactions and can be utilized in various reduction reactions. Such reactions are referred to as coupled reactions:

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⁺, NADH are the oxidized and reduced forms of the coenzyme; E₁ and E₂ are the enzymes.

Fig. 7.9. Structure (A) and chemical constitution (B) of the coenzymes NAD⁺ and NADP⁺

The two enzymatic Reactions Catalyzed by enzymes E₁ and E₂ are coupled with each other through NAD⁺, which serves as a substrate in both instances. For the first enzyme, the oxidized form NAD⁺ acts as a substrate, the hydrogen donor acts as the second substrate (an example of sequential reactions), and the reduced form of NAD serves as the product; for E₂, the reverse is true. As an example, consider the following coupled reactions, where E₁ is glyceraldehyde-3-phosphate dehydrogenase and E₂ is Lactate dehydrogenase: