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7.3Enzymes

Material changes in cells are all catalyzed by enzymes. Specifically, all chemical reactions in the living body are rigorously regulated by the synthesis, degradation and activity modulation of enzymes, which are normally made of proteins but may also contain prosthetic groups. RNA enzymes with catalytic action, referred to as ribozymes, are also known. Typical prosthetic groups include heme, which is contained in hemoglobin and cytochrome, and coenzymes such as NAD+. Some enzymes are bonded with metal ions such as Fe2+ and Mg2+. The enzymes with prosthetic groups are mainly involved in substrate binding and oxidation-reduction. Many coenzymes cannot be synthesized from scratch in the human body, and we therefore need to ingest precursors in the form of vitamins.

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Law of Thermodynamics: Free Energy Change and Equilibrium Constant

The basic law of thermodynamics is summarized as:

Enthalpy: H = U + PV
Gibbs free energy: G = H - TS

where U is the internal energy, P is the pressure, V is the volume, T is the temperature and S is the entropy.
Since physiochemical reactions normally occur at one atmosphere of pressure (1.013 x 105 Pa, or by a new standard, 1 x 105 Pa) and a constant temperature of around 298 K (although defined as a standard condition, there are several definitions), free energy change is expressed as:

G˚’ = ∆H˚’ - TS˚’

Where“˚ ”refers to the standard condition (a molar concentration of 1, and 25˚C), and“ ’ ”means that the reaction occurs at a pH of 7.0.

When ∆G˚’ is negative, the equilibrium of the reaction moves in a positive direction, which is expressed as:

G’ = ∆G˚’ + RTlnKeq

where Keq’ is the equilibrium constant of the reaction and R is the gas constant.
It should be noted that many biochemical reactions are considered reversible.

The actual reaction, not in equilibrium, is expressed as:

G’ = ∆G˚’ + RTln [Product] / [Reactant]

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7.3.1

Specificity and the Reaction Mechanism of Enzymes

Enzymes have two types of specificity: substrate specificity and reaction specificity. Enzymes engage in catalytic activity by recognizing certain substrates and attaching to them specifically. The reactions they catalyze are also predetermined. As an example, an enzyme called amylase acts on starch (many glucose units joined together by α-1,4 bonds; see Fig. 1-5 in Chapter 1) to dissociate maltose (a disaccharide formed from two units of glucose) but not glucose. Acid (H+) - an inorganic catalyst - randomly cuts the carbohydrate chains of starch, and produces glucose polymers of various lengths. In addition, since acids act on proteins and other high molecular substances other than starch, they are not highly specific. The high specificity of enzymes is made possible by their high-order structure; the form of the active center of enzymes allows them to easily bind with certain substrates, and amino-acid side chains are arranged so that enzymes can bind to the characteristic functional groups of substrates. Enzymes - a kind of protein - efficiently function as a catalyst because they have special amino acid residues in their active center, giving the enzymes, through interaction with other amino acid residues, a special level of reactivity not found in normal amino acid residues of the same kind. As an example, the triad of a protease called chymotrypsin is shown in Figure. 7-1. In this case, the particular serine residue that forms the active center attacks peptide bonds; this is made possible by the serine side chain becoming sub-ionized (e.g., -CH2-O-) by losing H+ to the sterically adjacent aspartic acid residue and the histidine residue. Due to such characteristics and for the maintenance of their steric structures, enzymes generally have optimal pH for activity. While the reaction speed increases with temperature, optimal temperatures also exist because the steric structure of enzymes is broken down in high-temperature conditions.

Triad constituting the active center of chymotrypsin

Fig. 7-1. Triad constituting the active center of chymotrypsin

The hydrogen nucleus (proton) of the hydroxyl group (OH group) in the 195th serine residue is strongly attracted to the lone pair of Ns in the 57th histidine residue, and the negatively charged O of the serine residue therefore attacks the slightly positively charged carbonyl carbon of the substrate’s peptide bond. This is the first stage of peptide bond hydrolysis. The symbol “→” indicates the direction of electrons.

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7.3.2

Enzyme Kinetics

In an enzymatic reaction, the initial rate of reaction, V, increases as the substrate S’s concentration, [S], rises; however, when the amount of enzyme added to a reaction system is fixed, V stops increasing at a certain point with rising values of [S] (Fig. 7-2). This extrapolation value is Vmax. In other words, V is saturated with respect to [S]. This saturation phenomenon is characteristic to catalytic reactions, and is explained by the substrate binding to a catalyst at a fixed ratio. Even when there is an abundance of substrate molecules, the number of substrate binding sites is limited, thus causing saturation.
Now, we discuss the kinetics of enzymatic reactions. The simple enzymatic reaction below is considered:

             k1     k2
E + S ⇄ ES → E + P
             k-1

In this case, the Michaelis-Menten equation generally applies:

V = Vmax/1 + Km/[S]

Relationship between substrate concentration and the initial rate of enzymatic reaction

Fig. 7-2. Relationship between substrate concentration and the initial rate of enzymatic reaction

where Vmax is the maximum initial reaction rate, and Km is the Michaelis constant (Km = (k-1 + k2) / k1). This equation is essentially a hyperbolic function that describes saturation phenomena. Since the equation was derived based on certain assumptions (see the Column in 7.4 regarding how the derivation was made), not all enzymatic reactions follow the assumptions. Nevertheless, many such reactions do follow the Michaelis-Menten equation, and Km has been used as an indicator of the affinity between enzymes and substrates (the smaller the value of Km, the higher the affinity).
One use for this equation is the classification of enzymatic inhibitors. Inhibitors are classified based on their effect on Km and Vmax, among which competitive inhibitors are taken as an example here. They have a structure very similar to that of substrates, and bind to the substrate binding sites of enzymes while remaining unaffected by enzymatic reactions. This inhibits the reaction of the original substrates, reflecting their role as competitive inhibitors. As an example, succinate dehydrogenase - an enzyme involved in the citric acid cycle - is inhibited by malonic acid (HOOC-CH2-COOH) - an allied substance of succinic acid (HOOC-CH2-CH2-COOH). In this case, malonic acid is a competitive inhibitor. The important point here is that, with the addition of an inhibitor, Km appears to increase but Vmax does not change. Mechanisms that influence enzymatic activity include allosteric regulation, which will be discussed later.

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7.3.3

Classification of Enzymes

There are various types of enzymatic reaction, which are classified by reaction type; the International Union of Biochemistry and Molecular Biology assigns a set of four numbers, called Enzyme Commission (EC) numbers, to each reaction. As an example, alcohol dehydrogenase reaction is given the numbers EC1.1.1.1. The first “1” indicates the reaction group to which it belongs (in this case, oxidoreductases), while the other three numbers represent more detailed classification. The other groups are 2 (transferases), 3 (hydrolases), 4 (lyases), 5 (isomerases) and 6 (ligases).

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