4.5Specificity of Enzymes and the Reaction Mechanism
All metabolic reactions within a cell occur through the catalytic actions of enzymes. A “catalyst” is any substance that increases the reaction rate without itself being converted before or after the reaction. However, it cannot accelerate the reaction without being completely involved in the reaction. In fact, the catalyst binds to the reactants (called the substrate), accelerates the reaction, and then repeatedly separates from the reaction products; thus, it continuously accelerates a specific reaction. Therefore, enzymes can only promote a specific biochemical reaction involving specific substances to which they can bind.
At the beginning of the 20th century, enzymes were recognized as important substances that held a key to the miracle of life. To better understand enzymes and their types, considerable efforts have been made to repeatedly purify and obtain enzymes in their highly active forms. Because enzymes are highly active even in trace amounts, almost nothing is left by the time they have been sufficiently purified. Highly sensitive techniques are currently available for analyzing enzymes, but at the beginning of the 20th century, purification of enzymes was extremely difficult. In 1926, urease was crystallized and found to be composed of proteins (see Column the bottom). However, at that time, proteins were neither clearly defined nor was their structure clearly understood. Research over the past few centuries has found that enzymes are macromolecular substances generally comprising proteins. Proteins are complexes of amino acids bound together by peptide bonds. Proteins will be described in greater detail after Chapter 6. An enzyme may sometimes contain components that are not proteins, and these are grouped under the term “prosthetic groups.” Representative prosthetic groups include heme, present in hemoglobin, cytochrome, and coenzymes such as NAD. Some enzymes can bind to metal ions such as iron or magnesium. These enzymes are mainly related to substrate-binding and redox reactions. Because many coenzymes cannot be synthesized in the body, they must be ingested as vitamins.
Crystallization of Enzymes
Column Fig. 4-1 Microscopic Photograph of a Protein Crystal
Crystal of the cytochrome b6f complex obtained from thermophilic cyanobacteria. It is a major protein complex that acts as an electron transfer component in the delivery of an electron from photosystem II to photosystem I. Its crystal structure was identified by the current Osaka University professor Genji Kurisu. Refer to Chapter 16 for details on photosynthesis.
Currently, it is well known that enzymes are proteins, but at the beginning of the 20th century, they were often not believed to be proteins. In 1926, James Sumner showed that enzymes are proteins. He first crystallized urease, which hydrolyzes urea to produce ammonia and carbon dioxide, from Canavalia beans to clarify its properties as a substance. Thereafter, many different enzymes have been crystallized. Furthermore, X-ray crystallography helped in establishing the arrangement of atoms in crystals. In 1958–1959, John Kendrew and Max Perutz described the crystal structures of myoglobin and hemoglobin. Proteins have a high molecular weight, and determination of their crystal structures required collection of data from many diffraction points and a powerful computer for processing these data. Another critical problem was the fact that X-rays damage proteins. Recently, data can be collected from multiple diffraction points using short-exposure X-ray irradiation and electron density distribution can be calculated. Using these methods, efforts are being made to explain the crystal structures of many proteins discovered by genome analysis.
Specificity of Enzymes and the Reaction Mechanism
Enzymes act on specific substances and catalyze specific reactions. This indicates that an enzyme has two types of specificity—a substrate specificity and a reaction specificity. An enzyme displays its catalytic activity by identifying and binding to its specific substrate. The catalytic reaction is also predetermined. For example, the enzyme called amylase acts on starch (glucose polymer) and produces maltose (a disaccharide consisting two glucose molecules), but it cannot isolate the glucose molecules. When considering acids (hydrogen ions) as examples of inorganic catalysts, glucose polymers of varied length are produced by severing the saccharide chains of starch at random. In other words, the reaction specificity of acids is low. Moreover, acids can act on substances other than starch such as proteins or macromolecular substances, and thus, they do not have high substrate specificity.
Fig. 4-5 Triple Catalysts composing the Active Center of Chymotrypsin
The hydrogen atom nucleus (proton) of the hydroxyl group (OH group) of serine residue 195 is strongly attracted to the lone electron of N of histidine residue 57. Hence, O of the serine residue has a negative charge and attacks the carbonyl carbon (slight positive charge) of the substrate’s peptide bond. This is the initial stage of peptide bond hydrolysis. The red arrows indicate electron transfer.
The high specificity of enzymes has been found to be supported by their steric structures. The active center of enzymes is shaped such that they bind particularly well with specific substrates, and the amino acid side chains are arranged such that binding with a specific functional group of the substrate can occur. Proteinaceous enzymes act effectively as catalysts because of special amino acid residues at the active centers, which impart a special reactivity as a result of their interactions with other amino acid residues. For example, the catalytic triad of chymotrypsin (Fig. 4-5). The figure indicates that of the amino acids comprising the enzyme, serine containing a hydroxyl group attacks the peptide bond as the active center. Hydrogen ions are extracted by aspartic acid and histidine, which are sterically associated amino acids. Thus, the serine side chain achieves an ionized state such as -CH2-O−. To maintain these characteristics and this steric structure, enzymes generally have a pH that is most suitable for their activity. Although the reaction rate increases with temperature, the steric structure is destroyed at high temperatures by denaturation. Thus, enzymes can function appropriately only at an optimal reaction temperature. A more detailed explanation of the steric structures of enzymes will be provided in Chapter 6.
The mechanism of enzymatic reactions has been studied since a long time by analyzing the protein composition of enzymes and their steric structures. At the beginning of the 20th century, the German scientists Leonor Michaelis and Maud Menten, who were studying an enzyme called invertase, later proposed a method for describing enzyme kinetics based on the following experimental facts.
In an enzymatic reaction, the initial reaction rate V is increased by increasing the concentration [S] of the substrate S. However, in cases where the amount of enzyme added to the reaction system is fixed, no matter how much you increase [S], V cannot increase over a certain limit (Fig. 4-6). This extrapolated value is called Vmax. In other words, V is saturated with regard to [S]. The phenomenon of saturation is characteristic of catalytic reactions and is explained as the binding of a catalyst and substrate. The phenomenon of saturation is observed because even if there is an abundance of substrate molecules, the number of substrate binding sites available for the catalyst is limited.
This is shown by a mathematical formula, and study of the rate of an enzymatic reaction is called kinetics. A simple enzymatic reaction is considered below. Invertase is an enzyme that hydrolyzes sucrose to produce glucose and fructose. Although a large quantity of water is involved in this reaction, it does not have an effect on the reaction rate, and hence, only sucrose (represented by [S]) is considered to be the substrate. The enzyme molecule is represented by E. Although there are two types of products formed, they are both represented by P. The concentration of the reaction products is considered in a range that does not affect the reaction rate. Important assumptions made here include the assumption that the enzyme and substrate bind reversibly and that a portion of the bonded substrate and enzyme will yield products through the primary reaction.
E + S ↔ ES → E + P
At this point, the following general equations are established.
These are called the Michaelis–Menten equations. Here Vmax refers to the maximum initial reaction rate, and Km refers to the Michaelis constant. Km represents [S] that gives an initial reaction rate of ½ Vmax. This equation is essentially a hyperbolic function demonstrating the phenomenon of saturation. The equation is derived on the basis of certain assumptions (Refer to Column on Page 55 for the derivation) that in reality do not always hold true for all enzymatic reactions. However, in practice, the equation holds true for many enzymatic reactions, and Km is used as an index showing affinity between the enzyme and substrate (smaller the Km, greater the affinity).
One application of this formula is the classification of inhibitors of enzymatic reactions. Although inhibitors are classified according to their effect on Km and Vmax, among those, only competitive inhibitors will be discussed here. When a substance with a structure similar to that of a substrate binds to the substrate binding site of the enzyme but does not participate in an enzymatic reaction, it interferes with the original substrate reaction and is called a competitive inhibitor. For example, succinate dehydrogenase, an enzyme in the citric acid cycle (see Fig. 4-3 and Fig. 16-3 of Chapter 16), is inhibited by malonic acid (HOOC-CH2-COOH), a substance analogous to succinic acid (HOO-CH2-CH2-COOH). Thus, the observed Km increases, but Vmax does not change, in the presence of the inhibitor.
In addition, allosteric regulation is another factor that exerts an effect on enzymatic activity. Allosteric regulation is commonly observed in enzymes composed of multimers. During this regulation, the steric structure of the constituent components change cooperatively when a regulator molecule binds to a site (the allosteric site) other than the active center. This causes the multimer to alternately switch between the high and low activity states depending on the large changes in the conformational state of the enzyme due to the allosteric regulator.
Derivation of the Michaelis−Menten Equation
In 1925, George Briggs and John Haldane derived the currently used Michaelis–Menten equation. In the simple enzymatic reaction mentioned in this textbook, dependence of the initial reaction rate on the concentration of the substrate is considered. If the total concentration of the enzyme is set as E0, then
E0 = [E] + [ES] ---------- (1)
At stable conditions assumed, no change is observed in the concentration of the intermediate ES,
which can be rewritten as follows:
[S][E] − Km[ES] = 0 ----------- (2)
>> [ES], the concentration of the separated substrate is considered the overall concentration of the substrate.
If [E] is removed from (1) and (2), then
By substituting (3) in the equation for the initial reaction rate V, the result obtained is as follows:
This gives Vmax = k2E0. Note that the enzyme E and the substrate S are in rapid equilibrium, and if k1,
k−1 >> k2, then km can give a dissociation constant for the enzyme and substrate of