Living organisms continuously transform energy and materials and are able to do so only because of the innumerable enzymes that they contain. Enzymes act as catalysts whose function is to make or break covalent bonds that otherwise could only be created or destroyed under conditions of temperature and pH that are incompatible with life. Catalysts act by reducing the stability of bonds and lowering the amount of energy required to break them. This energy is known as the activation energy; the greater it is, the less reactive the compound or system. A catalyst lowers the activation energy by combining with the substrate and forming an unstable intermediate whose rapid decomposition provides an alternative pathway with a lower energy barrier. Whether the activation energy is high or low, the overall change in free energy in the reaction is the same. Enzymes are proteins, and while they resemble inorganic catalysts in reducing the activation energy and in remaining unchanged at the end of the reaction, they are much more specific, more efficient, and more readily inactivated. They achieve their effects by providing a surface on which their substrates are specifically adsorbed and orientated. As a result of the formation of this enzyme-substrate complex, a substrate that is to be broken down becomes strained just at the point where fission is to take place. In condensation reactions, instead of being dependent on random collisions, the reactants are attracted and held by the enzyme in exactly the position needed for the reaction to occur.
Living organisms continuously tranform energy and materials and are able to do so only because of the innumerable enzymes that they contain. Enzymes act as catalysts whose function is to make or break covalent bonds which otherwise could only be created or destroyed under conditions of temperature and pH that are incompatible with life. For example, in order to oxidize glucose directly to carbon dioxide and water in vitro the temperature must be raised to several hundred degrees to provide enough energy to tear apart the atoms in the glucose molecule. Even the formation of water from hydrogen and oxygen, which releases so much energy that the reaction is explosive, will not occur unless it is sparked off. The energy provided by the spark breaks covalent bonds releasing oxygen and hydrogen atoms from their molecules, so enabling them to recombine as water, which has a lower energy content. The energy released during the reaction sets off the disintegration of other oxygen and hydrogen molecules and initiates a chain reaction.
Catalysts act by reducing the stability of bonds and consequently lowering the amount of energy required to break them. This energy is known as the activation energy and the greater it is the less reactive the compound or system. A catalyst lowers the activation energy by combining with the substrate and forming an unstable intermediate whose rapid decomposition provides an alternative pathway with a lower energy barrier. Whether the activation energy is high or low the overall change in free energy in the reaction will be the same (Figure 6.1).
Enzymes are proteins and, while they resemble inorganic catalysts in reducing the activation energy and in remaining unchanged at the end of the reaction, they are much more specific, more efficient and more readily inactivated. They achieve their effects by providing a surface on which their substrates are specifically adsorbed and orientated. As a result of the formation of this enzyme-substrate complex, a substrate which is to be broken down (lysed) becomes strained just at the point where fission is to take place. In condensation reactions instead of being dependent on random collisions the reactants are attracted and held by the enzyme in exactly the position needed for the reaction to occur.
While some enzymes are composed solely of protein, others require one or more non-protein substances of low molecular weight for their activity. If the non-protein moiety is firmly bound to the protein part it is known as a prosthetic group and the protein is a conjugated protein; but where the enzyme is only active in the presence of a discrete small organic molecule, separable by dialysis, the non-protein substance is known as a coenzyme.
The property which especially distinguishes enzymes from other catalysts is their specificity. Whereas inorganic catalysts may speed up more than one reaction, enzymes usually accelerate a reaction involving particular molecules or closely related types of molecule. The specificity of enzymes ensures the close control and coordination of reactions that are necessary for the existence of living organisms. Of the numerous chemical reactions that a compound may undergo, an enzyme reduces the activation energy for only one of them, i.e. enzymes possess reaction specificity. Different enzymes are needed to initiate different reactions involving the same compound. For example, an amino acid will undergo quite different reactions according to whether it is acted upon by an oxidase, a transaminase or a decarboxylase.
Apart from being specific for a particular reaction, enzymes show varying degrees of substrate specificity. This ranges from the absolute specificity shown by urease, which has urea as its one and only substrate, to group specificity in which an enzyme will act upon a general type of substrate, e.g. alcohols, esters or peptide bonds. The lipases have a broad specificity and will act on the esters of most fatty acids, while hexokinase catalyses the phosphorylation of a variety of aldohexoses.
Since enzyme action depends on the closeness with which the structure of the enzyme and its substrate complement each other, most enzymes are stereochemically specific. They act on only one of a pair of optical isomers or, if both should be attacked, one reacts much more readily than the other. For example, maltase is a group-specific enzyme that attacks several other α-glucosides as well as maltose but has no effect on β-glucosides.
5. Isomerases catalyse various intramolecular rearrangements such as the conversion of an aldose to a ketose sugar, or alteration of the position of a phosphate group, e.g. phosphohexoisomerase, phosphoglucomutase.
6. Ligases or synthetases catalyse the joining together of two molecules in a reaction that is coupled with the hydrolysis of a nucleoside triphosphate such as ATP (page 210), e.g. acetyl-CoA carboxylase, glycogen synthetase, succinic thiokinase.
According to the method of classification that has been adopted by the International Union of Biochemistry the systematic name of an enzyme consists of two parts, the first being the name of the substrate or substrates and the second indicating the type of reaction catalysed and ending in -ase. Thus the systematic name for hexokinase is ATP:hexose 6-phosphotransferase.
A considerable number of enzymes require the presence of one or more small organic molecules which participate in the overall reaction. These molecules are usually termed coenzymes, but since they react in stoichiometric proportions with the substrate(s), they are more accurately regarded as cosubstrates. The function of such molecules is to link reactions. They do this by acting as carriers of particular groups such as phosphate or acyl groups, or of reducing equivalents, e.g. electrons or hydrogen atoms (Table 6.1).
|Full name||Abbreviation||Group transferred||Corresponding vitamin|
|I. Hydrogen-transferring coenzymes|
|a. Nicotinamide–adenine dinucleotide||NAD||H+ + 2e||Nicotinamide|
|b. Nicotinamide–adenine dinucleotide phosphate||NADP||H+ + 2e||Nicotinamide|
|c. Flavin mononucleotide||FMN||2H||Riboflavin|
|d. Flavin adenine dinucleotide||FAD||2H||Riboflavin|
|f. Lipoic acid||2H + acyl||—|
|II. Group-transferring coenzymes|
|a. Pyridoxal phosphate||PALP||Amino||Pyridoxine (B6)|
|b. Tetrahydrofolate||CoF||Hydroxymethyl (formyl)||Folic acid|
|c. Biotin||—||Carboxyl (CO2)||Biotin|
|d. Cobalamin||B12||Carboxyl||Cobalamin (B12)|
|e. Coenzyme A||CoA||Acetyl and other acyl groups||Pantothenic acid|
|f. Thiamine pyrophosphate||TPP||Acetaldehyde||Thiamine|
The function of a coenzyme is well illustrated by the role of pyridoxal phosphate in the transfer of amino groups. The enzyme alanine aminotransferase (glutamate–pyruvate transaminase) catalyses the reaction of glutamate with pyruvate to form 2-oxoglutarate and alanine. In this reaction, the amino group of glutamate is transferred first to pyridoxal phosphate and then to pyruvate with the formation of alanine.
The pyridoxal phosphate returns to its original form at the end of the reaction. In the case of other coenzymes, a second enzyme reaction must occur before the coenzyme is reconverted to its original form. For example, with NAD (page 214), which is a coenzyme for many dehydrogenase enzymes, the oxidized form of the coenzyme is reduced in the first enzyme reaction to NADH which must then be reoxidized by a second enzyme-catalysed reaction before it is able to participate again in the first type of reaction.
Thus reduced NAD formed during glycolysis is usually reconverted to the oxidized form by passing its H atoms into the electron transport chain with the agency of a special NAD dehydrogenase. Alternatively if O2 is in short supply the NAD is regenerated by the reduction of pyruvate to lactate.
Pyridoxine, the parent substance of pyridoxal phosphate, is also known as vitamin B6 (page 165). Many coenzymes contain a derivative of one or other of the B vitamins as an essential part of their structure. Coenzymes may react with a number of different enzymes which are specific for different substrates but which catalyse the same general type of reaction.
The types of reaction which require a coenzyme include group transfers, isomerizations, oxidoreductions and reactions resulting in the formation of covalent bonds. Hydrolytic enzymes do not usually require a coenzyme.
A great deal of information about enzymes may be obtained by measuring the velocity of the reactions they catalyse under different conditions, i.e. by studying their kinetics. The velocity is measured in terms of the amount of substrate reacting or product formed in unit time under specified conditions, and it is usually expressed in micromoles of substrate transformed per minute. The quantity of enzyme which in 1 minute acts upon 1 micromole of substrate or produces 1 micromole of product under the prescribed conditions is said to possess 1 unit of enzyme activity.
If the progress of an enzyme reaction is plotted against time it is found that the velocity is initially high but soon begins to decrease so that a curve such as A in Figure 6.2 is obtained. If lower concentrations of enzyme are used similar curves (B) and (C) are obtained but the time taken to reach the same final concentration of product is increased.
This is a closed system and the falling off in the speed of the reaction can be accounted for by the disappearance of substrate S and the accumulation of products P so that eventually equilibrium conditions are established when
and the forward and backward reactions are occurring at an equal rate. The slowing may also result from other changes in conditions occurring as a result of the reaction, such as an alteration in pH. Since enzyme studies are almost always comparative, it is possible to design the experiments so as to ensure that these factors apply equally in all cases. This is usually achieved by measuring the initial velocity (Vi) of the reaction, i.e. the slope of the curve in the earliest stages when the substrate concentration [S] is virtually unchanged, insignificant amounts of product have been formed, the original pH is maintained and the enzyme is not denatured, i.e. conditions approximate to those operating in an open system in a steady state.
If a series of progress curves are plotted over a whole range of enzyme concentrations it is found that, provided the substrate is in excess, the initial velocity of the reaction (Vi) is directly proportional to the enzyme concentration and a straight line relationship is obtained.
Most enzyme reactions only occur at temperatures between 0 and 60°C. At the lower end of the range, enzyme reactions behave like ordinary chemical reactions and the rate increases as the temperature rises, the velocity being approximately doubled for every 10°C rise as a result of the increased kinetic energy of the reacting molecules. A typical curve relating the activity of an enzyme measured over a constant time, e.g. 5 or 10 minutes, with the temperature is shown in Figure 6.3 from which it may be seen that there is a definite optimum temperature which is usually related to the temperature of the environment of the cell from which the enzyme was derived. In mammals the optimum is about 37°C while, curiously, for many plant and bacterial enzymes it is higher than this. The rapid decrease in activity seen at temperatures above the optimum is due to heat denaturation, which occurs progressively as the temperature rises.
In view of the importance of pH in determining the ionization of proteins, it is not surprising that enzymes are extremely sensitive to it. Changes in their charge distribution may affect activity by altering either the overall conformation of the protein or the reactivity of amino acid side chain groupings involved in the active site. Each enzyme has a characteristic optimum pH although, if an enzyme acts on two different substrates, the optimum pH values may differ slightly. Pepsin is unusual in acting at very low pH values and has an optimum pH varying from 1.5 to 2.5 according to the protein being digested. Most enzymes have an optimum pH between 5 and 9 (Figure 6.4