Body proteins perform a number of functions, of which the most remarkable and highly specialized function is their abil-ity to act as biological catalysts. Catalytic proteins called enzymes, stimulate the rate of several reactions in the body without themselves getting consumed in the process. As biological catalysts, the enzymes, exhibit certain special charac-teristics which are rarely, if ever, exhibited by non-biological catalysts. These are:
1. The catalytic power of enzymes is extraordinary: they make occurrence of highly complex reactions possible under mild conditions of temperature and pH prevailing in the body.
2. Enzymes have a high degree of specifi city for the substrates. Most of the enzymes use only one substrate, although some enzymes have broader specifi city, with a capacity to act on more than one substrate.
3. Enzymes exhibit specifi city for the reactions also. They accelerate specifi c chemical reactions without formation of any by-products.
Enzymes act as functional units of the cellular metabolism. They catalyze innumerable body reactions whereby the nutrient molecules are degraded, chemical energy is conserved and transformed, and complex biomolecules are synthe-sized from simple precursors. Virtually, all the body reactions are mediated by enzymes. In fact, cellular reactions would not occur rapidly enough to sustain life if enzymes were not present.
This chapter outlines the properties and characteristics common to most enzymes. After going through this chapter, the student should be able to understand:
Concept of organic catalysts, the catalytic site and enzyme-substrate complex; role of cofactors and coenzymes in enzymatic catalysis.
Nomenclature and classifi cation of enzymes; mechanism of enzyme action; effect of various factors like temperature, pH, enzyme concentration, etc. on enzyme activity; qualifi cation of enzyme activity.
Michaelis-Menten theory of enzyme action.
Various types of enzyme inhibitions and their recognition; enzyme regulation through modulation of activity or by induction/repression.
Utility of enzyme and isoenzyme determination as a diagnostic tool.
E N Z YM E S
6
I. Properties of Enzymes A. Chemical Nature
Most known enzymes are proteins in nature. The typical enzyme is a globular protein that either is dissolved in
cytoplasm or in a cellular organelle; or is bound to a membrane, or is located extracellularly in interstitial space or vascular space; or, as in case of the digestive enzymes, in an external compartment. The catalytic activity of an enzyme depends on integrity of the protein structure. This is evidenced by the fact that various factors which disrupt the native conformation of the
Some enzymes require assistance of metal ion cofactors or organic coenzymes derived from vitamins. The coen-zymes function as reversibly bound co-substrates, or as permanently associated prosthetic groups.
2. Prosthetic groups: The prosthetic group on the other hand is bound permanently to the active site of its enzyme, either covalently or non-covalently. Disso-ciation of the prosthetic group results in an irreversible loss of catalytic activity of the enzyme. For example, biotin, is the prosthetic group for a group of enzymes called carboxylase. It is an integral component of these enzymes, being attached by an amide linkage with the apoenzyme component (i.e. apocarboxylase). Dissoci-ation of biotin from the apocarboxylase results in loss of activity of the carboxylase enzymes (Case 6.1).
The distinction between co-substrate and prosthetic group is, however, not absolute because a compound that enzyme polypeptide chain(s) cause loss of the enzyme
catalytic activity.
Enzyme, almost all of which are proteins, are highly selec-tive biocatalysts that accelerate reactions by factors up to 1015.
Some type of RNAs can also serve as enzymes. Such RNAs with catalytic activity are called the ribozymes. Most of them play specialized roles in gene expression (catalyze cleavage and synthesis of phosphodiester bonds), but are not involved in metabolic reactions.
B. Cofactors
Some enzymes are proteins, containing no chemical group other than amino acid residues (e.g. pancreatic ribo-nuclease trypsin, chymotrypsin, elastase). However, most enzymes associate with non-protein chemical components, called cofactors, which are required for their catalytic activ-ity. The complete active enzyme is called a holoenzyme, and it is made of the protein portion (apoenzyme) and the cofactor. The apoenzyme is inactive alone.
Holoenzyme Apoenzyme Cofactor Cofactors may be:
inorganic metal ions (Table 6.1) or
organic molecules, called coenzymes
Many coenzymes are derived from the dietary, water-soluble vitamins. An example is fl avin adenine dinucleo-tide (FAD) or fl avin mononucleodinucleo-tide (FMN), which are derived from ribofl avin (vitamin B2). In general, a coen-zyme actually participates in the overall reaction as another substrate, and mainly acts as a donor or acceptor of a particular chemical group. Specifi c coenzymes are concerned with transfer of specifi c groups, as shown in Table 6.2.
The coenzymes function as either co-substrates or prosthetic groups.
1. Co-substrates: The co-substrate associates with the active site of the enzyme only transiently for the purpose of the reaction. It is changed chemically during the reac-tion, and after completion of the reacreac-tion, the chemi-cally modifi ed co-substrate dissociates away and is free to participate in other enzymatic reaction cycle. For example, pyridoxal phosphate (a co-substrate in trans-amination reaction) brings about transfer of amino group from an amino acid to a keto acid by reacting with and serving as transient-carrier of the transferred amino group (Chapter 13).
Table 6.1. Metal ions as cofactors for specifi c enzymes (also called metalloenzymes)
Metal Enzyme
Ca2 Lipase
Cu2/Cu Cytochrome oxidase, tyrosinase, lysyl/oxidase, superoxide dismutase
Fe2/Fe3 Cytochrome oxidase, xanthine oxidase peroxidase, catalase
K Pyruvate kinase
Mg2 Hexokinase, phosphofructokinase, enolase, creatine kinase
Mn2 Arginase, glycosyl transferase, phosphoglucomutase
Ni2 Urease
Zn2 Carbonic anhydrase, alkaline phosphatase DNA polymerase, alcohol dehydrogenase
Table 6.2. Coenzymes and the entity transferred Coenzyme Entity transferred
Biotin Carbon dioxide
Coenzyme A Acyl group
FAD Hydrogen atoms
NAD Hydride ion (H–)
Pyridoxal phosphate Amino groups
Tetrahydrofolate One carbon groups (other than CO2) Thiamine pyrophosphate Hydroxy-ethyl
Coenzyme B12 Alkyl groups or hydrogen atoms FAD fl avin adenine dinucleotide, NAD nicotinamide adenine dinucleotide.
The enzyme specifi city resides in a relatively small region of the enzyme, called the active site. It is generally a crevice, pocket or cleft that is three dimensional, having the correct molecular dimensions and appropriate topol-ogy to accommodate specifi c substrate (Fig. 6.1; see also Box 6.1). The active site is formed by collection of differ-ent amino acid residues (termed active-site residues) that may or may not be adjacent in the primary sequence. The interactions between these residues and the substrate occur via the same forces that stabilize protein structure:
hydrophobic interactions, electrostatic interactions, hydro-gen bonding and van der Waals interactions. The active site residues are directly responsible for the catalytic action. They not only bind the substrate to form enzyme-substrate (ES) complex, but also provide specifi c interac-tions that stabilize the formation of the transition state (highest energy arrangement of atoms) for the chemical reaction to proceed and to form the reaction product.
Enzymes exhibit reaction specifi city and substrate spec-ifi city, which are determined by the geometric and elec-tronic character of the active site where the substrate binds and undergoes transformation.
E. Location within the Cell
Most enzymes are compartmentalized in specifi c organ-elles within the cell; for example, enzymes that catalyze degradation of fatty acids (i.e. -oxidation) are located in the mitochondrial matrix, whereas those involved in synthesis of fatty acids are present in the cytosol. Such arrangement helps to:
organize thousands of enzymes of the cell in distinct pathways,
provide favourable environment for cellular reactions,
Isolate the substrate or product of a given reaction from other competing reactions.
serves as a coenzyme for one enzyme can serve as a pros-thetic group for another enzyme.
The name “coenzyme” is applied to two different types of cofactors that function either as co-substrates or as true prosthetic groups. Interestingly, behaviour of co-substrate sound “promiscuous” since it associates with the active site of the enzyme only transiently. Prosthetic group sounds monogamous: it is tied-up with enzyme permanently.
Metalloenzymes: The enzymes that require inorganic metal ions as their cofactors are termed metalloen-zymes. Approximately two-third of the known enzymes are metalloenzymes. In most cases, the binding of metal ion is essentially required for the enzymatic activity (e.g.
zinc for carbonic anhydrase), whereas in some the enzyme is active even without the metal ion, but its activity increases greatly when the metal ion is added (e.g. chlo-ride and fl uochlo-ride ions activate salivary amylase).
C. Catalytic Effi ciency
Catalytic effi ciency of enzymes is very high—most of them bring about several-fold (103 to 1015 times) increase in the reaction rate. Typically, each enzyme molecule is capable of transforming 100 to 1000 substrate molecules into the corresponding product molecules each second. Carbonic anhydrase, the enzyme catalyzing formation of carbonic acid from water and carbon dioxide, is highly active; a single enzyme molecule can transform 36,000,000 sub-strate molecules each second.
D. Enzyme Specifi city: The Active Site
Enzymes possess most exquisite specifi city of all the cata-lysts known. They are highly selective for both the substrate and the reaction undergone by the substrate. Typically, each individual reaction requires its own enzyme, and if an enzyme is lacking, only one particular reaction is generally blocked. Many enzymes show absolute specifi city (e.g.
tyrosinase acting on tyrosine only) for the substrate, and this includes stereo-specifi city also. For example, D-amino acid oxidase binds only D-amino acids but not L-amino acids; fumarase hydrates fumarate to form malate, but the corresponding cis form of the former, maleic acid, is not acted upon. Other enzymes have relatively broad speci-fi city and act on several different analogs of a specispeci-fi c substrate; for example, glucose, mannose, fructose, glu-cosamine and 2-deoxyglucose are phosphorylated by hexokinase, but at different rates. (Glucokinase, on the other hand, is specifi c only for glucose.)
Fig. 6.1. Active site of an enzyme and enzyme-substrate complex (E enzyme, ES enzyme-substrate complex, S substrate).
Substrate
Active site
Enzyme
Enzyme-substrate complex +
E + S ES
the substrate for this conversion to the transition state. It is termed the free energy of activation (Gact), and it equals the free energy difference between the substrate and the tran-sition state. The Gact provides an energy barrier to the reaction—higher the barrier, the slower the reaction. The enzymes speed up the chemical reaction by lowering the magni-tude of the activation energy barrier. This results in decreasing the Gact. Lower Gact means increased concentration of
II. Mechanism of Enzyme Catalysis
The question, “how do enzymes work” can be answered from two different perspectives. The fi rst one describes the energy changes that occur during the catalyzed and the uncatalyzed reactions. The second deals with changes in the enzyme active site which facilitates catalysis.
A. Free Energy Changes During Chemical Reactions
For any chemical reaction to take place, the free energy content of the products should be lower than that of the substrates. In these cases, the free energy change of the reaction (G) has a negative sign and the reaction is said to be thermodynamically favourable (Chapter 8). However, not all reactions that are thermodynamically favourable occur spontaneously; the substrate has to be converted fi rst to a higher energy form (called transition state) before it can form the reaction product. The transition state is structurally an intermediate between the substrate and the product, and represents the highest energy arrangement of atoms. Therefore, it is unstable; once formed, it decom-poses almost immediately to form the reaction product (or sometimes the substrate again).
In the reaction coordinate diagram shown in Figure 6.2, the reaction is thermodynamically favourable but cannot occur spontaneously because the substrate has to be fi rst con-verted to the transition state. Some energy must be put into
Fig. 6.2. Diagrammatic representation of the free energy changes that occur during a chemical reaction. (a) Uncatalyzed, (b) Catalyzed, Gact free energy of activation. Enzymes speed up a chemical reaction by lowering the magnitude of activation energy barrier without affecting equilibrium of the reaction.
(b)
Free energy
Final state
Duration of reaction
Initial state ΔGact catalyzed ΔGact uncatalyzed Transition state (catalyzed reaction) Transition state (uncatalyzed reaction)
Energy change of reaction