Understanding Enzymes: Structure, Function, and Classification, Thesis of Chemistry

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Chapter – 1

General introduction to enzymes

SECTION 1.1 GENERAL INTRODUCTIONS

1.1.1. Enzyme introduction Enzymes are nature’s sustainable catalysts. They are biocompatible, biodegradable and are derived from renewable resources [1]. Enzymes constitute a large biological globular protein molecule responsible for thousands of metabolic processes that sustain life [2], and function as catalysts to facilitate specific chemical reactions within the cell. These reactions are essential for the life of the organism. The living cell is the site of tremendous biochemical activity called metabolism. This is the process of chemical and physical changes which go on continually in the living organism; enzyme facilitate life processes in essentially all life-forms from viruses to man. Enzymes have been naturally tailored to perform under different physiological conditions. Build-up of new tissues [3], replacement of old tissues [4], conversion of food into energy [5], disposal of toxic materials [6] , reproduction [7] almost all the activities that can be characterized as “life.” Enzymes act as life catalysts, substances that accelerate the rate of a chemical reaction. By reducing the activation energy ( E a) necessary to initiate the reaction, thus dramatically increasing the rate of reaction. Enzymes do not initiate reactions that would not naturally occur but they accelerate any reaction that is already underway. Enzymes enable the reaction to take place more rapidly at a safer, relatively low temperature that is consistent with living systems. During an enzyme-mediated reaction, the substrate physically attaches to the enzyme at its active site, allowing the substrate(s) to be converted to new product molecule(s). Most enzyme reaction rates are millions of times faster than those of comparable un- catalyzed reactions. Enzymes are neither consumed by the reactions they catalyze, nor

discovered a complex procedure for isolating pepsin. This precipitation technique devised by Northrop and Stanley has been used to crystallize several enzymes [12]. 1.1.3. Proteinaceous nature of enzymes Enzymes are proteins and are nature’s own biocatalyst and their function is determined by their complex structure. With the exception of a small group of catalytic RNA molecules, all enzymes are proteins which are made up of amino acids linked together by peptide bonds [13]. By the early 1800s, the proteinaceous nature of enzymes had been recognized. Knowledge of the chemistry of proteins drew heavily on improving techniques and concepts of organic chemistry in the second half of the 1800s; it culminated in the peptide theory of protein structure, usually credited to Fischer and Hofmeister. However, methods that had permitted the separation and synthesis of small peptides were unequal to the task of purifying enzymes. Indeed, there was no consensus that enzymes were proteins. After isolation of a series of crystalline proteolytic enzymes beginning with pepsin by Northrop et al., in 1930, the proteinaceous nature of enzymes was established. They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds. Their catalytic activity depends on the integrity of their native protein conformation. If an enzyme is denatured or dissociated into its subunits, catalytic activity is usually lost. Thus the primary, secondary, tertiary, and quaternary structures of protein enzymes are essential to their catalytic activity [14]. 1.1.4. Structure of enzymes Enzymes are proteins and, are agreeable to structural analysis by the methods of protein chemistry, molecular biology, and molecular biophysics. Like all proteins, enzymes are composed mainly of the 20 naturally occurring amino acids. The structures of enzymes can be elucidating by the physical methods such as

Spectroscopic methods [15], x-ray crystallography [16], and more recently, multidimensional NMR methods [17]. On the basis of arrangement of amino acids enzyme structure can be classified into following types, 1.1.4.1. Primary structure The structure and reactivity of a protein are defined by the identity of the amino acids that make up its polypeptide chain, this amino acid sequence of the peptide chains is the primary structure of the enzyme. 1.1.4.2. Secondary structure Secondary structure is due to the interaction of amino acids with each other in the same chain of protein. As a result the protein chain can fold up on itself in two ways, namely α-helix or β-sheet resulting secondary structures. 1.1.4.3. Tertiary structure The arrangement of secondary structure elements and amino acid side chain interactions that define the three-dimensional structure of the folded protein. So that specific contacts are made between amino acid side chains and between backbone groups. The resulting folded structure of the protein is referred to as its tertiary structure. 1.1.4.4. Quaternary structure and domains Many enzymes consist of more than one polypeptide chain (or subunit) that aggregate to confer catalytic activity. In some enzymes the subunits are identical, in others they differ in sequence and structure. This description of subunit arrangement in such enzymes is called the quaternary structure. A typical enzyme is not an entity completely folded as a whole, but may consist of apparently autonomous or semi- autonomous folding units called domains.

1.1.6. Sources of enzyme Enzymes occur in all living organisms and catalyze biochemical reactions necessary to support life [18]. A wide array of enzymes are extracted from plant sources; they have many advantages including cost of production and stability of products [19]. An ample range of sources are used for commercial enzyme production from a broad spectrum of plant species. Non-microbial sources provide a larger proportion of these, at the present time. Microbes are preferred to plants and animals as sources of enzymes because [20]:

• They are generally cheaper to produce.
• Their enzyme contents are more predictable and controllable,
• Regular supply due to absence of seasonal fluctuations and rapid growth of microorganisms on inexpensive media.
• Plant and animal tissues contain more potentially harmful materials than microbes, including phenolic compounds (from plants), endogenous enzyme inhibitors and proteases.
• Microbial enzymes are also more stable than their corresponding plant and animal enzymes and their production is more convenient and safer About fifty years ago, enzymes were being extracted strictly from animals like pig and cow from their pancreases [21]. Animal enzymes were multifold, they were not very stable at the low pH environment so that the enzyme product was destroyed before doing the job. To overcome this problem plant enzymes were discovered, most important one is extraction of peroxidase from horseradish roots occurs on a relatively large scale because of the commercial uses of the enzyme [22]. Peroxidase can also be extracted from soybean, it is also having the common features with horseradish peroxidase [23]. Some plants like Cruciferous vegetables, including broccoli ,

cabbage, kale and collard and turnip greens and papaya are rich in catalase [24-26]. Wheat sprouts contain high levels of catalase [27] and vegetarian sources of catalase include apricots, avocados, carrots [28]. Catalase is also present in some microbes and bacteria [29], Aspergillus niger culture also produces catalase enzyme [30]. 1.1.7 Naming and enzyme classification In general many enzymes have been named by adding the suffix “-ase” to the name of their substrate or to a word or phrase describing their activity. In 1961, according to the report of the first Enzyme Commission (EC) of International Union of Pure and Applied Chemistry (IUPAC), Enzymes are classified in to six types on the basis of reaction they catalyze [31]. They were assigned code numbers, prefixed by E.C., which contain four elements separated by points and have the following meaning as shown in Scheme 1.1.

Scheme 1.1: Naming of enzyme according to ‘International Union of Biochemistry’ formed a ‘Commission on Enzyme Nomenclature

1.1.8 Mechanism of enzyme action Enzymes are macromolecules that help to accelerate (catalyze) chemical reactions in biological systems. Some biological reactions in the absence of enzymes may be as much as a million times slower [32]. Any chemical reaction converts one or more molecules, called the substrate, into different molecule(s), called the product. Most of the reactions in biochemical processes require chemical events that are unfavorable or unlikely in the cellular environment, such as the transient formation of unstable charged intermediates or the collision of two or more molecules in the precise orientation required for reaction. In some of the Reactions like, digestion of food [33], send nerve signals [34], or contract a muscle simply do not occur at a useful rate without catalysis[35]. Enzyme overcomes these problems by providing a specific environment within which a given reaction can occur more rapidly. Enzymes are usually proteins – each has a very specific shape or conformation. Within this large molecule is a region called an active site , which has properties allowing it to bind tightly to the substrate molecule(s). The active site of the enzyme is shown in Figure 1.1. Substrate in active site

Figure. 1.1: Structure of enzyme showing the active state

As proposed by Charles-Adolphe Wurtz, an active site is a three dimensional cleft or crevice formed by groups that come from different parts of the amino acid sequence - residues far apart in the amino acid sequence may interact more strongly than adjacent residues in the sequence. The active site encloses a substrate and catalyzes its chemical transformation. The enzyme substrate complex was first discovered in 1880, is central to the action of enzymes. The enzyme–substrate interactions can be explained by the following theories. 1.1.8.1 Lock and key model In "lock and key" model the active site of the enzyme is complementary in shape to that of the substrate. The substrate is held in such a way that its conversion to the reaction products is more favorable. It was thought that the substrate exactly fitted into the active site of the enzyme molecule like a key fitting into a lock. In the Figure 1.2 "lock" refers to enzyme and "key" refers to its complementary substrate [36].

Figure. 1.2 Lock and key model for enzyme – substarte 1.1.8.2 Induced fit Lock and key model does not explain the stability of the transition state for it would require more energy to reach the transition state complex. To explain this concept Koshland in 1958, first proposed the induced-fit model, this suggests that the enzyme active site is conformationally fluid. Enzyme itself usually undergoes a change in conformation when the substrate binds, induced by multiple weak interactions and hydrophobic characteristics on the enzyme surface mold into a precise formation [37].

SECTION 1.2 ENZYME ASSAY

1.2.1. Introduction In recent years enzyme assays have greatly advanced in their scope and in the diversity of detection principles employed. Enzyme assays are laboratory methods to visualize enzyme activities. An enzyme assay consists of mixing the enzyme with a substrate in a solution of controlled pH with any additional substance whose effect is to be tested, incubating the reaction mixture at an appropriate temperature for the required time, stopping the reaction precisely, and then measuring the amount of reaction that has occurred. The amount of reaction that has taken place may be quantified by two ways, in terms of the disappearance of substrate or the appearance of product. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of enzymes, in particular for “white biotechnology’’ where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes [39, 40]. Enzyme assays can be classified into two types namely, 1.2.1.1 Continuous assay: Continuous assay gives a continuous reading of activity. 1.2.1.1 Discontinuous assay: In discontinuous assay the reaction is stopped and then the concentration of substrates or product is determined. The flow chart of enzyme assay and different analytical techniques available are shown in Scheme 1.

Scheme. 1.3: Flow chart of enzyme assay classification. Enzyme assays leads to the development of new analytical enzyme experiments in different fields like enzyme fingerprinting, cocktail fingerprinting, microarray experiments, enzyme coupled reactions, Bio- and nano sensors, ELISA and isotopic labeling studies. The immense knowledge of genetic information that has been accumulated over the past decade has further claimed the importance of enzyme. 1.2.2. The nature and origin of diversity in enzyme assay: The major consideration in the design of an enzyme assay method is that the amount of enzyme present should be the only variable which affects the reaction rate. This condition is more likely to be met if the assay procedure ensures optimal

μmol of enzyme, in other words the number of mol of product formed, substrate used per mol of enzyme per minute. This may not correspond to the number of mol substrate converted per enzyme active site per minute since an enzyme molecule may contain more than one active site. If the number of active site per mol is known the activity may be expressed as the catalytic centre activity, which corresponds to mol substrate used, or product formed per minute per catalytic center. The nomenclature commission of the International Union of Biochemistry recommended katal as the enzyme unit which is abbreviated as kat, in this the unit of time is expressed in terms of second rather than minute with international system of unit (SI Units). One katal corresponds to the conversion of 1 mol of substrate per second. It is larger quantity than the enzyme Unit. 1 kat = 60 mol min-1^ = 6 × 10^7 Units 1 Unit = 1 μmol min-1^ = 16.67 nkat 1.2.5. Related terminology 1.2.5.1 Maximum velocity V max of the reaction:

In enzyme kinetics, V max is defined as the maximum initial velocity of the

enzyme catalysed reaction under the given conditions or highest possible rate when the enzyme is saturated with the substrate [51]. It is the limiting value that Vo approaches as the substrate concentration approaches infinity. 1.2.5.2 Turnover number Turnover number (also termed kcat and abbreviated as TN) is defined as the maximum number of molecules of substrate that an enzyme can convert to product per catalytic site per unit of time (a turnover rate) or it is also defined as the number of moles of product formed per mole of cofactor per unit time [41] and can be calculated as follows,

cat [ E ] T

K = V max

Where, V max is the maximum rate or velocity of the reaction and [ E ] T is the total

enzyme concentration. For example, carbonic anhydrase has a turnover number of 400,000 to 600,000 s−1, which means that each carbonic anhydrase molecule can produce up to 600, molecules of product per second [42]. 1.2.5.3 Specific activity Specific activity is a term used in measuring enzyme kinetics. It is defined as the amount of substrate the enzyme converts (reactions catalyzed), per mg protein in the enzyme preparation [43], per unit of time or in other words the enzymatic activity per unit mass of enzyme [44]. Specific activity is a measure of enzyme purity. The value becomes larger as an enzyme preparation becomes more pure, since the amount of protein (mg) is typically less, but the rate of reaction stays the same.

1.2.5.4 Michaelis–Menten equation ( KM )

The Michaelis-Menten equation is the elementary equation of enzyme kinetics, even though it is originally derived for the simplest case of an irreversible enzyme reaction. A relationship between the value of Michaelis – Menten constant

( KM )for an enzyme and the physiological concentration of substrate was mentioned

over two decades ago. Cleland stated a general rule that enzyme will function with reactant concentrations in the region of their apparent Michaelis constants or above, when at the pathway they are a part of is operating at full capacity; otherwise the catalytic potential of the enzyme is wasted. Apart from this Fersht explained that the tight binding of substrate implies a stable, and therefore unreactive substrate. He

Hence K (^) m is equal to the substrate concentration at which the reaction rate is

half its maximum value. In other words, if an enzyme has small value of (^) K (^) m , it

achieves its maximum catalytic efficiency at low substrate concentrations. Hence, the smaller the value of K (^) m , the more efficient is the catalyst. The value of K (^) m for an

enzyme depends on the particular substrate. It also depends on the pH of the solution and the temperature at which the reaction is carried out. For most enzymes (^) K (^) m lies

between 10-1^ and 10-7^ M. 1.2.5.5 Catalytic power ( (^) K (^) Pow )

Catalytic power is the rate of an enzyme catalyzed reaction divided by the rate of the uncatalyzed reaction. Or catalytic power is the degree to which enzymes increase the rate of a chemical reaction [50]. The catalytic power of enzymes is due to the precise molecular interactions that occur at the active site, which lower the energy barrier and enable formation of the transition state.

Pow (^) K m K = V max

Where, V max is the maximum velocity of the reaction and K (^) M is the Michaelis –

Menten constant. 1.2.5.6 Catalytic efficiency Enzymes are important for a variety of reasons, most significantly because they are involved in many chemical reactions that help us to maintain our daily lives. Increasing the reaction rate of a chemical reaction allows the reaction to become more efficient, and hence more products are generated at a faster rate. This is known as catalytic efficiency of enzymes, which, by increasing the rates, results in a more efficient chemical reaction within a biological system. Enzyme efficiency is measured by,

[ 0 ]

2 max E kcat = k = V

Some time enzyme efficiency is referred to as specificity constant’ Kkcat^ M. It is an useful indicator for comparing the relative rates of an enzyme acting on alternative, competing substrates. 1.2.6. Factors affecting the enzyme activity: The catalytic activity of an enzyme is measured in terms of the rate of the reaction catalyzed. The reaction conditions for the extent of activities of an enzyme should be optimal, so that maximum possible rate of reaction is measured. The optimum reaction conditions refer to the type of buffer and its concentration, the pH, effect of inhibitors on enzyme activity, activators and substrate concentration(s). 1.2.6.1 Enzyme concentration An enzyme molecule binds its substrate (s), catalyzes a reaction, and releases the product (s). Each step in this process requires time—time to receive the raw materials, do what needs to be done to them, and release the product. So each enzyme molecule requires x amount of time to produce one unit of product. The more the enzyme molecules are available, however, the more will be the product that can be produced in x time. Thus the more enzyme is available, the more quickly the substrate can be converted into product. In general, as enzyme concentration increases, there is a proportional increase in reaction rate. 1.2.6.2 Substrate concentration The rate of an enzyme reaction depends on the substrate concentration. Coenzymes behave as substrates from the point of view of reaction kinetics. With increasing coenzyme concentration, the rate of the reaction becomes greater until it reaches the limiting value [52]. If the optimum reaction rate V of an enzyme reaction