Enzymes: Definition, Mechanisms and Classification | Microbiology

In this article we will discuss about:- 1. Definition of Enzymes 2. Mechanism of Enzyme Action 3. Enzyme Kinetics 4. Allosteric Enzymes 5. Classification 6. Components.

Definition of Enzymes:

Enzymes are highly specialized proteins which act as catalyst of biological system. Louis Pasteur was the first to recognize the importance of enzymes while studying the fermentation process and denoted it as “ferment”-an integral part of living cells.

It was Edward Buchner who in 1897 extracted the enzyme from yeast cells, responsible for fermentation of sugar to alcohol. In 1926, James B. Sumner isolated and crystallized urease and also postulated that all enzymes are proteins. Today we know, this is true but with exception of Ribozymes which is a catalytic RNA.

Enzymes are the large globular proteins with molecular weight ranging from 13,000 to millions Dalton. The catalytic efficiency of an enzyme depends upon its three dimensional conformation.

Moreover, these biocatalysts are highly specific for the reaction as well as other conditions e.g. each enzyme has its own optimum pH, temperature, etc. for maximum perfor­mance. Some enzymes require an additional moiety known as cofactors for their biological activity.

Mechanism of Enzyme Action:

Enzyme is active in catalytic action of biochemical reaction. They act on substrate and forms a complex after interactions with the enzyme is called active center. The enzyme and substrate forms a complex at the active centre.

This binding action makes both enzyme and substrate stable. The interaction between substrate and enzyme may be either ionic bonds and hydrogen bonds or Van der Waal forces. The active sites of enzyme have some special groups such as NH₂ COOH, -SH etc. which bind the substrate though above bonds to form a transitional (intermediate) compound called enzyme-substrate complex (ES).

This reaction is exergonic and releases some energy which raises energy level of the substrate molecule.

Thus, activating the substrate molecule and the phenomenon is known as activation energy or energy of activation as shown in Fig. 12.10:

Types of Mechanisms of Enzymes:

There are two types of mechanisms involved to explain substrate-enzyme complex formation; lock and key theory (template model), and induced-fit theory.

(i) Lock and Key Theory:

Emil Fischer (1894) explained the specific action of an enzyme with a single substrate using a theory of Lock and Key analog (Fig. 12.11). According to this theory, reaction of sub-state and enzyme is analogous to lock and key.

Enzyme is analogous to key, where the geometrical configuration of socket is fixed. Similarly substrate has also got fixed geo­metrical configuration like that of key. A particular lock can be opened or closed by a particular key. According to the particular substrate can be found at active site of particular enzyme forming substrate-enzyme complex.

Enzyme-substrate complex remains in tight fitting and active sites of enzymes are complementary to substrate molecules. Subsequently, enzyme-substrate complexes result in the transformation of substrate into the product formation due to activity of reaction sites.

Since product has lower free energy, it is released. Enzymes are fixed to receive another molecule of substrate and thus enzyme activity continues. In this analogy, the lock is the substrate and the key is the enzyme. Only the correctly sized key (substrate) fits into the key hole (active site) of the lock (enzyme).

Smaller keys, larger keys, or incorrectly positioned teeth on keys (incorrectly shaped or sized substrate molecules) do not fit into the lock (enzyme).

Only the correctly shaped key opens a particular lock as shown in Fig. 12.11:

(ii) Induced Fit Theory:

In 1958, Koshland modified the Fischer’s model for the formation of an enzyme-substrate complex to explain the enzyme property more efficiently. According the Fischer’s model the nature of the active site of enzyme is rigid, but it is able to be pre-shaped to fit the substrate.

Koshland explains that the enzyme molecule does not retain its original shape and structure, but the contact of the substrate induces some geometrical changes in the active site of the enzyme molecule. The enzyme molecule is made to fit completely the configuration and active centers of the substrate. At the same time, other amino acid residues may become buried in the interior of the molecule.

This theory can be explained by a hypothetical illustration as shown in Fig. 12.12. The hydrophobic and charged group both are involved in substrate binding. A phosphoserine (-P) and SH group of cysteine residue are involved in catalysis.

Residue of the other amino acid such as lysine (Lys) and methionine (Met) are not involved in either binding or catalysis. In the absence of substrate, the substrate binding group and catalytic group are far apart from each other.

But the contact of the substrate induces a conformational changes in the enzyme molecule and aligns both the groups for substrate binding and catalysis. Simultaneously, the spatial orientation of the other region also changed. This causes the lysine and methionine much closer.

Enzyme Kinetics:

A number of approaches are now avail­able to study the mechanism of enzyme action including knowledge of complete 3-D structure, site directed mutagenesis and protein engineering, still central approach is to determine the rate of reaction and how it is affected by different experimental param­eters-more precisely- ‘The enzyme kinetics’.

Enzyme kinetics follows the principles of general chemical reaction kinetics; how­ever, show a distinctive feature of saturating (Fig. 12.13). At lower substrate concentra­tion, the initial reaction velocity is propor­tional to substrate concentration (1^st order reaction). Further increase in substrate con­centration does not affect the reaction rate and the latter became constant (zero order reaction).

In hypothetical one substrate reaction Michaelis-Menten theory, enzymes first combine with substrate to from enzyme-substrate (ES) complex.

This ES complex then breaks in second step to release free enzyme and product as:

According to above equations, initial velocity of complete reaction equals the breakdown of enzyme substrate complex. Hence,

Vo = Kb (ES) (3)

Where, Vo = initial velocity,

(ES) = concentration of enzyme substrate complex

However, neither of the two parameters can be determined directly, and an alternative expression of Vo is required. This can be done by considering 2″” order rate equation for formation of ES from E and S.

d(ES)/dt = Ka(E) (S) = Ka[ET] – (ES)] (S) (4)

Where, Ka= second order rate constant

(ET) = total enzyme concentration

(ES) = concentration of enzyme substrate complex

Since, starting of reaction is being considered, formation of (ES) by reaction (2) may be neglected because in the beginning for reaction in forward direction, when (S) is high and (P) is zero.

Rate equation for degradation of ES can be expressed as sum of two reactions-first reaction yielding the product and second reaction yielding E + S. Then,

-d (ES) /dt = Ka (ES) + Kb (ES) (5)

However, in the steady state, when rate of formation of ES is equal to its breakdown, then equation 4 = 5

Thus:

(ES)/dt = -d(ES)/dt (6)

Or, Ka [ET]-[ES] (S) = Ka (ES) + Kb (ES)

Or, [S] [(ET)-(ES)]/(ES) = Ka + Kb/Ka = Km (7)

(Ka+Kb/Ka), i.e. expressed as Km is known as Michaehs-Menten constant. Rearranging the equation 7 gives,

(ES) = (ET) (S)/Km + (S) (8)

The value of (ES), when expressed in equation:

Vo = Kb (ES)

Vo = Kb (ET) (S)/ Km + (S) (9)

When substrate concentration is high, all enzymes in system are present as ES complex.

Hence enzyme will be saturated, and reach the maximum velocity (V max), given as:

Vmax = Kb (ET)

Putting this value in equation 9, we get

Vo = Vmax (S)/Km + (S) (10)

This is Michaelis-Menten equation i.e. the rate equation for a one substrate enzyme catalyzed reaction considering special case when initial reaction rate is exactly half of Vmax, then by equation 10

Vmax/2 = Vmax (S)/Km + (s)

Or, Km + (S) = 2 (S)

Or, Km = (S)

Thus Michaelis-Menten constant is equal to substrate concentration at which initial reaction velocity is half of maximum velocity.

Km varies according to substrate, pH, and temperature and is not a fixed value.

Reciprocating the equation 10, we get

1/Vo=Km + (S)/ Vmax (S)

Or, Wo = Km/Vmax (S) 4- 1 / Vmax (11)

It is Lineweaver-Burk equation, which gives a straight line when 1/Vo is plotted against 1/ S (Fig. 12.14)

Effect of pH and tempera­ture on enzymatic action:

En­zymes have an optimum pH and temperature (or a range) at which they are maximally active. The side chains in amino acid play the central role. The side chains in the active site may act as weak acid or base and their alignment de­pends on the state of ionization. Hence ionization state is impor­tant to decide the conformation of enzyme molecule and hence the activity.

Dissimilarly, higher temperatures result in the rearrangement of bonds. Because of this the conformation of active sites gets altered and activity is affected. Enzymes with more cysteine residues are thermally more stable because of the formation of -S-S bridges between the peptide chains.

Allosteric Enzymes:

Allosteric (allos = other; steros = site) are regulatory enzymes, their catalytic activity depends upon non-covalent binding of a specific metabolite at a site other then active site. In many multi- enzyme system, the end product inhibits the pathway by inhibiting an enzyme of initial reaction. This phenomenon is generally known as feedback inhibition.

The inhibited enzymes are allosteric enzymes, which accommodate the final product on allosteric sites. Interaction of modulator (end product) with allosteric site alters the three dimensional structure of catalytic site and thus affects it’s activity. Modulators may inhibit, or induce the target allosteric enzyme, hence called as negative and positive modulators respectively.

Classification of Enzymes:

Generally, the name of enzymes ends with the suffix ase. Until recently enzymes were named arbitrarily after the name of the discoverer. However, this is not a practical approach, because some of the enzyme have completely irrelevant names. Therefore, now a new system has been proposed to combat this difficulty and to include all the new enzymes being discovered.

This system has been recommended by International Committee for Nomenclature of Enzymes in 1973. This system includes six major classes including other subclasses according to the type of reaction catalyzed (Table 12.2).

Now-a-days, enzymes are generally assigned two names: the recommended name and a systematic name. Recommended name is the common name. It is small and easy to use.

Systematic name is given according to the reaction in the six classes of the enzymes:

Enzyme Components:

Generally, enzymes are solely made up of proteins. But many enzymes also contain a non protein portion which is necessary for the activity of the enzyme. The protein part of enzyme in these types of enzymes is called apoenzyme and the nonprotein part is called cofactor. The cofactor may be a metal ion or an organic molecule (coenzyme). The complete enzyme with both cofactor and apoenzyme is called holoenzyme.

In the enzyme with metal ion as cofactor are ions such as Mg⁺², Fe⁺², Fe⁺³, K⁺, etc.. These may serve as primary catalytic centres or a bridging group to bind substrate and enzyme together. Such enzyme with a metal ions as cofactor are sometimes called metalloenzymes e.g. phosphotrans ferases (which have Mg⁺² as metal cofactor), cytochromes and catalase (which have Fe⁺² and Fe⁺³ metal ion).

The coenzyme, which is a complex organic molecule, may be any of the vitamins (trace organic molecule that are vital to the function of all cells and required in the diet). When the coenzyme is very tightly bound to the enzyme molecules, it is usually called prosthetic group. Some of the important coenzymes are nicotinamide derivatives, flavin adenine dinucleotide (FAD) derivatives.