Enzymes
Enzymes are the proteins which catalyze biochemical reactions without being consumed in it. The enzymes are very specific in their action and thus separate enzymes exist for different reactions with some exceptions.
Properties of enzymes:
Enzymes are proteins and hence possess physical and chemical properties.
1) They are globular proteins with molecular weight of 13,000 - 500,000
2) They are soluble in water or salt solution formation of colloidal systems.
3) They act as amphoteric colloidal electrolytes and can be precipitated at their isoelectric points and with optimum concentration of salts.
4) They show electrophoretic mobility. They become inactive when heated to above 75°c due to denaturation.
5) They are inactivated by the alteration of pH and undue dilution. When enzymes are injected into blood stream, they produce antibodies.
Classification of enzymes:
Enzymes are classified into six classes and the standard nomenclature has been suggested for them by the commission on biochemical nomenclature of the “International Union of Biochemistry”. According to standard nomenclature, each enzyme is assigned a four digit code following the abbreviation EC (enzyme commission).
The various classes and subclasses of enzymes are as follows:
A) Oxidoreductase:
It catalyzes redox reactions involving transfer of hydrogen/oxygen atoms between molecules. These enzymes include:
1) Oxidase: It facilitates transfer of oxygen atom.
2) Oxygenases: It transfers O2 atom from molecular oxygen.
3) Peroxidases: It transfers electron to peroxidases
4) Dehydrogenase: It facilitates hybrid transfer.
Glucose oxidase is a popular example of this class of enzyme.
General reaction: AH2X + X → A + XH2
B) Transferase:
They catalyze the transfer of an atom or group of atoms like acyl-alkyl-glycosyl. For instance, glycosyl transferases catalyze transfer of sulphate group and amino transferases catalyze transfer of amino group. Asparyl amino transferase is a suitable example of this class.
General reaction: A - X + B → B - X + A
C) Hydrolases:
Hydrolases catalyze hydrolytic reactions or their reverse reactions. It include esterase, amidases, lipases, proteases, glycosidase etc. Amidase also help in peptide synthesis and esterase help in formation of esters. Similarly glycohydrolases are used for the synthesis of glycoprotein. Most of the hydrolytic enzymes are important for the pharmaceutical industries. Chymosin and rennin are suitable examples of this class.
General reaction: A - B + H2O → AH + BOH
D) Lyses:
Lyses catalyze reactions involved in the removal of a group of atoms from the substrate molecule. These enzymes include aldolases, decarboxylases, dehydralases and some pectinases. Histidine ammonia lyase is suitable example of this class.
General reaction: A - B → A + B
E) Isomerase:
Isomerase catalyzes the formation of isomers of substitute molecule and therefore includes epimerases and intermolecular transferases. Xylose isomerase is a suitable example of this class.
General reaction: X → X*
F) Ligases/ syntetase:
Ligases catalyze the formation of covalent bonds between two substrate molecules with the help of ATP or GTP molecules which provide the energy for reactions. Glutathione syntase is a suitable example of this class.
General reaction: A + B ↔ A - B
Enzyme specificity:
Enzymes, the organic catalyst differs from organic catalysts in their extraordinary specificity. The various types of enzyme specificity are:
A) Reaction specificity:
An enzyme catalyzes only a very few reactions frequently only one.
For e.g. Arginase and urease attacks arginine hydrogen peroxidase and urea respectively. Most enzymes can catalyze same type of reactions with several structurally related substrates. Reactions with alternate substrates take place if they are present in high concentration and also depend on their affinity to enzymes.
For example, Histidine acts on Histidine and it also acts on tryptophan when it is present in high concentration.
For example: In presence of acetyl CoA, the enzyme oxaloacetate is present then another specific enzyme i.e. malate dehydrogenase will catalyze it and forms different products i.e. malate.
B) Optical specificity:
Enzymes show absolute optical specificity for at least a portion of substrate molecules. The glycosidase which catalyze the hydrolysis of glycosidic bonds between sugars and alcohols, are highly specific to for the sugar portion but relatively non specific for the alcohol portion. Many substrates form 3 bands with the enzymes. This three point attachment contains and confirms symmetry on the symmetric molecules
C) Bond specificity:
A particular enzyme acts on a particular chemical bond.
Ex: glycosidase on glycosidase, alcohol dehydrogenase on alcohol, pepsin and trypsin on peptide.
D) Group specificity:
Particular enzyme acts on a particular chemical bond.
Ex: Chymotrypsin hydrolyses peptide bond in which the carbonyl group is contributed by the aromatic amino acid, such as tyrosine, tryptophan etc.
E) Oxidoreductase function in biosynthetic process and tends to use NADPH as reductant but those which function in degradative process tend to use NAD+ as oxidant.
Multienzyme complex:
A few examples of complex enzyme systems are known to exist. These are not independent molecules but occur as aggregates in a mosaic pattern, involving different enzymes. Pyruvic acid of E.coli is one such example.
This complex molecule has a molecular weight 4, 80,000 and consists of three enzymes, 24 moles of pyruvate decarboxylases, 24 moles of dihydrolipoic dehydrogenase and 8 submit of lipoyl reductase transacetylase. Each component of this complex enzyme is so arranged as to provide an efficient coupling of this individual reactions catalyzed by these enzymes.
Enzyme - substrate models:
The enzyme - substrate models defines the catalytic site of the enzyme. Some restricted region of the enzyme which is concerned with the process of catalysis is termed as active site or catalytic site. These models are described basically of two types:
A) Lock and Key model or rigid template model:
This model was originally proposed by Fischer. According to this model, the active site already exists in proper confirmations even in absence of substrate. Thus the active site, by itself, provides a rigid, preshaped template fitting with the shape and size of the substrate. The substrate fits into the active site of an enzyme as the key fits into the lock and hence it is called as lock and key model.
This model proposes that substrate binds with rigid preexisting, template of the active site and provide an additional group for binding other ligands. This model cannot explain change in presence of allosteric modulations. This model is still useful for understanding certain properties of enzymes
B) Koshland induced fit model or flexible model:
Due to the restrictive nature of lock and key model, another model was proposed by Koshland in 1963 which is known as induced fit model. The important feature of this model is the flexibility of the active site.
According to this model, the substrate induces a conformational change in the enzyme. This aligns amino acid residues or other groups on the enzymes in the correct spatial orientation for substrate binding, catalysis or both. At the same time, other amino acid residues may be buried in the interior of the molecule. Hydrophobic groups and charged groups both are involved in substrate binding. In the absence of substrate, the catalytic and the substrate binding groups are at several bond distances from one another. Approach of the substrate induces a conformational change in the enzyme. At the same time, the spatial orientation of other regions is also attested.
Enzyme induction:
The induction of enzyme starts with the synthesis of enzymes. From DNA, RNA is synthesizes by RNA polymerase. This process is called as transcription. The transcription process is similar to the replication of DNA, where new strand is formed from old DNA strand. From m-RNA, enzymes are formed by the translation process i.e. protein synthesis. All enzymes are proteins but all proteins are not enzymes. The best example of enzyme induction is lactose operon. Lactose utilization in E.coli was known to be controlled by three enzymes, whose genes are adjacent to each other on chromosome. One of these genes is β- galactosidase, which hydrolyses lactose and other β- galactosidase. When bacteria is grown with glucose as the sole carbon source, the levels of the lactose - utilizing enzymes are very low., with less then one molecule of β- galactosidase per cell on an average. However substitution of lactose for glucose in the medium leads to rapid enzyme induction. Removal of lactose from the culture slows down further synthesis of enzyme molecule.
Lactose Operon:
The lactose operon consists of three linked structural genes that encode enzymes of lactose utilization plus adjacent regulatory sites. The three structural genes - z, y and a encode β- galactoside, permease and thiogalactoside transacetylase. In the presence of an inducer, all three enzymes accumulate simultaneously, but to different levels. Lactose itself leads to the induction of lactose operon but the true intracellular inducer is allolactose. Transcription of the three structural genes is initiated near an adjacent site, the operator. Transcription yields a single polycistronic m-RNA. The i gene product is a macromolecular repressor which in the active form binds to the operator, blocking transcription.
The repressor also has a binding site for inducer. This repressor inactivation stimulates transcription of z, y and a, because dissociation of the repressor inducer complex from the operator removes a steric block to binding of RNA-polymerase at the initiation site. Thus, the introduction of lactose activates synthesis of gene products or enzymes involved in catabolism by removing a barrier to their transcription.
Enzyme regulation:
Types of regulations:
Enzymes are regulated in two ways:
1) Substrate level regulation:
Regulation that depends directly on the interaction of substrates and products with the enzymes is called as substrate level regulation. Increase in substrate concentration results in higher reaction rates and conversely, increase in product concentration reduces the rate at which the substrate is converted to product.
Example: Phosphorylation of glucose to generate glucose-6-phosphate. This reaction is the first step in glycolytic pathway. The enzyme hexokinase that catalyzes this reaction is inhibited by its product, glucose-6-phosphate. If utilization of glucose-6-phosphate is blocked for any reason, it will accumulate, inhibiting the hexokinase reaction and slowing down further entry to glucose in pathway.
The substrate level regulation is important control mechanism in cells, but it is not sufficient for regulation of most reactions or reaction sequences. For most pathways, enzymes are regulated by another mechanism called as allosteric regulation.
2) Allosteric regulation:
Let us consider a pathway for better understanding of this mechanism.
E1 E2 E3 E4
This pathway indicates that a cell converts some precursors A into some final product P via a series of intermediates B, C and D, in a sequence of reactions catalyzed respectively by enzymes E1, E2, E3. Product P could be an amino acid needed by the cell for protein synthesis and A could be some common cellular component that serves as the starting point for the specific reaction sequence leading to P.
If allowed to continue, this pathway has the capacity to convert large amount of A to P, with adverse effects resulting from a depletion of A or an excessive accumulation of P. Clearly, the best interest of the cell are served when the pathway is functioning neither to its maximum rate nor at some constant rate, but, at a rate that is carefully turned to the cellular need of P. Such regulation is possible in the above pathway because, the product P is a specific inhibitor of E1, the enzyme that catalyzes the first reaction in the sequence. This phenomenon is called as feedback inhibition. It is one of the most common mechanisms used by the cells to ensure that reaction sequences are adjusted to cellular needs.
Example: Synthesis of isoleucine amino acid from threonine amino acid. In this case, the first enzyme in the pathway, threonine deaminase is regulated by the concentration of isoleucine in the cell. If isoleucine is being used by the cell, its concentration will be low and under these conditions, threonine deaminase gets activated and the pathway functions to produce more isoleucine. If the need for isoleucine decreases, isoleucine will begin to accumulate and the increase in concentration will decrease the activity of threonine deaminase and hence to a reduce rate of isoleucine synthesis.
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