This action might not be possible to undo. Are you sure you want to continue?
The Active Sites of Enzymes And Some Common Features:
The substrates are bound to a specific region of the enzyme called the active site. Most enzymes are highly selective in the substrates that they bind. Indeed, the catalytic specificity of enzymes depends in part on the specificity of binding. The active site of an enzyme is the region that binds the substrates. It also contains the residues that directly participate in the making and breaking of bonds. These residues are called the catalytic groups. In essence, the interaction of the enzyme and substrate at the active site promotes the formation of the transition state. Although enzymes differ widely in structure, specificity, and mode of catalysis, a number of generalizations concerning their active sites can be stated: 1. The active site is a three-dimensional cleft formed by groups that come from different parts of the amino acid sequence indeed, residues far apart ins the sequence may interact more strongly than adjacent residues in the amino acid sequence. 2. The active site takes up a relatively small part of the total volume of an enzyme. Most of the amino acid residues in an enzyme are not in contact with the substrate, which raises the intriguing question of why enzymes are so big. Nearly all enzymes are made up of more than 100 amino acid residues, which gives them a mass greater than 10 kd and a diameter of more than 25 Å. The "extra" amino acids serve as a scaffold to create the three dimensional active site from amino acids that are far apart in the primary structure. Amino acids near to one another in the primary structure are often sterically constrained from adopting the structural relations necessary to form the active site. In many proteins, the remaining amino acids also constitute regulatory sites, sites of interaction with other proteins, or channels to bring the Substrates to the active sites. 3. Active sites are clefts or crevices. In all enzymes of known structure, substrate molecules are bound to a cleft or crevice. Water is usually excluded unless it is a reactant. The nonpolar character of much of the cleft enhances the binding of substrate as well as catalysis. Nevertheless, the cleft may also contain polar residues. In the nonpolar microenvironment of the active site, certain of these polar residues acquire special Page 1 Biotechnology dept.
Because the enzyme and the substrate interact by means of shortrange forces that require close contact.ENZYME ACTION properties essential for substrate binding or catalysis. van der Waals forces. Substrates are bound to enzymes by multiple weak attractions. 5. The modulator is a normal metabolite which when bind with allosteric site Page 2 Biotechnology dept. The noncovalent interactions in ES complexes are much weaker than covalent bonds. ES complexes usually have equilibrium constants that range from 10-2 to 10-8 M. Van der Waals forces become significant in binding only when numerous substrate atoms simultaneously come close to many enzyme atoms. Koshland.. To the regulatory site certain compounds called modifier or allosteric effectors are going to bind. The word ‘allosteric’ means ‘another site’. these enzymes also referred as Allosteric enzymes. The directional character of hydrogen bonds between enzyme and substrate often enforces a high degree of specificity. Jr. has proved to be highly stimulating and fruitful. in 1958. . and hydrophobic interactions mediate reversible interactions of biomolecules. 4. electrostatic interactions. Hence. which have energies between -50 and -110 kcal mol-1. The internal positions of these polar residues are biologically crucial exceptions to the general rule that polar residues are exposed to water. The specificity of binding depends on the precisely defined arrangement of atoms in an active site. we now know that enzymes are flexible and that the shapes of the active sites can be markedly modified by the binding of substrate. This process of dynamic recognition is called induced fit. As the meaning an allosteric enzymes have two sites. corresponding to free energies of interaction ranging from about -3 to -12 kcal mol-1. the enzyme and substrate should have complementary shapes. However. a substrate must have a matching shape to fit into the site. hydrogen bonds. The active sites of some enzymes assume a shape that is complementary to that of the transition state only after the substrate is bound. Regulatory site of enzymes: The enzymes having the other than the active site that site is called as regulatory site of enzyme. Emil Fischer's analogy of the lock and key expressed in 1890. as was postulated by Daniel E. namely regulatory site and a catalytic site or active site.
The two opposing effects create a preferred separation distance characterizing each atom — the van der Waals radius — which can be thought of as defining the surface of the molecule. this can add up to tens of kcal mol–1. the van der Waals attractive energy for two methylene groups is estimated to be 2 kcal mol–1. if two molecules approach each other too closely. The energy in van der Waals interactions is relatively low. in enzyme ligandcomplexes. CATALYTIC TACTICS AND INTERMOLECULAR FORCES The some more detailed way some of the molecular tactics and intermolecular forces that enzymes use to implement their catalytic strategies.ENZYME ACTION of an enzyme modified its kinetic properties. This dipolar interaction is a very shortrange phenomenon. Allosteric and substrate binding sites may or may not physically adjacent. VAN DER WAALS INTERACTIONS Molecules are clouds of electrons in which nuclei are embedded. weakening with the sixth power of the distance. . Allosteric enzymes are usually contain multiple sub units and the catalytic and allosteric site may be located on different subunit. the modulators may be either stimulatory or inhibitory in nature. For instance. which averages to zero in a nonpolar molecule as electrons explore the space available to them (defined by the wave function). At any instant in time. When two molecules approach each other. but can accumulate to become quite significant. these transient dipole moments become correlated. where there is a large degree of surface contact. However. resulting in attractive electrostatic interaction. there is a dipole moment. Just like the enzyme’s active site specific for its substrate. The allosteric site is specific for modulator. electrostatic repulsion dominates. Page 3 Biotechnology dept. • • • • The allosteric site may be one or more in an enzyme.
ENZYME ACTION HYDROGEN BONDS “Classical” hydrogen bonds occur when a hydrogen atom that is covalently bonded to an electronegative atom (oxygen or nitrogen) is only a short distance from an electronegative atom that has a lone electron pair (again. as well as groups provided from substrates or prosthetic groups. The enzyme ribonuclease forms hydrogen bonds with the uridine component of the substrate. The ionic character of hydrogen bonds makes their strength distance dependent but less sensitive (though not insensitive) to direction. as evidenced by the high rates of proton. has been invoked as acid or basic catalysts in enzymology. Estimates for the free energy of formation of typical hydrogen bonds in aqueous Hydrogen Bonds between an Enzyme and Substrate. . Page 4 Biotechnology dept. Two categories of proton transfer reactions may be considered: those to or from noncarbon atoms (heteroatoms) and those to or from carbon atoms. usually oxygen or nitrogen). The transfer of protons to or from heteroatoms is generally facile. ACID/BASE CATALYSIS Acid/base catalysis the participation of an enzyme-bound functional group in transferring protons is a nearly ubiquitous feature of enzyme reactions. Every ionizable protein functional group. suggesting that the proton s from the partial positive charge that resides on a hydrogen atom in the polar covalent bond to the electronegative donor atom and the partial negative charge that resides on the lone pair of electronegative acceptor atom. Experimentally determined distances between heavy atoms involved in a hydrogen bond show that they are slightly closer than the distance expected from their van der Waals radii.
threonine. esterases. constellations of charged amino acid side chains can create regions of positive or negative electrostatic potential.ENZYME ACTION ELECTROSTATICS The interaction of charges can have a strong influence on reaction rates. the enzyme breaks the reaction into partial reactions in which the group is transferred first to the enzymatic nucleophile and then to the ultimate acceptor. Hydrogen bonds and acid/base catalysis are electrostatic effects that are important enough to be treated in their own sections. Electrostatic fields due either to side-chain charges or dipole moments from the protein structure can interact with bound molecules. The side chains of serine. causing changes in pKa values and stabilizing developing charges in transition states and intermediates. but Page 5 Biotechnology dept. Nucleophilic catalysis is frequently involved in group transfer reactions. to name just a few. though little is gained by increasing the length of the helix beyond ~2 turns. beyond the catalytic effects of specifically positioned protons. These effects are sensitive to the dielectric constant that intervenes between the charge/dipole and the substrate/ transition state/intermediate. ELECTROPHILIC CATALYSIS The 20 genetically encoded amino acids provide an abundance of nucleophilic groups but no electrophilic groups (with the exception of a proton donated by an acid. cysteine. Similar effects can be produced by the amide groups of asparagine and glutamine side chains or the dipoles of the peptide groups in the protein backbone. and tyrosine have been identified as nucleophiles in a variety of enzymes. including proteases. . However. in which a group on one substrate is transferred to an acceptor site on another substrate. glutamate. NUCLEOPHILIC CATALYSIS Enzymes sometimes use nucleophilic side chains or nucleophilic prosthetic groups to form an adduct with the substrate or a substrate fragment. histidine. The combined effects of peptide dipoles can be concentrated by an helix. Rather than catalyzing the direct reaction between the two substrates. kinases. some argue that this is the primary origin of the catalytic power of enzymes. and can be amplified by excluding water . aspartate. and lyases.
It is the job of the electrophilic catalyst to stabilize this new lone pair. usually by delocalization in the case of organic prosthetic groups. Nonetheless. which subsequently reacts with the next substrate. necessitating the recruitment of electrophilic prosthetic groups. a large number of enzymes do redox chemistry. or metal ions. in a large number of cases a substrate oxidizes or reduces the enzyme directly. Emil Fisher proposed a model to explain the great specificity of enzymes. Electrophilic catalysts speed reactions by forming an adduct with a substrate and activating them for the loss of an electrophilic-leaving group such as CO2 or H+. or by direct charge neutralization in the case of metals. In 1890. the calytic site of the enzyme by itself is complementory in shape to that of the substrate. When an electron-deficient group leaves a molecule for instance. REDOX CATALYSIS A large number of biochemical reactions are net redox reactions. According to this concept. . Consequently. These can be either small organic molecules. in a deprotonation reaction it will leave behind an electron pair. Page 6 Biotechnology dept. THEORES OF MECHANISM OF ENZYME ACTION.ENZYME ACTION this is in a category of its own). there are many reactions that do not involve a net redox change but do require a reactive intermediate to be generated by redox chemistry. many biochemical reactions require catalysis by an electrophile. In addition to these. He explained the interaction between substrate and enzymes in terms of “Lock and Key”. Although some redox reactions occur directly between two substrates. Lock-and-Key Model of Enzyme-Substrate Binding. such as pyridoxal phosphate.
If the substrate [“Key”] become slightly modified. Koshland (1958) proposed the induced fit hypothesis. Page 7 Biotechnology dept. The enzymic reaction is possible if the substrate matches the active center as the key fits the lock. Induced-Fit Model of Enzyme-Substrate Binding. with the catalytic sites aligned and freely accessible.ENZYME ACTION Lock-and-Key Model of Enzyme-Substrate Binding. Also. the presence of a substrate at the active site may exclude water molecules and thus make the region more non-polar. In order to account for the above observation. can come into contact with the region of catalytic activity and some degree of reaction take place. the active site of the unbound enzyme is complementary in shape to the substrate. In this model. Therefore. in the lock and key mechanism. But not in the free enzyme during the binding of the substrate. it no longer fits the active center [“Lock”] and no reaction takes place. whether part of an appropriately bound substrate or not. a suitable reacting group. the structure of the substrate may be complementary to that of the active site in the enzyme substrate complex. which results in the required matching of structures. the active site is always structurally intact. According to this hypothesis. . Both of these factors could be responsible for some degree of change in the tertiary structure. Thus.
It is also possible that access to the catalytic groups of free enzyme might be blocked. which results in all the relevant groups in the substrate and enzyme coming together. in this mechanism. Only when the binding group of the substrate is recognized by the corresponding site of the enzyme and the binding process proceeds. Changing the pH changes the H bonds. FACTORS AFFECTING ENZYME ACTIVITY. The induced fit hypothesis requires that the active site be flexible and the substrate be rigid.ENZYME ACTION Induced-Fit Model of Enzyme-Substrate Binding. does conformational change take place. different catalytic components might be separated by a considerable margin in the free enzyme. Enzyme action is greatest within a narrow range of pH because all the enzymes are active. The active site forms a shape complementary to the substrate only after the substrate has been bound. the substrate can no longer bind to the active site and so enzyme action decreases. . allowing the enzyme to wrap itself around the substrate. Therefore. and thus the shape of the active site. minimizing the risk of a chance collision of a reactive group with both of them. the enzyme changes shape on substrate binding. Enzymes have an Page 8 Biotechnology dept. Also. thereby bringing together the corresponding catalytic sites and reacting groups. In this model. EFFECT OF pH: Enzymes are affected by changing in pH. The most favorable pH value the point where the enzyme is most active is known as the optimum pH.
EFFECT OF TEMPERATURE. Page 9 Biotechnology dept. This is because the amino acid side chains in the active site may act as weak acid and basses with critical functions that depend on their maintaining a certain state of ionization. For an enzyme molecule to be active as a catalyst. the reaction which it catalyses slow down and ultimately stops. . This specific ionic substrate would also exist over a limited pH range. or the coenzyme.ENZYME ACTION optimum pH at which their activity decreases. Some enzymes lose their activity when frozen. In some cases. is also capable of being ionized. At low temperature enzyme action is low because the movement of molecule is low. particularly at the active site Like all proteins. This state of affairs would obviously prevail only within a limited pH range.Ionization of the enzyme. substrate. The pH effect is mainly based on two factors: 1. enzyme molecules possess numerous ionizable groups whose state of ionizability depends on pH. Above which enzymes become denatured and lose activity which in turn is due to loss of the secondary and tertiary structure of the protein. EFFECT OF SUBSTRATE CONCENTRATION. However. the rate of an enzyme catalysed reaction increases as the temperature is raised. which would depend on the pka values of the groups concerned. like the enzyme. Ionization of the substrate. Thus optimum temperature of an enzyme may be defined as the temperature at which its activity is maximum. certain of these groups must be ionized while others must remain unionized. 2. As the enzyme inactivated. this happens only up to certain temperature commonly known as optimum temperature. Like most chemical reactions. Thus in such cases also it would be responsible to assume that only one ionic form of the substrate molecules might be capable of undergoing the reaction.
an enzyme that catalyzes the hydrolytic cleavage of peptide bonds. resulting in a stronger interaction. At later reaction stages. It does not catalyze a direct attack of water on the peptide bond. Ser195 is linked to His57 and Asp102 in a hydrogen-bonding network referred to as the catalytic triad. In nearly all cases. In the deacylation phase. allowing the His residue to act as an enhanced general base that can remove the proton from the Ser195 hydroxyl group. The reaction thus has two distinct phases.191) is a protease. In chymotrypsin. This enhanced interaction increases the pKa of His57 from ~7 (for free histidine) to 12. the ester linkage is hydrolyzed and the nonacylated enzyme regenerated. which determines the velocity of enzyme reactions. In the acylation phase. a very short-lived tetrahedral Page 10 Biotechnology dept. a subtle change in conformation compresses the hydrogen bond between His57 and Asp102.ENZYME ACTION Substrate concentration is one of the most important factor. This protease is specific for peptide bonds adjacent to aromatic amino acid residues (Trp. The reaction catalyzed by this enzyme illustrates the principle of transition-state stabilization and also provides a classic example of general acid-base catalysis and covalent catalysis. when initial velocity is plotted against substrate concentration. called a low-barrier hydrogen bond. Bovine pancreatic chymotrypsin (Mr 25. As the Ser195 oxygen attacks the carbonyl group of the substrate. a transient covalent acyl-enzyme intermediate is formed. a section of a rectangular hyperbola is obtained. instead. . The three-dimensional structure of chymotrypsin with functional groups in the active site emphasized. protonating the amino group in the displaced portion of the substrate (the leaving group). His57 also acts as a proton donor. the peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme. The Chymotrypsin Mechanism. Tyr). Chymotrypsin enhances the rate of peptide bond hydrolysis by a factor of at least 109. When a peptide substrate binds to chymotrypsin. Phe. Deprotonation prevents development of a very unstable positive charge on the Ser195 hydroxyl and makes the Ser side chain a stronger nucleophile.
Page 11 dept.ENZYME ACTION intermediate is formed in which the carbonyl oxygen acquires a negative charge. it reduces the energy required to reach these states. This charge. This is an example of the use of binding energy in catalysis. One of these hydrogen bonds (contributed by Gly193) is present only in this intermediate and in the transition states for its formation and breakdown. is stabilized by hydrogen bonds contributed by the amide groups of two peptide bonds in the chymotrypsin backbone. forming within a pocket on the enzyme called the oxyanion hole. Biotechnology .
ENZYME ACTION Page 12 dept. Biotechnology .
Lysozyme cleaves the (_1n4) glycosidic COO bond between the two types of sugar residue in the molecule. Biotechnology . Nacetylmuramic acid (Mur2Ac) and N-acetylglucosamine (GlcNAc).296) is a monomer with 129 amino acid residues. respectively. Six residues of the alternating Mur2Ac and GlcNAc in peptidoglycan bind in the active site. This was the first enzyme to have its threedimensional structure determined. that between a Mur2Ac residue in site D and a GlcNAc residue in site E. Lysozyme is a natural antibacterial agent found in tears and egg whites. More than five decades of lysozyme investigations have provided a detailed picture of the structure and activity of the enzyme. The reaction is a nucleophilic substitution. and F. with OOH from water replacing the GlcNAc at C-1 of Mur2Ac.ENZYME ACTION THE LYSOZYME MECHANISM. a carbohydrate found in many bacterial cell walls. often referred to as NAM and NAG. The hen egg white lysozyme (Mr 14. However. and an interesting story of how biochemical science progresses. D. The key catalytic amino acid residues in the active site are Glu35 and Asp52. The structure revealed four stabilizing disulfide bonds and a cleft containing the active site. With the active site residues identified and a detailed structure of the enzyme available. Only one of the bound glycosidic bonds is cleaved. in binding sites labeled A through F. definitive evidence for a particular mechanism eluded investigators for nearly Page 13 dept. by David Phillips and colleagues in 1965. the path to understanding the reaction mechanism seemed open in the 1960s. The substrate of lysozyme is peptidoglycan. Model building has shown that the lactyl side chain of Mur2Ac cannot be accommodated in sites C and E. restricting Mur2Ac binding to sites B.
The carbocation is stabilized by resonance involving the adjacent ring oxygen. The alternative mechanism involves two consecutive direct-displacement (SN2-type) steps. as well as by electrostatic interaction with the negative charge on the nearby Asp52. In step 1 . Asp52 attacks C-1 of Mur2Ac to displace the GlcNAc. In step 2 . the departing GlcNAc is protonated by general acid catalysis by Glu35. There are two chemically reasonable mechanisms that could generate the observed product of lysozymemediated cleavage of the glycosidic bond. water attacks at C-1 of Mur2Ac to displace the Asp52 and generate product. Page 14 dept.water attacks at C-1 of Mur2Ac to yield the product. located in a hydrophobic pocket that gives its carboxyl group an unusually high pKa. Glu35 acts as a general acid to protonate the departing GlcNAc. Phillips and colleagues proposed a dissociative (SN1 type) mechanism in which the GlcNAc initially dissociates in step 1 to leave behind a glycosyl cation (a carbocation) intermediate.ENZYME ACTION four decades. In step 2 . As in the first mechanism. Biotechnology . In this mechanism.
ENZYME ACTION Page 15 dept. Biotechnology .
is released. which is stabilized by the glutamate residue from a highly conserved motif VI (ENV). Biotechnology . the C5 position of the cytosine gets activated and becomes capable of performing a nucleophilic attack on the methyl group bound to the cofactor substrate AdoMet. Thereby. Then. yielding a covalent intermediate between the base and the enzyme. The enzyme facilitates the nucleolytic attack on the C6 atom by a transient protonation of the cytosine ring at the endocyclic nitrogen atom N3. The covalent complex between the methylated base and the DNA is resolved by deprotonation at the C5 position. Page 16 dept. which leads to the elimination of the cysteinyl group and the reestablishment of aromaticity. 1994) on the C6 position of the target cytosine. the methylated base together with the cofactor product.ENZYME ACTION THE DNA METHYL TRANSFERASE ACTIVITY. DNA methylation is initiated by a nucleophilic attack of an SH group from a catalytic cysteine residue located in the conserved amino acids sequence motif IV (GPPC) (Kumar et al. S-adenosyl-Lhomocysteine. According to this mechanism. the second arginine residue in motif VIII (RXR) plays an important role in the catalytic mechanism of DNA m5C methyltransferases. In addition to the residues already mentioned.
Page 17 dept. Biotechnology .ENZYME ACTION FIGURE: Schematic picture of the catalytic mechanisms proposed for DNA MTases and RNA MTases.
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue listening from where you left off, or restart the preview.