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Structural Biochemistry/Enzyme 1 Structural Biochemistry/Enzyme Overview Enzymes are macromolecules that help accelerate (catalyze) chemical reactions in biological systems. Some biological reactions in the absence of enzymes may be as much as a million times slower. Virtually all enzymes are proteins, though the converse is not true and other molecules such as RNA can also catalyze reactions. The most remarkable characteristics of enzymes are their ability to accelerate chemical reactions and their specificity for a particular substrate. Enzymes take advantage of the full range of intermolecular forces (van der waals interactions, polar interactions, hydrophobic interactions and hydrogen bonding) to bring substrates together in most optimal orientation so that reaction will occur. Also, enzymes can be inhibited by specific molecules by called competitive, uncompetitive, and noncompetitive inhibitors. Catalysis happens at the active site of the enzyme. It contains the residues that directly participate in the making and breaking of bonds. These residues are called the catalytic groups. Although enzymes differ widely in structure, specificity, and mode of catalysis a number of generalizations concerning their active sites can be made: 1. The 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 dequence may interact more strongly than adjacent residues in the 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 question of why enzymes are so big. Nearly all enzymes are made up of more than 100 amino acid residues. The "extra" amino acids serve as a scaffold to creat the three dimensional active site from the amino acids that are far apart in the primary structure. In many proteins the remaining amino acids also constitute regulatory sites, sites of interaction with other proteins, or channels to bring the substrate to the active sites. 3. Active sites are unique microenvironments. 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 microenvironment of the cleft enhances the binding of substrates as well as catalysis. Nevertheless, the cleft may also contain polar residues. Certain of these polar residues acquire special properties essential for substrate bidning or catalyis. 4. Substrates are bound to enzymes by multiple weak interations. Stated above 5. The specificity of binding depends on the precise defined arrangement of atoms in the active site. Because the enzyme and the substrate interact by means of short-range forces that require close contact, a substrate must have a matching shape to fit into the site. However, the active site of some enzymes assume a shape that is complementary to that of the substrate only after the substrate is bound. This process of dynamic recognition is called induced fit. Enzymes are highly specific and may require cofactors for catalysis. A cofactor is a non-protein chemical compound bound to a protein; there are 2 types of cofactors: Metals and organic/metalloorganic (which are derived from vitamins). An example of a metal cofactor is zinc and the enzyme, carbonic anhydrase, tightly binds the zinc at the active site. The process involves binding water to carbon dioxide and deprotonating it into carbonic acid. Then the carbonic acid becomes a bicarbonate ion due to the displacement of water. Catalysts can fasten the reaction speed by lowering the activation energy (not the transition state) of the process. The active site is a location on the enzyme which has complementary shape to the substrate. It is also where the amino acids with a complementary charge, polarity and shape to the ligand are. The enzyme function and catalysis result from the ability to stabilize the transition state in a chemical reaction. The transition state is the highest energy species in a reaction. It is a transitory molecular structure that is no longer the substrate but is not yet the product. It is the most seldom occupied species along the reaction pathway. The difference in free energy between the transition state and the substrate is called the Gibbs free energy of activation or

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Page 1: Structural Biochemistry/Enzymelibvolume7.xyz/.../biochemistry/enzymes/enzymestutorial1.pdf · 2014-11-24 · Structural Biochemistry/Enzyme 2 simply the activation energy. Thus we

Structural Biochemistry/Enzyme 1

Structural Biochemistry/Enzyme

OverviewEnzymes are macromolecules that help accelerate (catalyze) chemical reactions in biological systems. Somebiological reactions in the absence of enzymes may be as much as a million times slower. Virtually all enzymes areproteins, though the converse is not true and other molecules such as RNA can also catalyze reactions. The mostremarkable characteristics of enzymes are their ability to accelerate chemical reactions and their specificity for aparticular substrate. Enzymes take advantage of the full range of intermolecular forces (van der waals interactions,polar interactions, hydrophobic interactions and hydrogen bonding) to bring substrates together in most optimalorientation so that reaction will occur. Also, enzymes can be inhibited by specific molecules by called competitive,uncompetitive, and noncompetitive inhibitors.Catalysis happens at the active site of the enzyme. It contains the residues that directly participate in the making andbreaking of bonds. These residues are called the catalytic groups. Although enzymes differ widely in structure,specificity, and mode of catalysis a number of generalizations concerning their active sites can be made:1. The active site is a three dimensional cleft or crevice formed by groups that come from different parts of theamino acid sequence - residues far apart in the amino acid dequence may interact more strongly than adjacentresidues in the sequence.2. The active site takes up a relatively small part of the total volume of an enzyme. Most of the amino acid residuesin an enzyme are not in contact with the substrate, which raises the question of why enzymes are so big. Nearly allenzymes are made up of more than 100 amino acid residues. The "extra" amino acids serve as a scaffold to creat thethree dimensional active site from the amino acids that are far apart in the primary structure. In many proteins theremaining amino acids also constitute regulatory sites, sites of interaction with other proteins, or channels to bringthe substrate to the active sites.3. Active sites are unique microenvironments. In all enzymes of known structure, substrate molecules are bound to acleft or crevice. Water is usually excluded unless it is a reactant. The nonpolar microenvironment of the cleftenhances the binding of substrates as well as catalysis. Nevertheless, the cleft may also contain polar residues.Certain of these polar residues acquire special properties essential for substrate bidning or catalyis.4. Substrates are bound to enzymes by multiple weak interations. Stated above5. The specificity of binding depends on the precise defined arrangement of atoms in the active site. Because theenzyme and the substrate interact by means of short-range forces that require close contact, a substrate must have amatching shape to fit into the site. However, the active site of some enzymes assume a shape that is complementaryto that of the substrate only after the substrate is bound. This process of dynamic recognition is called induced fit.Enzymes are highly specific and may require cofactors for catalysis. A cofactor is a non-protein chemical compoundbound to a protein; there are 2 types of cofactors: Metals and organic/metalloorganic (which are derived fromvitamins). An example of a metal cofactor is zinc and the enzyme, carbonic anhydrase, tightly binds the zinc at theactive site. The process involves binding water to carbon dioxide and deprotonating it into carbonic acid. Then thecarbonic acid becomes a bicarbonate ion due to the displacement of water.Catalysts can fasten the reaction speed by lowering the activation energy (not the transition state) of the process. Theactive site is a location on the enzyme which has complementary shape to the substrate. It is also where the aminoacids with a complementary charge, polarity and shape to the ligand are.The enzyme function and catalysis result from the ability to stabilize the transition state in a chemical reaction. The transition state is the highest energy species in a reaction. It is a transitory molecular structure that is no longer the substrate but is not yet the product. It is the most seldom occupied species along the reaction pathway. The difference in free energy between the transition state and the substrate is called the Gibbs free energy of activation or

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Structural Biochemistry/Enzyme 2

simply the activation energy.Thus we can see the key to how enzymes operate: Enzymes accelerate reactions by decreasing the activation energy.The combination of substrate and enzyme creates a reaction pathway whose transition state is lower than that of thereaction in the absence of the enzyme. Because the activation energy is lower more substrate molecules have theenergy required to reach the transition state.It is important to note that enzymes have evolved specifically to recognize the transition states of chemical reactions.Therefore, enzymes do not bind to any reactive species before the species have actually begun to react; enzymes onlyrecognize and bind the transition states of such species. In fact, if enzymes were to bind to the reactants of a reaction"on sight", or immediately, this would result in an even higher activation energy than before! For this reason,enzymes recognize only the transition state and bind to reactive species only when this high-energy state has beenachieved. The fact that enzymes can recognize structures as specific and short-lived as transition states is a testamentto their incredible specificity and efficiency.Each enzyme is optimized for a particular reaction transition state. This ensures that enzymes will not compete witheach other and hinder cellular reactions instead of help them. Enzyme inhibition occurs when the activity of a givenenzyme is disrupted or interrupted in some fashion. Inhibitors can be molecules that have a similar shape, structure,or charge to the substrate in question so that the active site of an enzyme will "mistake" the inhibitor for thesubstrate. This affects the affinity of the enzyme for the substrate, as well as the rate of the overall reaction. Severaltypes of inhibition can occur in the cell; more detailed explanations on these can be found in the correspondingsections.Because of the active sites, enzymes are highly specific catalysts. These catalysts are governed by the ability tolower the free energy of thermodynamics to overcome transition states. The Michaelis-Menten Model describes thekinetic properties of many enzymes.The interaction between the substrate and the enzyme helps accelerate the reaction, and the specificity of enzymesresult in minimal side reactions.It is of great importance to note that an enzyme cannot alter the laws of thermodynamics and consequently cannotalter the equilibrium of the reaction. The amount of product formed for a reaction utilizing an enzyme is alwaysequal to the amount of product form of the same reaction occuring in the same reaction mixture without the enzyme.The enzyme just allows the reaction to reach its equilibrium faster. The equilibrium position is a function only of thefree-energy difference between reactants and products.

Lock and Key ModelThe "lock and key" model was first proposed by an organic chemist named Emil Fischer in 1894. In this model, the"lock" refers to an enzyme and the "key" refers to its complementary substrate. Each enzyme has a highly specificgeometric shape that is complementary to its substrate. In order to activate an enzyme, its substrate must first bind tothe active site on the enzyme. Only then will a catalytic reaction take place. However, like a lock and a key, theenzyme and substrate shape must be complementary and fit perfectly. Designed by evolution the active site forenzymes is generally highly specific in its substrate recognition and has the ability to distinguish betweensterioisomers.

Induced FitAccording to the Lock and Key Model, the geometric shape of both enzymes and substrates can not be changed as they are both predetermined. Thus, the binding of the substrate to the enzymes active site does not alter the shape of the enzyme. While this theory helped explain the specificity of the enzyme, it does not explain the stability of the transition state for it would require more energy to reach the transition state complex. Thus the induced fit model was proposed in which enzymes like proteins are flexible. The concept of induced fit is that when a substrate binds

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Structural Biochemistry/Enzyme 3

to the active site of an enzyme, there is a conformational change and structural adaptation that makes this bindingsite more complementary and tighter. In essence the substrate does not simply bind to a rigid active site but insteadthe macromolecules, weak interaction forces, and hydrophobic characteristics on the enzyme surface mold into a

precise formation so that there is an induced fit where the enzyme can perform maximum catalytic function.

Transition State Theory

Stabilization of the transition state by an enzyme.

Transition state theory states that in an enzymecatalysis, the enzyme binds more strongly to its"transition state complex rather than its ground statereactants." In essence, the transition state is morestable. The stabilization of the transition state lowersthe activation barrier between reactants and productsthus increasing the rate of reaction or enzymaticactivity as this will favor the increase of formation ofthe transition state complex.In the transition state theory, the mechanism ofinteraction of reactants is irrelevant. However, thecolliding molecules that take place in the reaction musthave sufficient amount of kinetic energy to overcome the activation energy barrier in order to react. In many cases,temperature, pH, or enzymes can be changed to facilitate the stabilization of the transition state as well asstatistically increasing the probability for molecules colliding and forming the transition state complex. For abimolecular reaction such as Sn2, a transition state is formed when the two molecules’ old bonds are weakened andnew bonds begin to form or the old bonds break first to form the transition state and then the new bonds form after.The theory suggests that as reactant molecules approach each other closely they are momentarily in a less stable statethan either the reactants or the products.

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Methods1. Some catalysts provide a charge to a molecule to make it more attractive to other reactants. Acids are an example

for this kind of catalyst. They give the reacting species a positive charge to attract the negative or partiallynegative reactant, increasing the chance for the two species to collide and react.

2. Some catalysts increase the local concentration of reactants so that they are more likely to collide.3. Some catalysts may modify the shape of one reactant to be more susceptible to other molecule.

Enzymatic Strategies and Examples1. Covalent Catalysis - Through the course of catalysis, a powerful nucleophile is temporarily attached to a part ofthe substrate. The nucleophile is contained in the active site. A proteolytic enzyme chymotrypsin is an excellentexample of this strategy.2. General Acid/Base Catalysis - Water often acts as a donor or acceptor, but in Acid/base catalysis, the moleculewhich donates or accepts a proton is NOT water. This strategy incorporates base and acid catalysis to shortenreaction times. In the case of Chymotrypsin, the enzyme uses a histidine residue as a base catalyst to enhance thenucloephilicity of serine analogous to how hisitidne residue in carbonic anhydrase facilitates the removal of a protonfrom a zinc bound water molecule to yield hydroxide.3. Catalysis by approximation - This method involves reactions where the molecule react with two substrates. Thetwo substrates are brought together to one area and this increases the rate of the reaction. NMP kinase for example,brings tow nucleotides together to improve the transferring of phosphoryl groups.4. Metal Ion Catalysis - Metal ions can be involved as a catalyst in many different ways. Zinc can help the formationof a nucleophile. It makes the pka of water change from approximately 14 to 7, which allows it to be protonated atneutral pH. It can also stabilize negative charges by acting as an electrophile in a complex. Metal ions are also usedto increase the binding energy of substrates, holding them together. A metal ion may also serve as a bridge betweenthe enzyme and substrate acting as a cofactor in cases of NMP kinases.

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Structural Biochemistry/Enzyme 5

Enzyme's Cofactors for Activity

The succinate dehydrogenase complex showing several cofactors, including flavin,iron-sulfur centers and heme.

The catalytic activity of enzymes dependson the presence of small molecules calledcofactors. The role of the catalytic activityvaries with the enzyme and its cofactors. Ingeneral, those cofactors can executechemical reactions which cannot beperformed by the standard 20 amino acids.An enzyme without cofactor is calledapoenzyme, however the one withcompletely catalytically active is calledholoenzyme.Cofactors can be divided into two individualgroups: Metal and Coenzymes. Metals areimportant for enzymes because they aremolecular assistants that play a vital role insome of the enzymatic reactions that fuel thebody metabolism. They also act to stabilizethe shapes of enzymes. For example, ironhelps the protein hemoglobin transportoxygen to organs in the body and copperhelps superoxide dismutase in sopping updangerous free radicals that accumulate inside the cells. Coenzymes are small organic molecules that often derivedfrom vitamins. Coenzymes can be either tightly or loosely bound to the enzyme. Tightly bound ones are calledprosthetic groups, while loosely bound coenzymes are like substrates and products, bind to the enzyme and getreleased from it. Enzymes that use the same coenzymes often perform catalysis by the similar mechanisms.

Enzyme Classification

Class Type of Reduction Examples

Hydrolases Catalyze hydrolysis reactions Estrases Digestive enzymes

Isomerases Catalyze isomerization (changing of a molecule into its isomer) Phospho hexo isomerase, Fumarase

Ligases Catalyze bond formation coupled with ATP hydrolysis. Citric acid synthetase

Lyases Catalyze a group elimination in order to form double bonds (or a ring structure). Decarboxylases Aldolases

Oxidoreductases Catalyze oxidation-reduction reactions Dehydrogenases Oxidases

Transferases Catalyze the transfer of functional groups among molecules. Transaminase Kinases

The classification of an enzyme is shown within the table as it's class and the type of reduction the enzyme goes through. An example of a name is glucose phosphotransferase. In this reaction ATP transfers one of it's phosphates to glucose: ATP + D-glucose -> ADP + D-glucose 6-phosphate. Since this process "transfers" a phosphate group to glucose, it is within the classification of transferases, hence the name "glucose phosphotransferase." Since many enzymes have common names that do not refer to their function or what kind of reaction they catalyze, a enzyme classification system was established. There are six classes of enzymes that were created with subclasses based on what they catalyze so that enzymes could easily be named. Depending on the type of reaction catalyzed, an enzyme can have various names. These classes are Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, and

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Ligases. This is the internation classification used for enzymes. For example, a common oxidoreductase isdehydrogenase. Dehydrogenase is known as an enzyme that oxidizes a substrate and transferring protons. Enzymesare normally used for catalyzing the transfer of functional groups, electrons, or atoms. Since this is the case, they areassigned names by the type of reaction they catalyze. This allowed for the addition of a four-digit number that wouldprecede EC(Enzyme Commission) and each enzyme could be identified. The reaction that an enzyme catalyzes mustbe know before it can be classified.Oxidoreductases catalyze oxidation-reduction reactions where electrons are transferred. These electrons are usuallyin the form of hydride ions or hydrogen atoms. When a substrate is being oxidized it is the hydrogen donor. Themost common name used is a dehydrogenase and sometimes reductase will be used. An oxidase is referred to whenthe oxygen atom is the acceptor.Transferases catalyze group transfer reactions. The transfer occurs from one molecule that will be the donor toanother molecule that will be the acceptor. Most of the time, the donor is a cofactor that is charged with the groupabout to be transferred.Hydrolases catalyze reactions that involve hydrolysis. This cases usually involves the transfer of functional groups towater. When the hydrolase acts on amide, glycosyl, peptide, ester, or other bonds, they not only catalyze thehydrolytic removal of a group from the substrate but also a transfer of the group to an acceptor compound. Theseenzymes could also be classified under transferaes since hydrolysis can be viewed as a transfer of a functional groupto water as an acceptor. However, as the acceptor's reaction with water was discovered very early, it's considered themain function of the enzyme which allows it to fall under this classification.Lyases catalyze reactions where functional groups are added to break double bonds in molecules or the reversewhere double bonds are formed by the removal of functional groups.Isomerases catalyze reactions that transfer functional groups within a molecule so that isomeric forms are produced.These enzymes allow for structural or geometric changes within a compound. Sometime the interconverstion iscarried out by an intramolecular oxidoreduction. In this case, one molecule is both the hydrogen acceptor and donor,so there's no oxidized product. The lack of a oxidized product is the reason this enzyme falls under this classification.The subclasses are created under this category by the type of isomerism.Ligases are used in catalysis where two substrates are litigated and the formation of carbon-carbon, carbon-sulfide,carbon-nitrogen, and carbon-oxygen bonds due to condensation reactions. These reactions are couple to the cleavageof ATP.

The Michaelis-Menten ModelThe Michaelis-Menten model is used to describe the kinetic properties of many enzymes. In this model, anenzyme(E)combines with a substrate(S)to form an enzyme-substrate(ES)complex, and proceed to form aproduct(P)or to dissociate into E and S.

The rate of formation of product,V0, can be calculated by the Michaelis-Menten equation:

Vmax is the reaction rate when the enzyme is completely saturated with substrate. KM is the Michaelis constant,which is the substrate concentration at the half of the maximum reaction rate. The kinetic constant kcat isa called theturnover number, which is the number of substrate molecules converted into produce per unit time at a singlecatalytic site when the enzyme is saturated with substrate. It often count for most enzyme between 1 and 104persecond.

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Allosteric enzymes is an important class of enzymes. Its catalytic activity can be regulated. It has multiple activesites which display cooperativity, as evidenced by a sigmoidal dependence of reaction velocity on substrateconcentration. We also find that K max is the substrate concentration in which the overall reaction rate at thatparticular time is half of V max. V max on the other hand, is the maximum reaction rate in which the active site iscompletely saturated with substrate. As a result of this physical characteristic, we see that no matter how muchsubstrate is consequently added, the relative rate of the reaction remains unchanged as additional substrate do notcontribute to any kinetic interaction with binding the active site. The affinity also eventually does not change as moresubstrate is increased and the reaction goes towards equilibrium.

Replicative DNA polymeraseThere have been studies of the three multi-subunit DNA polymerase enzymes in the nucleus. This provides insightsinto the makeup of the replication machinery in eukaryotic cells. The first DNA polymerase structure to by solvedcrystallographically was the Klenow fragment of E. coli DNA polymerase I. This crystallization revealed a structurethat was likened to the palm, fingers, and thumb of a right hand. Studies of the Klenow fragment showed that DNAwas bound within the cleft and that the fingers and thumb architecture is conserved in many of the polymerasefamilies. The polymerase active site residues are located in the palm domain. The fingers are important fornucleotide binding, and the thumb domain binds the DNA.

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DNA polymerases adds nucleotides to the 5' endof a strand of DNA <Allison, Lizabeth A.>

<Allison, Lizabeth A. Fundamental MolecularBiology. Blackwell Publishing. 2007. p.112>. If a

mismatch is accidentally incorporated, thepolymerase is inhibited from further extension.

Proofreading removes the mismatched nucleotideand extension continues.

References

http:/ / www. tutorvista. com/ content/ biology/ biology-iii/cellular-macromolecules/ enzymes-classification. php

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Article Sources and Contributors 9

Article Sources and ContributorsStructural Biochemistry/Enzyme  Source: http://en.wikibooks.org/w/index.php?oldid=2005790  Contributors: Asitangg, Babelovs, Calibuon, Ccatolico, Danagustin, Eln001, Hmhoang, Jamespou, Jek027, Jewon, Jyl059, Najili, Ndang817, PV Equals nRT, Panic2k4, Rgao, RiQuach, Rmsamawi, Salaviza, Sasy, Slwee, T4truong, Vilau, 3 anonymous edits

Image Sources, Licenses and ContributorsFile:Induced_fit_diagram.svg  Source: http://en.wikibooks.org/w/index.php?title=File:Induced_fit_diagram.svg  License: Public Domain  Contributors: User:FvasconcellosImage:Enzyme catalysis delta delta G.png  Source: http://en.wikibooks.org/w/index.php?title=File:Enzyme_catalysis_delta_delta_G.png  License: unknown  Contributors: Original uploaderwas Zephyris at en.wikipediaImage:Succinate Dehydrogenase 1YQ3 Electron Carriers Labeled.png  Source:http://en.wikibooks.org/w/index.php?title=File:Succinate_Dehydrogenase_1YQ3_Electron_Carriers_Labeled.png  License: GNU Free Documentation License  Contributors: Richard Wheeler(Zephyris)File:Michaelis-Menten-Vereinfachung.svg  Source: http://en.wikibooks.org/w/index.php?title=File:Michaelis-Menten-Vereinfachung.svg  License: unknown  Contributors: User:Matthias M.Image:DNA polymerase.svg  Source: http://en.wikibooks.org/w/index.php?title=File:DNA_polymerase.svg  License: Creative Commons Attribution-Sharealike 2.5  Contributors:User:Madprime

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