Biologic polymers that catalyze the chemicalreactions Enzymes are neither consumed norpermanently altered as a consequence oftheir participation in a reaction With the exception of catalytic RNAmolecules, or ribozymes, enzymes areproteins In addition to being highly efficient, enzymesare also extremely selective catalysts
Class Reactions catalyzed Oxidoreductases oxidation-reduction Transferases transfer of moieties such as glycosyl,methyl, or phosphoryl groups Hydrolases catalyze hydrolytic cleavage Lyases add/remove atomsto/from a double bond Isomerases geometric or structuralchanges within a molecule Ligases joining together of twomolecules coupled to thehydrolysis of ATP
Cofactors can be subdivided into two groups: metalsand small organic molecules Cofactors that are small organic molecules are calledcoenzymes. Most common cofactor are also metal ions If tightly bound, the cofactors are called prostheticgroups Loosely bound Cofactors serve functions similar tothose of prosthetic groups but bind in a transient,dissociable manner either to the enzyme or to asubstrate
Tightly integrated into the enzyme structureby covalent or non-covalent forces. e.g;◦ Pyridoxal phosphate◦ Flavin mononucleotide( FMN)◦ Flavin adenine dinucleotide(FAD)◦ Thiamin pyrophosphate (TPP)◦ Biotin◦ Metal ions – Co, Cu, Mg, Mn, Zn Metals are the most common prostheticgroups
Enzymes that contain tightly bound metal ionsare termed – Metalloenzymes Enzymes that require metal ions as looselybound cofactors are termed as metal-activatedenzymes Metal ions facilitate◦ Binding and orientation of the substrate◦ Formation of covalent bonds with reaction intermediates◦ Interact with substrate to render them more electrophilicor nucleophilic
Coenzymes serve as recyclable shuttles—orgroup transfer agents—that transport manysubstrates from their point of generation to theirpoint of utilization. The water-soluble B vitamins supply importantcomponents of numerous coenzymes chemical moieties transported by coenzymesinclude hydrogen atoms or hydride ions, methylgroups (folates), acyl groups (coenzyme A), andoligosaccharides (dolichol).
Takes the form of a cleft or pocket Takes up a relatively small part of the totalvolume of an enzyme Substrates are bound to enzymes by multipleweak attractions The specificity of binding depends on theprecisely defined arrangement of atoms in anactive site The active sites of multimeric enzymes arelocated at the interface between subunits andrecruit residues from more than one monomer
Two models have been proposed to explain how anenzyme binds its substrate: the lock-and –key model andthe induced-fit model.Lock-and-Key Model of Enzyme-Substrate Binding In this model, theactive site of the unbound enzyme iscomplementary in shape to thesubstrate."lock and key model" accounted for theexquisite specificity of enzyme-substrate interactions, the impliedrigidity of the enzymes active sitefailed to account for the dynamicchanges that accompany catalysis.
Induced-Fit Model ofEnzyme-Substrate BindingIn this model, the enzymechanges shape onsubstrate binding. Theactive site forms a shapecomplementary to thesubstrate only after thesubstrate has been bound.When a substrateapproaches and binds toan enzyme they induce aconformational change, achange analogous toplacing a hand (substrate)into a glove (enzyme).
Enzymes are catalysts and increase the speed of a chemicalreaction without themselves undergoing any permanent chemicalchange. They are neither used up in the reaction nor do theyappear as reaction products. The basic enzymatic reaction can be represented as follows where E represents the enzyme catalyzing the reaction, S thesubstrate, the substance being changed, and P the product ofthe reaction. The mechanism of action of enzymes can be explained by twoperspectives- Thermodynamic changes Processes at the active site
All enzymes accelerate reaction rates byproviding transition states with a lowered∆G F for formation of the transition states.The lower activationenergy means that moremolecules have therequired energy toreach the transitionstate.
1. Catalysis by proximity – for the molecules to reactthey must come within bond-forming distance ofone another. When an enzyme binds substratemolecules at its active site, it creates a region ofhigh local substrate concentration. Enzyme-substrate interactions orient reactive groups andbring them into proximity with one another.2. Acid base catalysis – the ionizable functionalgroups of aminoacyl side chains of prostheticgroups contribute to catalysis by acting as acidsor bases
Catalysis by strain – enzymes that catalyze the lyticreactions involve breaking a covalent bond typicallybind their substrates in a configuration slightlyunfavorable for the bond that will undergocleavage Covalent Catalysis – involves the formation of acovalent bond between the enzyme and one ormore substrates which introduces a new reactionpathway whose activation energy is lower
In general, there are four distinct types ofspecificity: Absolute specificity - the enzyme willcatalyze only one reaction. Group specificity - the enzyme will act onlyon molecules that have specific functionalgroups, such as amino, phosphate andmethyl groups Linkage specificity - the enzyme will act on aparticular type of chemical bond regardless ofthe rest of the molecular structure Stereo chemical specificity - the enzyme willact on a particular steric or optical isomer.
Enzyme kinetics is the field of biochemistryconcerned with the quantitative measurement ofthe rates of enzyme-catalyzed reactions and thesystematic study of factors that affect theserates. The rate of a chemical reaction is described bythe number of molecules of reactant(s) that areconverted into product(s) in a specified timeperiod. Reaction rate is always dependent on theconcentration of the chemicals involved in theprocess and on rate constants that arecharacteristic of the reaction.
The catalytic action of an enzyme, its activity, is measured bydetermining the increase in the reaction rate under preciselydefined conditions—i. e., the difference between the turnoverof the catalyzed reaction and uncatalyzed reaction in aspecific time interval Normally, reaction rates are expressed as the change inconcentration per unit of time (mol 1–1 s–1). The catalytic activity of an enzyme isindependent of the volume, the unit used for enzymes isusually turnover per unit time, expressed in -katal (kat, mol s–1).However, the international unit U is still more commonly used(µmol turnover min–1;1 U = 16.7 nkat).
Numerous factors affect the reaction rate- Temperature The reaction rate increases with temperature to amaximum level, then abruptly declines withfurther increase of temperature Most animal enzymes rapidly become denaturedat temperatures above 40oC The optimal temperatures of the enzymes inhigher organisms rarely exceed 50 °C The Q10, or temperature coefficient, is the factorby which the rate of a biologic process increasesfor a 10 °C increase in temperature.
For mammals andother homoeothermicorganisms, changes inenzyme reaction rateswith temperatureassume physiologicimportance only incircumstances such asfever or hypothermia.
The rate of almost all enzyme-catalyzedreactions exhibits a significant dependenceon hydrogen ion concentration Most intracellular enzymes exhibit optimalactivity at pH values between 5 and 9. The relationship of activity to hydrogen ionconcentration reflects the balance betweenenzyme denaturation at high or low pH andeffects on the charged state of theenzyme, the substrates, or both.
Except for Pepsin, acid phosphatase and alkaline phosphatase, mostenzyme have optimum pH between 5 to 9
As the amount of enzyme isincreased, the rate of reactionincreases. If there are moreenzyme molecules than areneeded, adding additionalenzyme will not increase therate. Reaction rate thereforeincreases as enzymeconcentration increases butthen it levels off.
At lower concentrations, the active sites on mostof the enzyme molecules are not filled becausethere is not much substrate. Higherconcentrations cause more collisions between themolecules. The rate of reaction increases(Firstorder reaction). The maximum velocity of a reaction is reachedwhen the active sites are almost continuouslyfilled. Increased substrate concentration afterthis point will not increase the rate. Reactionrate therefore increases as substrateconcentration is increased but it levels off (Zeroorder reaction)
The shape of thecurve that relatesactivity to substrateconcentration ishyperbolic.First orderreactionZero order reaction
The Michaelis-Menten equation is aquantitative description of the relationshipamong the rate of an enzyme- catalyzedreaction [v1], the concentration of substrate[S] and two constants, V max and km (whichare set by the particular equation). The symbols used in the Michaelis-Mentenequation refer to the reaction rate [v1],maximum reaction rate (V max), substrateconcentration [S] and the Michaelis-Mentenconstant (km).
The dependence of initial reaction velocity on [S] and Km may beillustrated by evaluating the Michaelis-Menten equation underthree conditions.(1) When [S] is much less than km, the term km + [S] is essentiallyequal to km. Since V max and km are both constants, their ratio isa constant (k). In other words, when [S] is considerably belowkm, V max is proportionate to k[S]. The initial reaction velocitytherefore is directly proportionate to [S].
(2) When [S] is much greater than km, the termkm + [S] is essentially equal to [S]. Replacing km+ [S] with [S] reduces equation to Thus, when [S] greatly exceeds km, the reactionvelocity is maximal (V max) and unaffected byfurther increases in substrate concentration.
(3) When [S] = km Equation states that when [S] equals km, theinitial velocity is half-maximal. Equation alsoreveals that km is a constant and may bedetermined experimentally from—thesubstrate concentration at which the initialvelocity is half-maximal.
A Linear Form of the Michaelis-MentenEquation Is Used to determine km & V maxInvert factorand simplify
A plot of 1/vi as y as a function of 1/[S] as xtherefore gives a straight line whose yintercept is 1/ V max and whose slope is km /V max. Such a plot is called a double reciprocalor Lineweaver-Burk plot
The Michaelis constant Km is the substrateconcentration at which vi is half the maximalvelocity (Vmax/2) attainable at a particularconcentration of enzyme It is specific and constant for a given enzymeunder defined conditions of time , temperatureand p H Km determines the affinity of an enzyme for itssubstrate, lesser the Km for is the affinity andvice versa Km value helps in determining the true substratefor the enzyme.
Inhibitors are chemicals that reduce the rate ofenzymic reactions The are usually specific and they work at lowconcentrations They block the enzyme but they do not usuallydestroy it Many drugs and poisons are inhibitors ofenzymes in the nervous system Inhibitors of the catalytic activities of enzymesprovide both pharmacologic agents and researchtools for study of the mechanism of enzymeaction.
Irreversible inhibitors: Combine with the functionalgroups of the amino acids in the active site,irreversibly Reversible inhibitors: These can be washed out of thesolution of enzyme by dialysis.Applications of inhibitors Negative feedback: end point or end productinhibition Poisons snake bite, plant alkaloids and nerve gases Medicine antibiotics, sulphonamides, sedatives andstimulants
Classification Inhibitors can be classified based upon theirsite of action on the enzyme, on whether they chemically modify theenzyme, or on the kinetic parameters they influence.
A competitive inhibitor Has a structure similar to substrate(Structural Analog) Occupies active site Competes with substrate for active site Has effect reversed by increasing substrateconcentration Vmax remains same but Km is increased
Drug Enzyme Inhibited Clinical UseDicoumarol Vitamin K EpoxideReductaseAnticoagulantSulphonamide Pteroid Synthetase AntibioticTrimethoprim DihydrofolatereductaseAntibioticPyrimethamine DihydrofolatereductaseAntimalarialMethotrexate DihydrofolatereductaseAnticancerLovastatin HMG Co A Reductase Cholesterol LoweringdrugAlpha Methyl Dopa Dopa decarboxylase AntihypertensiveNeostigmine Acetyl Cholinesterase Myasthenia Gravis
Noncompetitive inhibitors bind enzymes at sitesdistinct from the substrate-binding site Generally bear little or no structural resemblanceto the substrate Binding of the inhibitor does not affect binding ofsubstrate Formation of both EI and EIS complexes istherefore possible The enzyme-inhibitor complex can still bindsubstrate, its efficiency at transforming substrateto product, reflected by Vmax, is decreased.
In the presence of acompetitive inhibitor,Vmax can still be reachedif sufficient substrate isavailable, one-half Vmax requires ahigher [S] than beforeand thus Km is larger.With noncompetitiveinhibition, enzyme rate(velocity) is reduced forall values of [S],includingVmax and one-half Vmax butKm remains unchanged
Cyanide inhibits cytochrome oxidase Fluoride inhibits Enolase and hence glycolysis Iodoacetate inhibits enzymes having SHgroups in their active sites BAL ( British Anti Lewisite, dimercaprol) isused as an antidote for heavy metalpoisoning◦ Heavy metals act as enzyme poisons by reactingwith the SH groups◦ BAL has several SH groups with which the heavymetal ions bind and thereby their poisonous effectsare reduced
Irreversible inhibition Structural analog of the substrate isconverted to more effective inhibitor with thehelp of enzyme to be inhibited The new product irreversibly binds to theenzyme and inhibits further reaction e.g;◦ Ornothine decarboxylase – is irreversibly inhibitedby difluormethyl ornithine, as a result multiplicationof parasite is arrested . Used against trypanosomein sleeping sickness
Allopurinol is oxidized by Xanthine oxidase toalloxanthine which is a strong inhibitor ofXanthine Oxidase Aspirin action is based on suicide inhibition◦ Acetylates a serine residue in the active center of cyclooxygenase . Thus PG synthesis is inhibited soinflammation subsides Disulfiram – used in treatment of alcoholism◦ Drug irreversibly inhibits the enzyme aldehydedehydrogenase preventing further oxidation ofacetaldehyde which produces sickening effects leadingto aversion to alcohol.
Allosteric means ―other site‖EActive siteAllosteric siteThese enzymes have onecatalytic site (Active site)where the substrate bindsand another separateallosteric site where themodifier binds.The allosteric sites may ormay not be physicallyadjacent.The binding of the modifiermay enhance (Positivemodifier) or inhibit(Negative modifier) theenzyme activity.
Inhibitor is not a substrate analogue Partially reversible, when excess substrate isadded Km is usually increased(K series enzymes) Vmax is reduced(V series enzymes) When the inhibitor binds the allostericsite, the configuration of the active site ischanged so that the substrate can not bindproperly. Most allosteric enzymes possess quaternarystructure.
Inhibitor moleculeSubstratecannot fit into the active siteInhibitor fits into allosteric siteWhen the inhibitor ispresent it fits into itssite and there is aconformational changein the enzyme moleculeThe enzyme’s molecularshape changesThe active site of thesubstrate changesThe substrate cannotbind with the substrate.The reaction slowsdownWhen the inhibitor concentration diminishes theenzyme’s conformation changes back to its activeform. This is not competitive inhibition but it isreversible
Phosphofructokinase This enzyme an active sitefor fructose-6-phosphate molecules to bind withanother phosphate group It has an allosteric site for ATP molecules, theinhibitor When the cell consumes a lot of ATP the level ofATP in the cell falls No ATP binds to the allosteric site ofphosphofructokinase The enzyme’s conformation (shape) changes andthe active site accepts substrate molecules
Phosphofructokinase The respiration pathway accelerates and ATP (thefinal product) builds up in the cell As the ATP increases, more and more ATP fits intothe allosteric site of the phosphofructokinasemolecules The enzyme’s conformation changes again andstops accepting substrate molecules in the activesite Respiration slows down
Cell processes consist of series of pathwayscontrolled by enzymes A B C D E FeFeDeCeA eBEach step is controlled by a different enzyme (eA, eB, eC etc)This is possible because of enzyme specificity
The first step (controlled by eA) is oftencontrolled by the end product (F) Therefore negative feedback is possibleA B C D E FInhibitioneFeDeCeA eBThe end products are controlling their ownrate of productionThere is no build up of intermediates (B,C,D and E).Usually such end productinhibition is effected allosterically
Accumulatedproduct bindsat a site otherthan the activesite to bringaboutconformationalchanges, so asto inhibit thebinding of thesubstrate. Therate of reactiondeclines.
Regulation of enzyme activity is needed to maintainhomeostasis Many human diseases, including cancer, diabetes,cystic fibrosis, and Alzheimers disease, arecharacterized by regulatory dysfunctions triggered bypathogenic agents or genetic mutations The flux of metabolites through metabolic pathwaysinvolves catalysis by numerous enzymes, activecontrol of homeostasis is achieved by regulation ofonly a small number of enzymes Regulatory enzymes are usually the enzymes that arethe rate-limiting, or committed step, in a pathway,meaning that after this step a particular reactionpathway will go to completion.
Two General Mechanisms that Affect EnzymeActivity: 1) control of the overall quantities of enzymeor concentration of substrates present 2) alteration of the catalytic efficiency of theenzyme
The overall synthesis and degradation of aparticular enzyme, also termed its turnovernumber, is one way of regulating the quantity ofan enzyme. The amount of an enzyme in a cell can beincreased by increasing its rate ofsynthesis, decreasing the rate of itsdegradation, or both.
Induction -an increase caused by an effectormolecule This can manifest itself at the level of geneexpression, RNA translation, and post-translational modifications The actions of many hormones and/or growthfactors on cells leads to an increase in theexpression and translation of "new" enzymes notpresent prior to the signal. Inducible enzymes of humans include tryptophanpyrrolase, aminotransferase, enzymes of the ureacycle, HMG-CoA reductase, Glucokinase andcytochrome P450.
An excess of a metabolite may curtailsynthesis of its cognate enzyme viarepression. Both induction and repression involve ciselements, specific DNA sequences locatedupstream of regulated genes, and trans-acting regulatory proteins.
The degradation of proteins constantly occurs in the cell Protein degradation by proteases is compartmentalized in thecell in the lysosome (which is generally non-specific), or inmacromolecular complexes termed proteasomes Degradation by proteasomes is regulated by a complexpathway involving transfer of a 76 aa polypeptide, ubiquitin,to targeted proteins. Ubiquination of protein targets it fordegradation by the proteasome. Proteolytic degradation is an irreversible mechanism dysfunctions of the ubiquitin-proteasome pathway contributeto the accumulation of aberrantly folded protein speciescharacteristic of several neurodegenerative diseases
Zymogen Activation- Certain proteins are synthesizedand secreted as inactive precursor proteins known asproproteins. The proproteins of enzymes are termedproenzymes or zymogens. Selective proteolysis converts a proprotein by one ormore successive proteolytic "clips" to a form thatexhibits the characteristic activity of the matureprotein, eg, its enzymatic activity. The digestive enzymes pepsin, trypsin, andchymotrypsin (proproteins = pepsinogen,trypsinogen, and chymotrypsinogen, respectively),several factors of the blood clotting and blood clotdissolution cascades, complement and Kinin systemare examples of Zymogen activation.
Compartmentation ensures metabolicefficiency & simplifies regulation Segregation of metabolic processes into distinctsubcellular locations like the cytosol or specializedorganelles (nucleus, endoplasmic reticulum, Golgiapparatus, lysosomes, mitochondria, etc.) isanother form of regulation
These enzymes function through reversible, non-covalent binding of a regulatory metabolite at a siteother than the catalytic, active site. When bound, these metabolites do not participate incatalysis directly, but lead to conformational changesin one part of an enzyme that then affect the overallconformation of the active site (causing an increase ordecrease in activity, hence these metabolites aretermed allosteric activators or allosteric inhibitors Most of the Allosteric enzymes have multiple subunits
Cooperativity - in relation to multiple subunitenzymes, changes in the conformation of one subunit leadsto conformational changes in adjacent subunits. Thesechanges occur at the tertiary and quaternary levels of proteinorganization and can be caused by an allosteric regulator. Homotropic regulation - when binding of one molecule to amulti-subunit enzyme causes a conformational shift thataffects the binding of the same molecule to another subunit ofthe enzyme. Heterotropic regulation - when binding of one molecule to amulti-subunit enzyme affects the binding of a differentmolecule to this enzyme (Note: These terms are similar tothose used for oxygen binding to hemoglobin)
Allosteric enzymes do exhibit saturation kinetics at high[S], but they have a characteristic sigmoidal saturationcurve rather than hyperbolic curve when vo is plottedversus [S] (analogous to the oxygen saturation curves ofmyoglobin vs. hemoglobin). Addition of an allostericactivator (+) tends to shift the curve to a more hyperbolicprofile (more like Michaelis-Menten curves), while anallosteric inhibitor (-) will result in more pronouncedsigmoidal curves. The sigmoidicity is thought to resultfrom the cooperativity of structural changes betweenenzyme subunits (again similar to oxygen binding tohemoglobin).
NOTE: A true Km cannot be determined forallosteric enzymes, so a comparativeconstant like S0.5 or K0.5 is used.
Another common regulatory mechanism is thereversible covalent modification of an enzyme.Phosphorylation, whereby a phosphate istransferred from an activated donor (usually ATP)to an amino acid on the regulatory enyme, is themost common example of this type of regulation.Frequently this phosphorylation occurs inresponse to some stimulus (like a hormone orgrowth factor) that will either activate or inactivatetarget enzymes
Phosphorylation of one enzyme can lead tophosphorylation of a different enzyme which in turnacts on another enzyme, and so on. An example ofthis type of phosphorylation cascade is theresponse of a cell to cyclic AMP and its effect onglycogen metabolism. Use of a phosphorylation cascade allows a cell torespond to a signal at the cell surface and transmit theeffects of that signal to intracellular enzymes (usuallywithin the cytosol and nucleus) that modify a cellularprocess. This process is generically referred to as being part ofa signal transduction mechanism
A-forms, most active B-forms, less active
Dietary cholesterol decreases hepatic synthesis ofcholesterol, this feedback regulation does notinvolve feedback inhibition. HMG-CoA reductase, the rate-limiting enzyme ofcholesterol genesis, is affected, but cholesteroldoes not feedback-inhibit its activity. Regulation in response to dietary cholesterolinvolves curtailment by cholesterol or acholesterol metabolite of the expression of thegene that encodes HMG-CoA reductase (enzymerepression).