Module 2: Enzymes Objectives 1. To know the distinguishing properties of enzymes. 2. To know what is enzyme kinetics. 3.

To identify the different types of enzyme inhibition. 4. To know the mechanisms of enzyme action. 5. To know how are enzymes regulated. 6. To know how are enzymes used in clinical diagnosis.

San Beda College of Medicine Mendiola Biochemistry E.C. 2.7.1.1

IUB Enzyme Nomenclature

E.C. 2.7.1.1 First digit class 2: transferase Second digit subclass 7: transfer of phosphoryl group Third digit sub-subclass 1: alcohol is phosphoryl acceptor Fourth digit for specific enzyme

SIX IUB CLASSES OF ENZYMES 1. Oxidoreductases ENZYMES

Biologic polymers that catalyze the chemical reactions that make life as we know it possible. Protein catalysts (except for RNA molecules or ribozymes). Catalytic activity alters speed of chemical reactions Neither consumed nor permanently altered after during a reaction. lowers free energy of activation Stabilizes high energy intermediates o catalyze oxidation-reduction reactions

2. Transferases
o Catalyze transfer of group from donor compound to acceptor.

3. Hydrolases
o Catalyze hydrolysis of bonds.

ENZYME STRUCTURES
Holoenzyme: The complete, active enyzme . The holoenzyme has a protein portion (APOENZYZME) and a COFACTOR A tightly bound cofactor is called a PROSTHETIC GROUP

4. Lyases
o Catalyze cleavage of C C, C O, C-N bonds by means other than hydrolysis or oxidation; group elimination to form double bonds.

ENZYME NOMENCLATURE
The commonly used names for most enzymes described the type of reaction catalyzed, Followed by the suffix ase. o eg. dehydrogenase : removes hydrogen atom o isomerases : catalyze rearrangement in configuration. Other ways of naming enzymes: Based on the source of the enzyme o eg. Pancreatic ribonuclease Based on the mode of regulation o eg. Hormone-sensitive lipase Based on the distinguishing characteristic of their mechanism o eg. Serine protease

5. Isomerases
o catalyze intramolecular rearrangements.

6. Ligases
o catalyze joining of two molecules coupled with the hydrolysis of a pyrophosphate bond in ATP or similar triphosphate .

ENZYME KINETICS
Field of biochemistry concerned with quantitative measurement of the rates of enzyme-catalyzed reactions and the systematic study of factors that affect these rates. Important in understanding how stress affects homeostasis. Enzyme-catalyzed reactions are 103 1011 faster than uncatalyzed reactions. Enzymes do this by decreasing the free energy of activation.

INTERNATIONAL UNION OF BIOCHEMISTS (IUB System)

Unambiguous system of enzyme nomenclature. Each enzyme has a unique name and code number that identify the type of reaction catalyzed and the substrates involved. Removes ambiguity and confusion o Eg. Common name : Hexokinase IUB -ATP:D-hexose-6-phosphotransferase

[Vmax] maximum velocity maximal number of subtrate molecules converted to product per unit time. [Km] Michaelis constant substrate concentration at which Vi is half the maximal velocity (Vmax/2) o high Km = low substrate affinity o low Km = high substrate affinity

LINEWEAVER-BURK EQUATION
o o From Hans Lineweaver and Dean Burk. Derived from Michaelis-Menten equation by simply taking its reciprocal for solving :
ma * +

Activation energy
o Amount of energy required to bring the reactants to the transition state. Describes both direction in which a chemical reaction will tend to proceed and the concentration of reactants and products that will present equilibrium. The G for a chemical reaction equals the sum of free energies of formation of the reaction products Gp minus the sum of free energies of formation of substrates Gs. The difference between the energies of the reactant (initial state) and the energies of the products (final state). If the free energy formation of formation of the products is lower than that of the substrates, the signs of G will be negative meaning that theres a favoured reaction in the direction from left to right. This reaction is referred to as spontaneous reaction.

Gibbs free energy (G)

o

or

Standard free energy change (Go)

o

o o o

Yields a double reciprocal plot Y intercept is 1/vmax X intercept is -1/Km

Note: Michaelis Menten Equation yields a hyperbolic curve whereas Lineweaver-Burk will give a straight line. SUBSTRATE SPECIFICITY Geometric Complementarity
o Substrate-binding site in enzyme is complementary in shape to the substrate.

The formula for G is:

G= -RTlnKeq
Where; [R] Gas constant (1.98 cal/molk or 8.31 J/molK) [T] Absolute temperature in Kelvin [Keq] concentrations of of reactions of products divided by the product of substrates raised to the power of their stoichiometry. o If G is negative, the concentration of the products at equilibrium will exceed than that of substrates. If G is positive, the formation of the substrates will be favoured. Expresses velocity as a function of substrate concentration.

Electronic Complementarity
o Amino acid residues that form the binding site are arranged to interact specifically with the substrate in an attractive manner.

MICHAELIS MENTEN EQUATION

o

Where; [S] Substrate concentration

COENZYMES Stereospecificity
o Enzymes are highly specific both in binding chiral substrates and in catalyzing their reactions eg. Enzymes involved in glucose metabolism are specific for D-glucose o o Organic compounds that take part in enzymatic reactions. Are chemically changed by the enzymatic reactions in which they participate; must be returned to their original state in order to complete the catalytic cycle Serves as recyclable shuttles or group transfer agents. Mostly derived from water-soluble B vitamins.

THREE-POINT ATTACHMENT OF SUBSTRATE TO ENZYME

CATALYTIC TRIAD OF AA: Consists of serine, histidine, and aspartate 1. Histidine bridges serine and aspartate 2. A covalent adduct is formed between the active site serine and the carbonyl group of a peptide bond, which displaces the amino group from the peptide bond 3. Polypeptide is released form active site

ISOENZYMES
Physically distinct versions of a given enzyme, each of which catalyze the same reaction Usually are oligomeric enzymes with different protomers in various combinations; one tissue produces one protomer predominantly and another tissue, a different protomer Arises from gene duplication.

ACTIVE SITE
Region of an enzyme concerned with substrate binding and catalysis.

MODELS OF ACTIVE SITE LOCK AND KEY or RIGID TEMPLATE MODEL (Fisher)
o Catalytic site is presumed pre-shaped to fit the substrate

INDUCED FIT MODEL (Koshland)

o Binding of the substrate induces a conformational change in the enzyme that results in a complementary fit once the substrate is bound

Clinical applications of enzymes

Measurement of enzymes is used diagnostically

Some enzymes are used as therapeutic agents

Streptokinase used to clear clots in the lower extremities and after a Myocardial Infarction Asparaginase used for the treatment of some types of adult leukemia Methotrexate used in cancer chemotherapy; inhibits dihydrofolate reductase Allopurinol used in the treatment of gout; inhibits xanthine oxidase Statins used to lower plasma cholesterol; inhibits HMG CoA reductase Aspirin analgesic and antiinflammatory; inhibits cyclooxygenase, the enzyme for the synthesis of prostaglandins Penicillin antibiotic; inhibits transpeptidase, the enzyme involved in cell wall synthesis.

hydrophobic bonds that maintains 2o and 3o structure denaturation loss of catalytic activity

Many drugs are enzyme inhibitors

o

o o o

ENZYME-LINKED IMMUNOASSAYS
Used for rapid screening and quantification of the presence of a protein in a given sample as in enzyme-linked immunosorbent assays (ELISA) Used to detect proteins that lack catalytic activity. 2. pH

Enzymes from humans generally exhibit stability at temperature up to 45o 55o C Q10 or temperature coefficient Factor by which the rate of a biologic process increases for a 10oC increase in temperature For most biologic processes, Q10 = 2; rate doubles for a 10o C rise Affects enzyme activity by: Enzyme denaturation at low or high pH Alteration in charged state of enzyme and/or substrate Change in the charge of group which is distal to region where substrate bound but which is needed to maintain 3o and 4o structure.

ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

Use antibody covalently linked to a reporter enzyme such as alkaline phosphatise or horse radish peroxidise. Use absorbance via light or fluorescence. Used in pregnancy testing. Also called collision theory. For two molecules to react, they must: o Approach within bond-forming distance of one another or collide o Must possess sufficient kinetic energy to overcome the energy barrier for reaching the transition state Anything that increases the frequency and the energy of collision between substrates will increase the rate of the reaction

KINETIC THEORY

OPTIMUM pH OF SOME ENZYMES

FACTORS THAT AFFECT REACTION RATE 1. Temperature

Increase temperature increase kinetic energy of molecules increase motion and frequency with which molecules collide 2. Reactant concentration Frequency with which molecules collide is directly proportionate to their concentration

FACTORS THAT AFFECT RATES OF ENZYMECATALYZED REACTIONS

1. TEMPERATURE Initially, reaction rate increases as temperature rises (due to increase kinetic energy of reacting molecules) Eventually, further increase in temperature breaks hydrogen and

3. SUBSTRATE CONCENTRATION Velocity of reaction increases as concentration of substrate is increased (first order kinetics), however there will come a point when further increases in substrate will not increase velocity (zero order)

LINEWEAVER BURK PLOT also know as DOUBLE RECIPROCAL PLOT Converts hyperbolic curve to a straight line

4. ENZYME CONCENTRATION Initial velocity of reaction is directly proportional to concentration of enzyme, providing substrate is in excess. When the substrate concentration is equal to Km value, the initial velocity (vi) is halfmaximal.

ENZYME INHIBITORS

Km can be considered an inverse measure of the affinity of the enzyme for the substrate. The lower the Km, the higher is the affinity.

COMPETITIVE INHIBITOR Question: From the table below, which substrate has a greater affinity for hexokinase?
ENZYME Hexokinase SUBSTRATE Km (mM) Glucose 0.15 Fructose 1.5 Answer: Glucose because it has a lower Km value than that of Fructose. o o o Resembles the substrate (substrate analogs). compete for the same binding site on the enzyme A mutually exclusive process: when the inhibitor is bound, substrate is unable to bind and vice versa. A competitive inhibitor acts by decreasing the number of free enzyme molecules available to bind substrate. Raises the apparent m ( m) for the substrate At high substrate concentration, Vmax is unchanged.

SIGNIFICANCE OF Km AND Vmax

Vmax is related to the turnover number of an enzyme, a quantity equal to the catalytic constant kcat . o o

Turnover number is the number of moles of substrate that react to form product per mole of enzyme per unit time.
The diagram above shows the kinetic scheme for competitive inhibition.

Note: A very high concentration of substrate lessens the effect of the competitive inhibitor, unless the inhibitor has a much higher binding activity for enzyme than does the substrate. The inhibitor molecule cannot be converted to a product because it does not have a functional groups normally acted on by the enzyme.

IRREVERSIBLE ENZYME INHIBITOR

Forms a non-dissociable complex with the enzyme o Cyanide is a classic example of an irreversible enzyme inhibitor; inhibits cytochrome oxidase, an enzyme in the respiratory chain The kinetic effect of irreversible inhibitors is to decrease the concentration of active enzyme, thus decreasing the maximum possible concentration of ES complex.

NON-COMPETITIVE INHIBITOR
o o o o Bears no structural resemblance to substrate. Binds to a different site on the enzyme. Both inhibitor and substrate can bind simultaneously to the enzyme molecule. Do not affect Km; lowers Vmax.

ENZYME INHIBITORS AS DRUGS

If the drug requirement is to increase the intracellular concentration of the substrate, either a competitive or non-competitive inhibitor will serve, since both will inhibit the utilization of substrate, so that it accumulates. If the drug requirement is to decrease the intracellular concentration of the product, then the inhibitor must be non-competitive. As unused substrate accumulates, the increasing substrate concentration overcomes the effect of the competitive inhibitor. Increasing the concentration of substrate does not affect a non-competitive inhibitor. For a competitive inhibitor, the lines converge above the x axis, and the value of [I] where they intersect is -Ki

The diagram above shows the kinetic scheme for noncompetitive inhibition

Note: The presence of the inhibitor does not affect substrate binding but does interfere with the catalytic functioning of the enzyme. The actual mechanism of action for the inhibitor varies with the catalytic functioning of the enzyme.

UNCOMPETITIVE INHIBITOR
Binds only to ES complexes at locations other than the catalytic site. Substrate binding modifies enzyme structure, making inhibitor-binding site available. Inhibition cannot be reversed by substrate. Apparent Vmax decreased; Km is decreased.

IRREVERSIBLE INHIBITION
Forms covalent or a very strong non covalent bonds with the enzyme. Forms a very stable complex Inhibition of cyclooxygenase by aspirin, which involves acetylation of a serine residue .

Note: Uncompetitive is similar to noncompetitive since it allows substrates to bind to the active site however it differs since this type of inhibition binds only to the ES complex.

DRUGS WHOSE STRUCTURE RESEMBLE SUBSTRATES

Captopril for treatment of hypertension and congestive heart failure, which inhibit Angiotensin-Converting Enzyme (ACE) Simvastatin for hypercholesterolemia, which inhibit HMG CoA Reductase, the rate-limiting enzyme of cholesterol synthesis

Involve covalent catalysis and a transient, modified form of enzyme. Enzyme alternates between two forms E and F. Also called double displacement reactions.

BISUBSTRATE REACTIONS
Enzymatic reactions involving two substrates and yielding two products (Bi-Bi reactions) Both substrates are present at saturating levels.

Account for ~ 60% of known biochemical reactions Are either: o TRANSFERASE reactions in which the enzyme catalyzes the transfer of a specific functional group X from one of the substrate to the other o OXIDATION REDUCTION reactions in which reducing equivalents are transferred between two substrate

CONTROL OF ENZYME AVAILABILITY

Control of enzyme quantity Enzyme induction Enzyme repression Enzyme turnover Enzyme compartmentation Formation of macromolecular complex Partial proteolysis Covalent modification Regulation by allosteric effectors Cells can synthesize specific enzymes in response to the presence of specific inducers In many instances, the substrate of the enzyme also serves as inducer E.g., lactose inducing the synthesis of b-galactosidase

1. SEQUENTIAL REACTIONS Reactions in which all substrates must combine with the enzyme before a reaction can occur and products be released Also called single displacement reaction because group undergoing transfer is usually passed directly, in a single step, from one substrate to the other

ENZYME INDUCTION

ENZYME REPRESSION
Curtailment of enzyme synthesis by the presence in the reaction medium of the metabolite being synthesize (product feedback repression) o E.g., In S. typhimurium, addition of His represses synthesis of enzymes of histidine synthesis o Upon removal or exhaustion of the essential intermediate from the medium, enzyme synthesis resumes (derepression) Combined process of enzyme synthesis and degradation

TYPES
A. Ordered / Compulsary o compulsory order of substrate addition to the enzyme o A must first combine with E before B can combine with EA complex. B. Random o no preference for order of substrate addition o Either substrate A or substrate B may combine first with the enzyme to form EA or EB complex. 2. PING PONG REACTIONS One or more products are released before all substrates have been added.

ENZYME TURNOVER

Conversion of Chymotrypsinogen to Chymotrypsin

Susceptibility of enzyme to proteolytic degradation: Depends upon its conformation which in turn is dependent on the presence or absence of substrate, coenzyme or metal ions Also affected by physiologic, hormonal or dietary manipulation Examples: o Arginase synthesis after a protein-rich diet o Arginase degradation - during starvation o Tryptophan oxygenase synthesis presence of glucocorticoid o Tryptophan oxygenase degradation presence of tryptophan

ENZYME COMPARTMENTATION
Localization of specific metabolic process in the cytosol or in cellular organelles E.g., fatty acid oxidation occurs only in the mitocondria; fatty acid synthesis occurs in the cytosol Requires shuttle mechanisms for translocation of metabolites across compartmental barriers

Covalent modification o Involves phosphorylation of specific serine residues in the enzyme o Enzymes are phosphorylated or dephosphorylated in response to extracellular signals such as hormones or growth factors o Enzymes are interconvertible to two activity state high or low catalytic efficiency o eg. In muscles, o phosphorylation o activate glycogen o phosphorylase and o inhibits glycogen o synthase o Kinase catalyze phosphorylation o Phosphatase catalyze dephosphorylation

FORMATION OF MACROMOLECULAR COMPLEX

Organization of a set of enzyme that catalyze a protracted sequence of metabolic reaction so as to coordinate the enzymes and channel metabolites along metabolic path.

CONTROL OF ENZYME ACTIVITY

Partial proteolysis o Conversion of an inactive proenzyme (zymogen) to a catalytically active form o Mechanism utilized by proteases and other digestive enzymes and enzymes involved in blood coagulation o Cleavage at N-terminal between Arg 15 and Ile 16 o Results in the formation of the catalytic site and the substratebinding pocket.

Regulation by allosteric effectors o Involves control of enzyme activity by binding of a substance (effector) to an allosteric site that is physically distinct from the catalytic site.

o o

Negative allosteric effector inhibits the enzyme Positive allosteric effector activates the enzyme

FEEDBACK INHIBITION
Inhibition of activity of an enzyme in a biosynthetic pathway by an end-product of the pathway Form of allosteric inhibition

Multiple Feedback Inhibitions in a Branched Biosynthetic Pathway

1. Cumulative feedback inhibition o Inhibitory effect of 2 or more endproducts on a single regulatory enzyme is strictly additive 2. Concerted or multivalent feedback inhibition o Complete inhibition occurs only when 2 or more end-products are present in excess 3. Cooperative feedback inhibition o A single end-product present in excess inhibits the regulatory enzyme but inhibition when 2 or more endproducts are present far exceeds the additive effects of cumulative feedback inhibition

Comparison between Covalent Modification and Allosteric Regulation

Similarities: 1. Provides for short-term regulation 2. Provides for a reversible means of regulation 3. Acts without altering gene expression 4. Acts on an early enzyme of a protracted metabolic sequence 5. Acts at an allosteric rather than catalytic site 6. Effect is rapid Differences o Covalent modification: Involves several proteins and ATP Under direct neural and hormonal control o Allosteric regulation: Involves single protein Lack hormonal and neural features

ALLOSTERIC REGULATION
Allosteric regulation enables a cell to adjust an enzymatic activity almost instantaneously in response to changes in the concentration of a metabolite since it does not require an intermediate enzyme Binding of an effector at an allosteric site usually changes the conformation at the active site Enzyme can exist in two conformation: R (relaxed) state or T (tense) state Allosteric activator binds preferentially to the R state and stabilizes the conformation that is more effective at binding the substrate Allosteric inhibitor acts by stabilizing the T state

PROXIMITY AND ORIENTATION EFFECT

The binding of the substrate to the enzyme brings together the substrate molecule and the catalytic group at the active site of the enzyme in perfect arrangement for the given reaction to occur

Immobilization of substrates on an enzyme active site can increase collision probability, juxtaposition of reactive groups and also influence the precise alignment of orbitals needed for the reaction to occur In an aqueous medium, the charged groups of the substrate are neutralized by the dipolar water molecules When the substrate becomes embedded in the hydrophobic environment of the active

DESOLVATION EFFECT

site, the formation of hydrogen bonds and electrostatic bonds with the reacting groups in the enzyme produces polarizing effect on the electrons of the substrate facilitating the reaction

COVALENT CATALYSIS
Involves the formation of an intermediate state in which the substrate is covalently attached to a nucleophilic group of the enzyme - OH group of serine e-NH2 group of lysine - SH group of cysteine

STRAIN EFFECT
A conformational change subsequent to substrate binding may lead to distortion of some portion of the substrate

MECHANISM OF ACTION OF SERINE PROTEASES Mechanism of action of lysozyme

Lysozyme is an enzyme that ruptures certain bacterial cells by cleaving the polysaccharide chains that makes up their cell wall. Its mechanism of action involves straininduced destabilization of the sugar when bound to the active site SERINE PROTEASES Includes the digestive enzymes, chymotrypsin, trypsin and elastase and clotting factor, thrombin Uses a highly reactive seryl residue for the formation of a covalent acyl-enzyme intermediate CHYMOTRYPSIN Catalyzes hydrolysis of peptide bonds in which the COOH group is contributed by an aromatic amino acid (Phe, Tyr, Trp) or by one with a bulky non-polar R group (Met) Also catalyzes the hydrolysis of certain esters

GENERAL ACID AND GENERAL BASE CATALYSIS

General acid any substance that is capable of releasing a proton in an aqueous solution General base any substance that is capable of binding a proton in an aqueous solution Chemical bonds are formed by electrons Formation or breakage of bonds requires the migration of electrons Reacting groups either acts as an electron donor or electron acceptor; or as nucleophile or electrophile Nucleophile electron-rich substance that reacts with an electron-deficient substance Electrophile electron-deficient substance that reacts with an electron-rich substance The task of a catalyst often is to make a potentially reactive group more reactive by increasing its electrophilic or nucleophilic character In many cases, the simplest way to do this is to add or remove a proton Proteins contain numerous ionizable groups that can serve as general acid or general base and which can be positioned precisely with respect to the substrate in the active site allowing the proximity effect to come into play

Ser 195 plays an important role in the catalytic mechanism Diisopropylfluorophosphate inhibits chymotrypsin by attaching covalently to the serine residue Reactivity of Ser 195 is not a property of serine residues in general but has been brought about by its juxtaposition with His 57 and Asp 102 Forms a charge-relay network that functions as a proton shuttle CHYMOTRYPSIN, TRYPSIN AND ELASTASE Have similar amino acid sequence, 3dimensional structure and catalytic mechanism Exhibit very different specificities Chymotrypsin acts on peptide bonds of amino acid with an aromatic ring (Phe, Tyr, Trp) Trypsin acts on peptide bonds of amino acid with positively charged R group (Lys, Arg)

Elastase acts on peptide bonds of amino acid with small R group (Ala, Ser) Have a substrate-binding pocket that the enzyme use to identify the residue for which it is specific ASPARTIC PROTEASE Includes the digestive enzyme pepsin, the lysosomal cathepsin and the protease produced by the human immunodeficiency virus (HIV) Catalysis involves two conserved aspartyl residues which act as acid-base catalysts FRUCTOSE 2,6-BISPHOSPHATASE Enzyme involved in gluconeogenesis Catalyzes the hydrolytic release of phosphate from carbon of fructose 2,6 bisphosphate METAL IONS Are present in 25% of all enzymes Classes of metal ion-requiring enzymes 1. Metalloenzymes - Contain tightly bound metal ions, most commonly transition metal ions such as Fe++, Fe+++, Cu++, Mn++, Co++, Zn++ 2. Metal-activated enzymes - Loosely binds metal ions from solutions, usually the alkali and alkali earth metal ions Na+, K+, Mg++, Ca++ TYPES OF TERNARY COMPLEXES

CARBONIC ANHYDRASE

ROLE OF METAL IONS Serve as 3-dimensional template for orientation of basic groups on enzyme or substrate Accept electrons via sigma or pi bonds to activate electrophile or nucleophile Mask a nucleophile and thus prevent an otherwise likely side reaction Coordination sphere of metal may bring together enzyme and substrate or form chelate producing distortion in either the enzyme or substrate

SUMMARY TRUE OR FALSE? Coenzymes are small, heat-stable molecules containing a nucleotide grouping that are often involved in enzyme-catalyzed transfer reactions. TRUE Enzymes that contain prosthetic groups can often be separated into a protein part, the holoenzyme, which is inactive, and a nonprotein part False. holoenzyme itself is a whole enzyme and it is active. Enzymes may have one or more active centers where their substrates bind and undergo chemical change TRUE Phosphatases are enzymes that synthesize phosphate esters False. They remove phosphate. Liver and heart lactate dehydrogenase are identical False. H (heart) and M (liver) subunits. Isoenzymes are distinct molecular forms of enzymes catalyzing the same reaction TRUE The addition of a competitive inhibitor to an enzyme reaction will increase the apparent Km for substrate TRUE The value of Vmax determined for an enzyme is proportional to the amount of enzyme used when measuring values of the initial rate v False. the initial velocity belongs to the 1st order kinetics.