UNIT I: Protein Structure and Function Enzymes

Overview Virtually all reactions in body mediated by enzymes, which are protein catalysts that increase rate of reactions without being changed Enzymes direct all metabolic events

Nomenclature Each enzyme has two names: A. Recommended name Short, recommended name, convenient for use More complete, systematic name used when enz must be identified without ambiguity A. Recommended name Most commonly used enz’s end with “-ase” attached to substrate (e.g., glucosidase, sucrase, urease), or to description of action performed (e.g., lactate dehydrogenase, adenylyl cyclase) Some enz’s retain original trivial names e.g., trypsin, pepsin

B. Systematic name The international union of biochem & mol biol (IUBMB) set a system in which enzymes are divided into 6 major classes, each with numerous subgroups Suffix –ase is attached to description of chemical reaction catalyzed e.g., D-glyceraldehyde 3-phosphate: NAD oxidoreductase. IUBMB names unambiguous & informative, but cumbersome in general use

Figure 5.1. Examples of the six major classes of the international classification of enzymes (THF is tetrahydrofolate).

III. Properties of enzymes Enzymes are protein catalysts, increase velocity of a chemical reaction, and not consumed Some types of RNA can act like enzymes, usually catalyzing cleavage & synthesis of phosphodiester bonds. RNAs with catalytic activity = ribozymes, less common than protein catalysts Active sites A special pocket, contains aa side chains that create a 3D surface complementary to S Active site binds S  ES complex that is converted to EP  dissociates to E + P

Figure 5.2. Schematic representation of an enzyme with one active site binding a substrate molecule.

B. Catalytic efficiency - Most E catalyzed reactions are highly efficient, 103-108 x faster than uncatalyzed. Typically an E molecule  transforms ~ 100-1000 S molecules  P each second Number of S molecules  P per sec is “turnover number” C. Specificity - E’s are highly specific, interacting with one or few S & catalyze only one type of chemical reaction

D. Cofactors - Some E’s associate with a non-protein cofactor for activity e.g., metal ions (Zn2+ or Fe2+), and organic molecules a.k.a co-enzymes, that are often derivatives of vitamins e.g., NAD+ contains niacin, FAD contains riboflavin, coenz A contain pantothenic acid Holoenzymes = E + its cofactor Apoenzyme = protein portion of the holoenzyme In absence of appropriate cofactor, apoenz. typically show no biologic activity - Prosthetic group = tightly bound coenz that does not dissociate from enz (e.g., biotin bound to carboxylase)

E. Regulation - E activity can be regulated i.e., E can be activated or inhibited, i.e., rate of P formation responds to needs of cell F. Location within the cell - Many E’s localized in specific organelles - Compartmentalization isolates reaction S or P from other competing reactions. This provides favorable environ for reaction, & organizes the 1000’s of E’s in a cell into purposeful pathways

Figure 5.3. The intracellular location of some important biochemical pathways.

How enzymes work Mechanism of E action can be viewed from 2 different perspectives: - Catalysis in terms of energy changes that occur during reaction, i.e., E’s provide an alternate, energetically favorable reaction pathway different from uncatalyzed one - How active site chemically facilitates catalysis

A. Energy changes occurring during the reaction - All chemical reactions have an energy barrier separating reactants and products = free energy of activation  energy difference b/w reactants and high energy intermediate that occurs during formation of product A ↔ T* ↔ B - T* is the transition state = high energy intermediate

Figure 5.4 Effect of an enzyme on the activation energy of a reaction

Free energy of activation: - Difference in free energy b/w reactant and T*, because of high free energy of activation, rates of uncatalyzed chemical reactions are often slow 2. Rate of reaction: For molecules to react, they must contain sufficient energy to overcome energy barrier of transition state In absence of E, only a small proportion of molecules may possess enough energy to achieve T*. Rate of reaction is determined by number of energized molecules The lower free energy to pass through T*, & the faster the reaction.

3. Alternate reaction pathway: - An E allows a reaction to proceed under conditions prevailing in cell by providing a pathway with a lower free energy of activation - E does not change free energies of R’s or P’s, and so does not change equilibrium of reaction

B. Chemistry of active site - Active site is a complex molecular machine employing a diversity of chemical mechanisms to facilitate R  P. A number of factors responsible for catalytic efficiency of E’s e.g., 1. Transition state stabilization: active site often acts as a flexible molecular template that binds S in a geometric structure resembling activated T*. By stabilizing S in T*, the E greatly increases the conc. of reactive intermediates that can be converted to P, thus, accelerates reaction

2. Other mechanisms: active site can provide catalytic groups that enhance probability that T* is formed. - In some E’s, groups can participate in general acid-base catalysis e.g., aa residues provide or accept protons - In other E’s, catalysis may involve transient formation of covalent enzyme-substrate complex

Enzymes that form covalent intermediates

3. Visualization of the transition state Figure 5.5. Schematic representation of energy changes accompanying formation of enzyme-substrate complex and subsequent formation of a transition-state complex.

V. Factors affecting reaction velocity E’s can be isolated and their properties studied in vitro. Different E’s show different responses to [S], Temp, pH A. Substrate concentration 1. Maximal velocity: rate or velocity of a reaction (v) is the # S molecules  P per unit time; it is usually expressed as μmol of P formed per minute. Rate of E-catalyzed reaction increases with S conc. until a maximal velocity (Vmax) is reached. Leveling off of reaction rate at high [S] reflects saturation with S of all available binding sites on E molecules present

2. Hyperbolic shape of the enzyme kinetics curve - Most E’s show Michaelis-Menten kinetics, in which plot of initial velocity, v0, against [S] is hyperbolic - In contrast, allosteric E’s frequently show sigmoidal curve

Figure 5.6. Effect of substrate conc. on reaction velocity.

B. Temperature Increase of velocity with temperature. As a result of increased # of molecules having sufficient energy to pass over energy barrier and form P’s Decrease of velocity with higher temperatures. As a result of temp-induced denaturation of E.

Figure 5.7 Effect of temperature on an enzyme-catalyzed reaction.

C. pH Effect of pH on ionization of active site: conc. of H+ affects reaction velocity in several ways. 1st, catalytic process usually requires E and S have specific chemical groups in ionized or unionized state in order to interact e.g., amino group of E be in protonated form (-NH3+). At alkaline pH, this group is deprotonated and rate of reaction declines Effect of pH on E denaturation. Extremes of pH can  denaturation, because structure of catalytically active protein molecule depends on ionic character of aa side chains

3. The pH optimum varies for different enzymes: the pH at which maximal E activity is achieved is different for different E’s, & often reflects [H+] at which E functions in body e.g., pepsin, a digestive E in stomach, is maximally active at pH 2, whereas other E’s, designed to work at neutral pH are denatured by such an acidic environ

Figure 5.8 Effect of pH on enzyme-catalyzed reactions.

VI. Michaelis-Menten equation Reaction model - E reversibly combines with S to form ES complex that subsequently breaks down to P, regenerating free E. The model, involving one S molecule: E + S ↔ ES → E + P B. Michaelis-Menten equation - Describes how reaction velocity varies with [S] k1 k2 K-1

Assumptions made in deriving Michaelis-Menten eq: V0 = initial velocity Vmax = maximal velocity Km = Michaelis constant = (k-1 + k2)/k1 [S] = substrate conc v0 Vmax [S] Km + [S] = Assumptions made in deriving Michaelis-Menten eq: Relative concentrations of E and S: [S] is much greater than [E], so % of total S bound by E at any one time is small Steady-state assumption: [ES] does not change with time i.e., rate of formation of ES = breakdown of ES (to E + S & to E + P) Initial velocity: only v0’s are used in analysis of E reactions. i.e., rate of reaction is measured as soon as E and S are mixed. At that time conc of P is very small and so, rate of back reaction (P  S) can be ignored

C. Important conclusions about Michaelis-Menten kinetics Characteristics of Km: - Km is characteristic of an E and its particular S, and reflects affinity of E for that S. - Km is numerically = [S] at which reaction velocity is ½ Vmax. Km does not vary with conc of E Small Km: reflects a high affinity of E for S, as low conc of S is needed to half-saturate the E- i.e., reach a velocity that is ½ Vmax Large Km: reflects a low affinity of E for S. As high [S] is needed to half saturate the E.

Figure 5.9 Effect of substrate concentration on reaction velocities for two enzymes: enzyme 1 with a small Km, and enzyme 2 with a large Km.

2. Relationship of velocity to enzyme concentration - Rate of reaction α [E] at all S conc’s. e.g., if [E] is halved, initial rate of reaction (v0), & that of Vmax, are reduced to ½ that of the original. Order of reaction: When [S] is much less than Km, velocity of reaction is ~ proportional to [S]. Rate of reaction is said to be 1st order wrt S. When [S] is much greater than Km, velocity is constant and = Vmax. Rate of reaction is then independent of [S], and is said to be zero order wrt [S].

Figure 5.10 Effect of substrate concentration on reaction velocity for an enzyme catalyzed reaction.

D. Lineweaver-Burke plot When v0 is plotted against S, it is not always possible to determine when Vmax has been achieved because of the gradual upward slope of the hyperbolic curve at high [S] If 1/v0 is plotted vs. 1/[S], a straight line is obtained. This plot = Lineweaver-Burke plot (a.k.a double-reciprocal plot) can be used to calculate Km & Vmax, as well as to determine mechanism of action of E inhibitors

1. The eq. describing Lineweaver-Burke plot is: Vmax [S] = Km + 1 Vmax - Where intercept on x-axis = -1/Km, & intercept on y-axis = 1/Vmax

Figure 5.11. Lineweaver-Burke plot.

VII. Inhibition of enzyme activity Any S that can diminish velocity of E catalyzed reaction is inhibitor (I) Reversible inhibitors bind to E through non-covalent bonds. Dilution of E-I complex results in dissociation of reversibly bound I, and recovery of E activity Irreversible inhibition occurs when an inhibited E does not regain activity on dilution of E-I complex Commonly encountered types: Competitive Non-competitive

A. Competitive inhibition I binds reversibly to same site of S, i.e., competes with S for that site Effect on Vmax: effect of competitive I is reversed by increasing [S]. At sufficiently high [S], reaction velocity reaches Vmax observed in absence of I. Effect on Km: competitive I increases apparent Km for a given S. i.e., in presence of competitive I, more S is needed to achieve ½ Vmax Effect on Lineweaver-Burke plot: plots of inhbited & uninhibited reactions intersect on y-axis at 1/Vmax (Vmax is unchanged). Inhibited and uninhibited reactions show different x-axis intercepts i.e., apparent Km is increased in presence of competitive I.

Figure 5.12. A. Effect of a competitive inhibitor on the reaction velocity (vo) versus substrate [S] plot. B. Lineweaver-Burke plot of competitive inhibition of an enzyme.

4. Statin drugs as examples of competitive inhibitors: This group of antihyperlipidemic agents competitively inhibits 1st committed step in cholesterol synthesis This reaction is catalyzed by hydroxymethylglutaryl CoA reductase (HMG CoA reductase) Statin drugs e.g., atorvastatin (Lipitor) & simvastatin (Zocor) are structural analogs of natural S for this E, & compete effectively to inhibit HMG CoA reductase  inhibit de novo cholesterol synthesis, thereby lowering plasma cholesterol levels.

Figure 5.13. Lovastatin competes with HMG CoA for the active site of HMG CoA reductase.

B. Non-competitive inhibition Occurs when I and S bind at different sites on E. The non-competitive I can bind either free E or ES complex, thereby preventing reaction from occurring Effect on Vmax: non-competitive inhibition can not be overcome by increasing conc of S, i.e., non-competitive inhibition decreases Vmax Effect on Km: non-competitive I’s do not interfere with binding of S to E. So, E shows same Km in presence or absence of the non-competitive inhibitor HMG CoA  Mevalonic acid

3. Effect on Lineweaver-Burke plot: non-competitive inhibition is differentiated by noting Vmax decrease, whereas Km is unchanged in presence of non-competitive inhibitor 4. Examples of non-competitive inhibitors: some I’s act by forming covalent bonds with specific groups of E’s. e.g., lead forms covalent bonds with sulfhydryl side chains of cysteine in proteins. Ferrochelatase catalyzes insertion of Fe2+ into protoporphyrin (a precursor of heme) is sensitive to inhibition by lead. Other e.g.’s are certain insecticides whose neurotoxic effects result from their irreversible binding at catalytic site of acetylcholiesterase (that cleaves the neurotransmitter acetylcholine)

Figure 5.14. A. Effect of a noncompetitive inhibitor on the reaction velocity (vo) versus substrate [S] plot. B. Lineweaver-Burke plot of noncompetitive inhibition of an enzyme.

C. Enzyme inhibitors as drugs E.g., the widely prescribed ß-lactam antibiotics e.g., penicillin & amoxycillin inhibit enzymes involved in bacterial CW synthesis Drugs may also act by inhibiting extracellular reactions e.g., angiotensin-converting enzyme (ACE) inhibitors. They lower blood pressure by blocking the E that cleaves angiotensin I to form the potent vasoconstrictor, angiotensin II. These drugs, e.g., captopril, enalapril, lisinopril, cause vasodilation & so reduction in blood pressure

Figure 5.15 A noncompetitive inhibitor binding to both free enzyme and enzyme-substrate complex.

VIII. Regulation of enzyme activity Regulation of reaction velocity of E’s is essential to coordinate numerous metabolic processes Rate of most E’s responsive to changes in [S], as intracellular level of many S’s is in range of Km. an increase in [S]  increase in reaction rate  return [S] to normal. Some E’s with specialized regulatory functions respond to allosteric effectors or covalent modification, or show altered rates of E synthesis when physiologic conditions are changed

A. Allosteric binding sites Allosteric E’s regulated by molecules = effectors (also modifiers), that bind non-covalently at a site other than active site These E’s are composed of multiple subunits, & regulatory site may be present on a subunit that is not itself catalytic Presence of allosteric effector can alter affinity of E to its S, or modify maximal catalytic activity of E, or both Effectors that inhibit E activity = negative effectors, that increase E activity = positive effectors Allosteric E’s usually contain subunits and frequently catalyze the committed step early in a pathway

1. Homotropic effectors: when S itself serves as effector, effect is said to be homotropic. Most often allosteric S functions as a positive effector Presence of S molecule at one site enhances catalytic properties of other S-binding sites i.e., binding sites exhibit cooperativity These E’s show sigmoidal curve when reaction velocity (v0) is plotted agains [S] Positive & negative effectors of allosteric E’s can affect either Vmax or Km, or both

Figure 5.16. Effects of negative or positive effectors on an allosteric enyzme. A. Vmax is altered. B. The substrate concentration that gives half maximal velocity (K0.5) is altered.

2. Heterotropic effectors: effector different from S, effect = heterotropic. -e.g., consider the following feedback inhibition - E that converts A to B has an allosteric site that binds the end-product. If conc. of end product increases (e.g., not used as rapidly as synthesized), the initial enz in the pathway is inhibited

Feedback inhibition provides cell product it needs by regulating flow of S molecules through the pathway that synthesizes the product e.g., glycolytic E phosphofructokinase is allosterically inhibited by citrate, which is not a S for the E.

B. Regulation of enzymes by covalent modification Many E’s may be regulated by covalent modification, frequently addition or removal of phosphate groups from specific ser, thr, or tyr residues Protein phosphorylation is recognized as one of primary ways in which cell processes regulated 1. Phosphorylation & dephosphorylation: phospho. reactions are catalyzed by a family of E’s = protein kinases that use ATP as P donor. P groups are cleaved by phosphoprotein phosphatases

Figure 5.18. Covalent modification by the addition and removal of phosphate groups.

2. Response of enzyme to phosphorylation: depending on specific E, the P-form may be more or less active than unphospho-form. e.g., P-form of glycogen phosphorylase (degrades glycogen) has increased activity, whereas addition of P to glycogen synthase (synthesizes glycogen) decreases activity

C. Induction & repression of enzyme synthesis Cells can also regulate amount of E present- usually by altering rate of E synthesis. Increased (induction) or decreased (repression) of E synthesis leads to an alteration in the total population of active sites Efficiency of existing E molecules is not affected E’s subject to regulation of synthesis are often those needed at one stage of development or under selected physiologic conditions E.g., elevated levels of insulin as a result of high blood glucose levels can cause an increase in synthesis of key enzymes involved in glucose metabolism

- In contrast, E’s that are in constant use are usually not regulated by altering rate of synthesis Alterations in E levels by induction or repression of protein synthesis are slow (hours to days), compared with allosterically regulated changes in E activity, which occur in seconds to minutes.

Figure 5.19. Mechanisms for regulating enzyme activity. Substrate inhibition: e.g., Phosphofructokinase-1 inhibition by high ATP

IX. Enzymes in clinical diagnosis Plasma E’s can be classified into 2 major groups - 1st a relatively small group of E’s are actively secreted into blood by certain cell types e.g., liver secretes zymogens (inactive precursors) of E’s involved in coagulation - 2nd a large # of E species are released from cells during normal cell turnover. These E’s almost always function intracellularly, & no physiologic use in plasma In healthy individuals, levels of these E’s are fairly constant, and represent a steady in which rate release from damaged cells into plasma is balanced by equal rate of removal of the E protein from plasma

Presence of elevated E activity in plasma may indicate tissue damage that is accompanied by increased release of intracellular E’s. Note: - Plasma is fluid, non-cellular part of blood. Lab assays of E activity most often use serum, obtained by centrifugation of whole blood after it has been allowed to coagulate. Plasma is a physiologic fluid, serum is prepared in lab

Figure 5.20. Release of enzymes from normal and diseased or traumatized cells

Alteration of plasma enzyme levels in disease states Many diseases that cause tissue damage  increased release of intracellular E’s into plasma Activities of many of these E’s routinely determined for diagnostic purposes in diseases of heart, liver, skeletal muscle, etc. Level of specific E activity in plasma frequently correlates with extent of tissue damage So, determining degree of elevation of an E activity in plasma is often useful in evaluating prognosis for patient

B. Plasma enzymes as diagnostic tools Some E’s show relatively high activity in only one or few tissues. Presence of such E’s in plasma : damage to corresponding tissue. E.g., alanine aminotransferase (ALT) is abundant in liver. Appearance of elevated levels of ALT in plasma signals possible damage to hepatic tissue Increases in plasma levels of E’s with a wide tissue distribution provide a less specific indication of site of cellular injury. Lack of tissue specificity limits diagnostic value of many E’s Ala < pyruvate, a-KG > Glu

C. Isoenzymes and diseases of the heart - Most isozymes are E’s that catalyze same reaction. But, they do not necessarily have same physical properties because of genetically determined differences in aa sequence Isoenzymes different numbers of charged aa’s, and may be separated from each other by electrophoresis Different organs frequently contain characteristic proportions of different isozymes Pattern of isozymes found in plasma may serve as means to identify site of tissue damage. E.g., plasma levels of creatine kinase (CK) & lactate dehydrogenase (LDH) commonly determined in diagnosis of myocardial infarction. Particularly useful when electrocardiogram is difficult to interpret, e.g., when there have been previous episodes of heart disease

Quaternary structure of isoenzymes - Many isozymes contain different subunits in various combinations. e.g., CK occurs as 3 isozymes. Each is a dimer composed of 2 polyp’s (B & M) subunits associated in 1 of 3 combinations: CK1 = BB, CK2 = MB, CK3 = MM. each CK shows a characteristic electrophoretic mobility

Figure 5.21 Subunit structure and electrophoretic mobility and enzyme activity of creatine kinase isoenzymes.

2. Diagnosis of myocardial infarction: Myocardial muscle is the only tissue that contains > 5% of total CK activity as CK2 (MB) isozyme. Appearance of this hybrid isozyme in plasma is virtually specific for infarction of the myocardium Following an acute MI, this isozyme appears ~ 4-8 hours following onset of chest pain & reaches a peak of activity at ~ 24 h NOTE: Lactate dehydrogenase (LDH) activity is also elevated in plasma following infarction, peaking 36-40 h after onset of symptoms - LDH activity is, thus, of diagnostic value in patients admitted > 48 h after infarction- a time when plasma CK2 may provide equivocal results

Figure 5.22 Appearance of creatine kinase (CK) and lactate dehydrogenase (LDH) in plasma after a myocardial infarction.

3. Newer markers for MI: Troponin T and Troponin I are regulatory proteins involved in myocardial contractility They are released into plasma in response to cardiac damage Elevated serum troponins are more predictive of adverse outcomes in unstable angina or MI than conventional assay of CK2

Summary E’s = protein catalysts, increase v of chemical reactions by lowering energy of transition state, not consumed E’s contain special pocket = active site that has aa side chains which create 3D surface complementary to S. Active site binds S  ES complex  EP  E + P E’s allow reactions to proceed rapidly under conditions prevailing in cells by providing alternate reaction path with lower free energy of activation E’s do not change free energy of reactants or products, so do not change equilibrium of reaction Most E’s show Michaelis-Menten kinetics, a plot of v0 vs. [S]  hyperbolic shape Any substance that can diminish v of E-catalyzed reaction is inhibitor. Two common types: competitive (increases apparent Km) & non-competitive (decreases Vmax)

Multi-subunit allosteric E’s frequently show a sigmoidal curve Frequently catalyze committed (rate limiting) step(s) of a pathway Allosteric E’s are regulated by effectors (modifiers) that bind non-covalently at a site other than active site. Effectors can be +ve (accelerate E-catalyzed reaction) or –ve (slow down) An allosteric effector can alter affinity of E for its S, or modify maximal catalytic activity of E, or both.