Week 5-Enzymes.pdf

ENZYMES Assist. Prof. Dr. Derya DİLEK KANÇAĞI Room Number: 511 E-mail: [email protected] Office Hour: Wednesday 13.00-15.00 Fundamental conditions for life… 1. 2. Self replication Chemical reactions catalyzed by enzymes, the most remarkable and highly specialized of the proteins a. b. Li...

ENZYMES Assist. Prof. Dr. Derya DİLEK KANÇAĞI Room Number: 511 E-mail: [email protected] Office Hour: Wednesday 13.00-15.00 Fundamental conditions for life… 1. 2. Self replication Chemical reactions catalyzed by enzymes, the most remarkable and highly specialized of the proteins a. b. Living systems make use of energy from the environment Without catalysis, chemical reactions could not occur on a useful time scale, and thus could not sustain life. Enzymes: 1. A deficiency or even a total absence of one or more enzymes → disease 2. Excessive activity of an enzyme → disease 3. Measurements of the activities of enzymes in blood plasma, erythrocytes, or tissue samples are important in diagnosis 4. Drug interactions affecting enzyme activity 5. Practical tools in chemical engineering, food technology, and agriculture. Central Principles in Enzymes Principle 1 Principle 2 • Enzymes are powerful biological catalysts. • Enzymes exhibit a very high degree of specificity. – Rate accelerations by enzymes are often far greater than those by synthetic or inorganic catalysts. Like all catalysts, enzymes increase reaction rates, lowering reaction activation barriers. Enzymes do not affect the equilibria of reactions. – Each enzyme catalyzes only one chemical reaction, or sometimes a few closely related reactions. Reaction activation barriers are thus lowered selectively. Central Principles in Enzymes Principle 3 Principle 4 • Enzymatic reactions occur in specialized pockets called active sites. • Two concepts explain the catalytic power of enzymes. – These pockets are similar to ligand binding sites, except that a reaction occurs there—the conversion of a substrate, a molecule that is acted on by an enzyme, to a product. – First, enzymes bind most tightly to the transition state of the catalyzed reaction, using binding energy to lower the activation barrier. Second, enzyme active sites are organized by evolution to facilitate multiple mechanisms of chemical catalysis simultaneously. Central Principles in Enzymes Principle 5 • Many enzymes are regulated. – Regulatory mechanisms include reversible covalent modification, binding of allosteric modulators, proteolytic activation, noncovalent binding to regulatory proteins, and elaborate regulatory cascades. Enzymes are often subject to multiple methods of regulation, which allows for exquisite control of every chemical process that occurs in a cell. An introduction to enzymes Enzymes • Biological catalysis was first recognized and described in the late 1700s, in studies on the digestion of meat by secretions of the stomach. • In 1897, Eduard Buchner: – Demonstrated cell-free yeast extracts could ferment sugar to alcohol – Showed fermentation was promoted by molecules that continued to function when removed from cells • Frederick W. Kühne later gave the name enzymes (from the Greek enzymos, “leavened”) to the molecules detected by Buchner This work marked the end of vitalistic notions advanced by Louis Pasteur that biological catalysis was a process inseparable from living systems. Most Enzymes Are Proteins • • • • Catalytic activity depends on the integrity of the native protein conformation – – If an enzyme is denatured or dissociated into its subunits, catalytic activity is usually lost. The catalytic activity of each enzyme is intimately linked to its primary, secondary, tertiary, and quaternary protein structure. Molecular weight = ranges from 12,000 to >1 million Some enzymes require no chemical groups for activity other than their amino acid residues. Some enzyme require additional chemical components: – Cofactor = 1+ inorganic ions, such as Fe2+, Mg2+, Mn2+, or Zn2+ – Coenzyme = complex organic or metalloorganic molecule that act as transient carriers of specific functional groups • Most are derived from vitamins, organic nutrients required in small amounts in the diet. With the exception of a few classes of catalytic RNA molecules, enzymes are proteins. Inorganic Ions as Cofactors Table 6-1 Some Inorganic Ions That Serve as Cofactors for Enzymes Ions Enzymes Cu2+ Cytochrome oxidase Fe2+ or Fe3+ Cytochrome oxidase, catalase, peroxidase K+ Pyruvate kinase Mg2+ Hexokinase, glucose 6-phosphatase, pyruvate kinase Mn2+ Arginase, ribonucleotide reductase Mo Dinitrogenase Ni2+ Urease Zn2+ Carbonic anhydrase, alcohol dehydrogenase, carboxypeptidases A and B Coenzymes as Transient Carriers of Atoms or Functional Groups Table 6-2 Some Coenzymes That Serve as Transient Carriers of Specific Atoms or Functional Groups Coenzyme Examples of chemical groups transferred Dietary precursor in mammals Biocytin CO2 Biotin (vitamin B7) Coenzyme A Acyl groups Pantothenic acid (vitamin B5) and other compounds 5′-Deoxyadenosylcobalamin (coenzyme B12) H atoms and alkyl groups Vitamin B12 Flavin adenine dinucleotide Electrons Riboflavin (vitamin B2) Lipoate Electrons and acyl groups Not required in diet Nicotinamide adenine dinucleotide Hydride ion (:H–) Nicotinic acid (niacin, vitamin B3) Pyridoxal phosphate Amino groups Pyridoxine (vitamin B6) Tetrahydrofolate One-carbon groups Folate (vitamin B9) Thiamine pyrophosphate Aldehydes Thiamine (vitamin B1) Describing Enzymes and Their Additional Chemical Components • Prosthetic group = coenzyme or metal ion that is very tightly or covalently bound to the enzyme protein – Some enzymes require both a coenzyme and one or more metal ions for activity • Holoenzyme = complete catalytically active enzyme together with its bound coenzyme and/or metal ions • • Apoenzyme or apoprotein = the protein part of a holoenzyme Some enzyme proteins are modified covalently by phosphorylation, glycosylation, and other processes. Many of these alterations are involved in the regulation of enzyme activity. Enzymes Are Classified by the Reactions They Catalyze • Enzymes are divided into seven classes, each with subclasses, based on the type of reaction catalyzed • Each enzyme has a four-part enzyme commission number (E.C. Number) and a systematic name • Most enzymes have trivial names Table 6-3 International Classification of Enzymes Class number Class name Type of reaction catalyzed 1 Oxidoreductases Transfer of electrons (hydride ions or H atoms) 2 Transferases Group transfer 3 Hydrolases Hydrolysis (transfer of functional groups to water) 4 Lyases Cleavage of C—C, C—O, C—N, or other bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds 5 Isomerases Transfer of groups within molecules to yield isomeric forms 6 Ligases Formation of C—C, C—S, C—O, and C—N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor 7 Translocases Movement of molecules or ions across membranes or their separation within membranes *In chemistry, a trivial name is a non-systematic name for a chemical substance. How Enzymes Work Enzyme-Catalyzed Reactions Take Place within the Active Site • • Active site = provides a specific environment in which a given reaction can occur more rapidly Substrate = the molecule that is bound to the active site and acted upon by the enzyme Under biologically relevant conditions, uncatalyzed reactions tend to be slow — most biological molecules are quite stable in the neutral-pH, mild-temperature, aqueous environment inside cells The surface of the active site is lined with amino acid residues with substituent groups that bind the substrate and catalyze its chemical transformation. Often, the active site encloses a substrate, sequestering it completely from solution. The enzyme-substrate complex is central to the action of enzymes. Enzymes Affect Reaction Rates, Not Equilibria • Simple enzymatic reactions can be written as E + S ⇌ ES ⇌ EP ⇌ E + P Where E, S, and P represent the enzyme, substrate, and product and ES and EP are transient complexes of the enzyme Reaction equilibrium: A reaction is at equilibrium when there is no net change in the concentrations of reactants or products. Ground State and Transition State • Ground state = starting point for either the forward or reverse reaction – the contribution to the free energy of the system by an average molecule (S or P) under a given set of conditions • Transition state (‡) = the point at which decay to substrate or product are equally likely Any reaction, such as S ⇌ P, can be described by a reaction coordinate diagram Energy Changes in a Reaction Coordinate Diagram • Biochemical standard free-energy change = ∆G′° = the standard freeenergy change at pH 7.0 (biochemical systems commonly involve H+ concentrations far below 1 M) • Activation energy (∆G‡) = difference between the ground state energy level and the transition state energy level – A higher activation energy corresponds to a slower reaction. Temperature of 298 K; partial pressure of each gas, 1 atm, or 101.3 kPa; concentration of each solute, 1 M Catalysts Lower the Activation Energy and Increase the Reaction Rate To undergo reaction, the molecules must overcome this barrier and therefore must be raised to a higher energy level. At the top of the energy hill is a point at which decay to the S or P state is equally probable (it is downhill either way). This is called the transition state, often symbolized by a double dagger (‡). That barrier consists of the energy required for alignment of reacting groups, formation of transient unstable charges, bond rearrangements, and other transformations required for the reaction to proceed in either direction. Catalysts Do Not Affect Reaction Equilibria • Any enzyme that catalyzes the reaction S → P also catalyzes the reaction P → S • Enzymes accelerate the interconversion of S and P • Enzymes are not used up in the process • The equilibrium point is unaffected – The reaction reaches equilibrium much faster when the appropriate enzyme is present, because the rate of the reaction is increased. Reaction Intermediates • Reaction intermediate = any species on the reaction pathway that has a finite chemical lifetime – Example: ES and EP complexes Rate-Limiting Steps • Rate-limiting step = the step in a reaction with the highest activation energy that determines the overall rate of the reaction when several steps occur in a reaction • Activation energies are barriers to chemical reactions Enzymes Lower Activation Energies Selectively • Enzymes have developed to lower activation energies selectively to increase rates for reactions needed for cell survival Reaction Rates and Equilibria Have Precise Thermodynamic Definitions • Reaction equilibria are linked to the standard free-energy change for the reaction, ∆G′° • Reaction rates are linked to the activation energy, ∆G‡ Thermodynamics Relates Keq and ∆G ° • Equilibrium constant, Keq = describes an equilibrium such as S ⇌ P • Under standard conditions: [P] K′eq = [S] • From thermodynamics: ∆G′° = −RT ln K′eq where R is the gas constant, 8.315 J/mol·K, and T is the absolute temperature, 298 K (25 ∘C). The Relationship between K eq and ∆G ° Table 6-4 Relationship between K′eq and ∆G′° K′eq ∆G′° (kJ/mol) 10–6 34.2 10–5 28.5 10–4 22.8 10–3 17.1 10–2 11.4 10–1 5.7 1 0.0 101 –5.7 102 –11.4 103 –17.1 Rate Constants and Rate Equations • The rate of any reaction is determined by the concentration of reactant(s) and the rate constant, k • For the unimolar reaction S → P, a rate equation expresses the rate of the reaction V = K[S] Where V is the velocity or rate of the reaction and [S] is the concentration of the substrate First-Order Reactions • First-order reaction = rate depends only on the concentration of S • The factor k is a proportionality constant that reflects the probability of reaction under a given set of conditions (pH, temperature, and so forth) • k has units of reciprocal time, such as s–1 Second-Order Reactions • Second-order reaction = rate depends on the concentration of two different compounds or the reaction is between two molecules of the same compound • k has units of m–1s–1 • The rate equation becomes v = k[S1][S2] The Relationship between Rate Constants and Activation Energy • From transition-state theory K= kt –∆g‡/rt e h Where k is the Boltzmann constant and h is Planck’s constant • The relationship between the rate constant k and the activation energy ∆G‡ is inverse and exponential A Few Principles Explain the Catalytic Power and Specificity of Enzymes • Enzymes enhance rates in the range of 5 to 17 orders of magnitude Table 6-5 Some Rate Enhancements Produced by Enzymes Cyclophilin 105 Carbonic anhydrase 107 Triose phosphate isomerase 109 Carboxypeptidase A 1011 Phosphoglucomutase 1012 Succinyl-CoA transferase 1013 Urease 1014 Orotidine monophosphate decarboxylase 1017 Interactions between Enzymes and Substrates • Binding energy, ∆GB = energy derived from noncovalent enzyme-substrate interaction • Mediated by hydrogen bonds, ionic interactions, and the hydrophobic effect • Major source of free energy used by enzymes to lower the activation energy • Catalytic functional groups on an enzyme may form a transient covalent bond with a substrate and activate it for reaction, or a group may be transiently transferred from the substrate to the enzyme. • • Covalent interactions between enzyme and substrate lower the activation energy Metal ions facilitate additional mechanisms of catalysis that do not involve covalent interactions. Noncovalent Interactions between Enzyme and Substrate Are Optimized in the Transition State • "Lock and key” hypothesis = enzymes are structurally complementary to their substrates • An enzyme completely complementary to its substrate would be a very poor enzyme Enzymes Must Be Complementary to the Reaction Transition State • The full complement of interactions between substrate and enzyme is formed only when the substrate reaches the transition state Optimal interactions between substrate and enzyme occur only in the transition state. The Role of Binding Energy in Catalysis • • • Real enzymes work on an analogous principle. Some weak interactions are formed in the ES complex, but the full complement of such interactions between substrate and enzyme is formed only when the substrate reaches the transition state. The free energy (binding energy) released by the formation of these interactions partially offsets the energy required to reach the top of the energy hill. The sum of the unfavorable activation energy ∆G‡ and the favorable binding energy ∆GB results in a lower net activation energy Weak binding interactions between the enzyme and the substrate drive enzymatic catalysis – The weak interactions formed only in the transition state are those that make the primary contribution to catalysis. The Active Site of an Enzyme • The requirement for multiple weak interactions to drive catalysis is one reason why enzymes (and some coenzymes) are so large. – An enzyme must provide functional groups for ionic, hydrogen bond, and other interactions, and must precisely position these groups so that binding energy is optimized in the transition state. • Optimized binding energy in the transition state is accomplished by positioning a substrate in a cavity (the active site), removed from H2O. Enzyme Specificity • Specificity = ability to discriminate between a substrate and a competing molecule – Given by binding energy – In general, specificity is derived from the formation of many weak interactions between the enzyme and its specific substrate molecule. If an enzyme active site has functional groups arranged optimally to form a variety of weak interactions with a particular substrate in the transition state, the enzyme will not be able to interact to the same degree with any other molecule. Binding Energy Overcomes the Barrier to Reaction • Barrier to reaction, ∆G‡ includes: – The entropy of molecules in solution, which reduces the possibility that they will react together – The solvation shell of hydrogen-bonded water that surrounds and stabilizes most biomolecules in aqueous solution – The distortion of substrates that must occur in many reactions – The need for proper alignment of catalytic functional groups on the enzyme Binding energy can be used to overcome all these barriers. Rate Enhancement by Entropy Reduction • Entropy reduction = large restriction in the relative motions of two substrates that are to react • Binding energy constrains substrates in the proper orientation to reaction Substrates can be precisely aligned on the enzyme, with many weak interactions between each substrate and strategically located groups on the enzyme clamping the substrate molecules into the proper positions. Studies have shown that simply constraining the motion of two reactants can produce rate enhancements of many orders of magnitude. Desolvation of the Substrate • Desolvation = replacement of the solvation shell of structured water around the substrate with weak bonds between substrate and enzyme – Replaces most or all hydrogen bonds between the substrate and H2O that would otherwise impede reaction. Distortion of the Substrate • Substrate must undergo distortion (electron redistribution) to react – Causes an unfavorable free-energy change • Binding energy compensates thermodynamically for this The Induced Fix Mechanism Reversible binding • • • Induced fit = mechanism by which the enzyme itself undergoes a conformational change when the substrate binds, induced by multiple weak interactions with the substrate – Enhances catalytic properties Induced fit serves to bring specific functional groups on the enzyme into the proper position to catalyze the reaction. The conformational change also permits formation of additional weak bonding interactions in the transition state. In either case, the new enzyme conformation has enhanced catalytic properties. Covalent Interactions and Metal Ions Contribute to Catalysis • Once a substrate is bound to an enzyme, properly positioned catalytic functional groups aid in the cleavage and formation of bonds by a variety of mechanisms: – General acid-base catalysis These mechanisms are distinct from those based on binding energy, because they generally involve transient covalent interaction with a – Covalent catalysis substrate or group transfer to or from a substrate. – Metal ion catalysis General Acid-Base Catalysis • Protons are transferred between an enzyme and a substrate or intermediate – Charged intermediates can often be stabilized by the transfer of protons to form a species that breaks down more readily to products. • Specific acid-base catalysis = uses only the H+ (H3O+) or OH– ions present in water • General acid-base catalysis = mediated by weak acids or bases other than water General acid-base catalysis becomes crucial in the active site of an enzyme, where water may not be available as a proton donor or acceptor. Amino Acids in General Acid-Base Catalysis • • • Several amino acid side chains can and do take on the role of proton donors and acceptors These groups can be precisely positioned in an enzyme active site to allow proton transfers, providing rate enhancements of the order of 102 to 105. This type of catalysis occurs on the vast majority of enzymes. Covalent Catalysis • Covalent catalysis = transient covalent bond forms between the enzyme and the substrate – Catalysis only results when the new pathway has a lower activation energy than the uncatalyzed pathway – All new steps must be faster than the uncatalyzed reaction • Hydrolysis of a bond: H2O A ⎯ B ⎯⎯⎯ →A + B • Hydrolysis of a bond between a and b in the presence of a covalent catalyst: H2O A ⎯ B + X: ⎯⎯ → A ⎯ X + B ⎯⎯⎯ → A + X: + B Nucleophilic functional groups are those which have electron-rich atoms able to donate a pair of electrons to form a new covalent bond. Metal Ion Catalysis • Metals: – Help orient the substrate for reaction – Stabilize charged reaction transition states – Mediate oxidation-reduction reactions by reversible changes in the metal ion’s oxidation state • Nearly 1/3 of all known enzymes require 1+ metal ions for catalytic activity Enzyme Kinetics as an Approach to Understanding Mechanism Enzyme Kinetics • Enzyme kinetics = the discipline focused on determining the rate of a reaction and how it changes in response to changes in experimental parameters Substrate Concentration Affects the Rate of Enzyme-Catalyzed Reactions • Pre–steady state = initial transient period during which ES builds up – • Steady state = period during which [ES] and other intermediates remain constant – • The pre-steady state is frequently too short to be observed easily, lasting only the time (often microseconds) required to convert one molecule of substrate to product The reaction quickly achieves a steady state Steady-state kinetics = the traditional analysis of reaction rates Initial Velocities of Enzyme-Catalyzed Reactions • Initial rate (initial velocity), V0 = tangent to each curve taken at time = 0 – Reflects a steady state • At the beginning of the reaction, [S] is regarded as constant A key factor affecting the rate of a reaction catalyzed by an enzyme is the concentration of substrate, [S]. Effect of [S] on the V0 of an Enzyme-Catalyzed Reaction • The plateau-like V0 region is close to the maximum velocity, vmax Vmax – the maximum rate of the reaction, when all the enzyme’s active sites are saturated with substrate. Km (also known as the Michaelis constant) – the substrate concentration at which the reaction rate is 50% of the Vmax. Km is a measure of the affinity an enzyme has for its substrate, as the lower the value of Km, the more efficient the enzyme is at carrying out its function at a lower substrate concentration. Maximum activity General Theory of Enzyme Action Proposed by Michaelis and Menten • Step 1 – enzyme and substrate combine to form a complex in a reversible, relatively fast step: • Step 2 – the complex breaks down to yield the free enzyme and the reaction product in a slower step: • Because the second step limits the overall reaction rate, the overall rate is proportional to [ES] The Saturation Effect • Vmax is observed when virtually all the enzyme is present as the ES complex – Further increases in [S] have no effect on rate – Responsible for the plateau observed The Relationship between Substrate Concentration and Reaction Rate Can Be Expressed with the Michaelis-Menten Equation • The curve expressing the relationship between [S] and V0 can be expressed by the MichaelisMenten equation: Vmax [S] V0 = Km + [S] Where V0 is the initial velocity, vmax is the maximum velocity, [S] is the initial substrate concentration, and Km is a constant called the Michaelis constant The Michaelis-Menten Equation • The Michaelis-Menten equation is the rate equation for a one-substrate enzyme-catalyzed reaction: Vmax [S] V0 = Km + [S] • V0, Vmax, [S], and Km are readily measured experimentally Deriving the Michaelis-Menten Equation, Step 2 • Steady-state assumption = the rate of formation of ES is equal to the rate of its breakdown • When equated for the steady state: k1([Et] – [ES])[S] = k–1 [ES] + k2[ES] Michaelis-Menten Kinetics Can Be Analyzed Quantitatively • An algebraic transformation of the Michaelis-Menten equation converts the hyperbolic curve into a linear form: V0 = Vmax[S] Km + [S] 1 Km + [S] = V0 Vmax[S] Deriving the Lineweaver-Burk Equation • Simplifying this equation gives the Lineweaver-Burk equation: 1 Km + [S] = V0 Vmax[S] 1 Km [S] = + V0 Vmax[S] Vmax[S] 1 Km 1 = + V0 Vmax[S] Vmax A Double-Reciprocal, or Lineweaver-Burk, Plot • For enzymes obeying the Michaelis-Menten relationship, a plot of 1/V0 versus 1/[S] yields a straight line Kinetic Parameters Are Used to Compare Enzyme Activities • All enzymes that exhibit a hyperbolic dependence of V0 on [S] follow Michaelis-Menten kinetics where: Km = [S] when V0 = ½Vmax The most important exceptions to MichaelisMenten kinetics are the regulatory enzymes Interpreting Km and Vmax • Km can vary for different substrates of the same enzyme Table 6-6 Km for Some Enzymes and Substrates Enzyme Substrate Km (mM) Hexokinase (brain) ATP D-Glucose D-Fructose 0.4 0.05 1.5 Carbonic anhydrase HCO3– 26 Chymotrypsin Glycyltyrosinylglycine N-Benzoyltyrosinamide 108 2.5 β-Galactosidase D-Lactose 4.0 Threonine dehydratase L-Threonine 5.0 Interpreting Km and Vmax Both the magnitude and the meaning of Km and Vmax can vary greatly from enzyme to enzyme. • Km can vary for different substrates of the same enzyme Table 6-6 Km for Some Enzymes and Substrates Enzyme Substrate Km (mM) Hexokinase (brain) ATP D-Glucose D-Fructose 0.4 0.05 1.5 Carbonic anhydrase HCO3– 26 Chymotrypsin Glycyltyrosinylglycine N-Benzoyltyrosinamide 108 2.5 β-Galactosidase D-Lactose 4.0 Threonine dehydratase L-Threonine 5.0 The Dissociation Constant, Kd • For reactions with two steps: k2 + k–1 Km = k1 • When k2 is rate-limiting, k2 << k–1, and Km reduces to k–1/k1, which is defined as the dissociation constant, Kd, of the ES complex – Under these conditions, Km represents a measure of the affinity Interpreting Vmax The quantity Vmax depends upon the rate-limiting step of the enzyme catalyzed reaction. • The number of reactions steps and the identity of the rate-limiting step(s) varies from enzyme to enzyme • For a two-step Michaelis-Menten mechanism, where k2 is rate-limiting Vmax = k2[Et] • When product release, EP → E + P, is rate-limiting: The General Rate Constant, kcat • General rate constant, kcat = describes the limiting rate of any enzyme-catalyzed reaction at saturation – If one step in a multistep reaction is clearly limiting, kcat equals the rate constant for the step – More complex when several steps are rate-limiting • In the Michaelis-Menten equation, kcat = Vmax/[Et]: V0 = Vmax[S] Km + [S] V0 = kcat[Et][S] Km + [S] The Constant kcat Is a First-Order Rate Constant Two enzymes catalyzing different reactions may have the same kcat (turnover number) • kcat = turnover number = the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated Table 6-7 Turnover Number, kcat, of Some Enzymes Enzyme Substrate kcat (s–1) Catalase H2O2 40,000,000 Carbonic anhydrase HCO3– 400,000 Acetylcholinesterase Acetylcholine 14,000 β-Lactamase Benzylpenicillin 2,000 Fumarase Fumarate 800 RecA protein (an ATPase) ATP 0.5 Comparing Catalytic Mechanisms and Efficiencies • Comparing the ratio kcat/km for two reactions is the best way to compare catalytic efficiencies or turnover • Specificity constant = the rate constant for the conversion of E + S to E + P • When [S] << km: V0 = kcat [Et][S] Km *How many substrate molecules are transformed into products per unit time by a single enzyme. *The affinity of the substrate to the active site of the enzyme The Constant kcat/Km Is a Second-Order Rate Constant • kcat/Km has units of M–1s–1 with an upper limit of 108 to 109 M–1s–1 Table 6-8 Enzymes for Which kcat/Km Is Close to the Diffusion-Controlled Limit (108 to 109 M–1s–1) Enzyme Substrate kcat (s–1) Km (M) kcat/Km (M–1s–1) Acetylcholinesterase Acetylcholine 1.4×104 9×10–5 1.6×108 Carbonic anhydrase CO2 HCO3– 1×106 4×105 1.2×10–2 2.6×10–2 8.3×107 1.5×107 Catalase H2O2 4×107 1.1×100 4×107 Crotonase Crotonyl-CoA 5.7×103 2×10–5 2.8×108 Fumarase Fumarate Malate 8×102 9×102 5×10–6 2.5×10–5 1.6×108 3.6×107 β-Lactamase Benzylpenicillin 2.0×103 2×10–5 1×108 Many Enzymes Catalyze Reactions with Two or More Substrates • Nearly 2/3 of all enzymatic reactions have 2 substrates and 2 products • Example types of reactions: – A group is transferred from one substrate to the other – One substrate is oxidized while the other is reduced Some Enzyme Reactions Involve a Ternary Complex • Substrates can bind in a random sequence or in a specific order Some Enzyme Reactions Do Not Form a Ternary Complex • The first substrate is converted to product and dissociates before the second substrate binds – Example: ping-pong, or double-displacement, mechanism Steady-State Kinetic Analysis of Bisubstrate Reactions • Michaelis-Menten steady-state kinetics can distinguish between pathways that have a ternary intermediate and pathways that do not Intersecting Lines Indicate the Formation of a Ternary Complex Parallel Lines Indicate a Ping-Pong (Double-Displacement) Pathway Enzyme Activity Depends on pH • The pH range over which an enzyme undergoes changes in activity can provide a clue to the type of amino acid residue involved. • Enzymes have an optimum pH (or pH range) at which their activity is maximal; at higher or lower pH, activity decreases. This is not surprising. Amino acid side chains in the active site may act as weak acids and bases only if they maintain a certain state of ionization. Enzymes Are Subject to Reversible or Irreversible Inhibition • Enzyme inhibitors = molecules that interfere with catalysis, slowing or halting enzymatic reactions • 2 classes of enzyme inhibitors: – Reversible – Irreversible Reversible Inhibition • Types of reversible inhibition: – Competitive inhibition – Uncompetitive inhibition – Mixed inhibition – Noncompetitive inhibition Competitive Inhibition • Competitive inhibitor: – Competes with the substrate for the active site of an enzyme – Type of reversible inhibition Many competitive inhibitors are structurally similar to the substrate and combine with the enzyme to form an unreactive EI complex. Competitive Inhibitors Alter the Michaelis-Menten Equation • Competitive inhibition can be measured by steady-state kinetics • The Michaelis-Menten equation becomes where α=1+ [I] KI V0 = Vmax[S] αKm + [S] and KI = [E][I] [EI] Alpha determines mechanism. Its value determines the degree to which the binding of inhibitor changes the affinity of the enzyme for substrate. Its value is always greater than zero. KI is the inhibition constant Competitive Inhibitors Affect the Apparent Km, but Not the Vmax • “Apparent” Km = the determined variable αKm experimentally • When [S] >> [I], the reaction exhibits normal Vmax • In the presence of inhibitor, the [S] at which V0 = ½Vmax, the apparent Km, increases by the factor α A Lineweaver-Burk Plot Reveals Competitive Inhibition lines intersect at the y axis Uncompetitive Inhibition • Uncompetitive inhibitor: – Binds at a site distinct from the substrate active site – Unlike a competitive inhibitor, binds only to the ES complex – Type of reversible inhibition Uncompetitive Inhibitors Alter the Michaelis-Menten Equation • The Michaelis-Menten equation becomes V0 = where α′ = 1 + [I] K′I Vmax[S] Km + α′[S] and K′I = [ES][I] [ESI] Uncompetitive Inhibitors Affect Both the Apparent Km and the Vmax • The measured Vmax decreases because at high [S], V0 approaches Vmax/α′ • Apparent Km decreases because the [S] required to reach ½Vmax decreases by the factor α′ A Lineweaver-Burk Plot Reveals Uncompetitive Inhibition lines are parallel Mixed Inhibition • Mixed inhibitor: – Binds at a site distinct from the substrate active site – Binds to either E or the ES complex – Type of reversible inhibition Mixed Inhibitors Alter the Michaelis-Menten Equation • The Michaelis-Menten equation becomes V0 = Vmax[S] αKm + α′[S] Mixed Inhibitors Usually Affect Both the Apparent Km and the Vmax • Vmax is affected because the effective [E] on which Vmax depends decreases • Apparent Km may increase or decrease depending on which enzyme form the inhibitor binds most strongly A Lineweaver-Burk Plot Reveals Mixed Inhibition lines intersect to the left of the y axis Noncompetitive Inhibition • Noncompetitive inhibitor: – Special case of α = α′ – Affects the Vmax but not the Km Irreversible Inhibition • Irreversible inhibitor = bind covalently with or destroy a functional group on an enzyme that is essential for the enzyme’s activity, or form a highly stable noncovalent association Suicide Inactivators • Suicide inactivator = mechanism-based inactivators = undergo the first few steps until it is converted into a compound that combines irreversibly with the enzyme – Class of irreversible inhibitors These compounds are relatively unreactive until they bind to the active site of a specific enzyme. Transition-State Analogs • Transition-state analogs= stable molecules designed to resemble transition states – Type of irreversible inhibitors – Bind to an enzyme more tightly than does the substrate in the ES complex Chemical compounds with a chemical structure that resembles the transition state of a substrate molecule in an enzyme-catalyzed chemical reaction. Examples of enzymatic reactions The Chymotrypsin Mechanism Involves Acylation and Deacylation of a Ser Residue • Protease = an enzyme that catalyzes the hydrolytic cleavage of peptide bonds – – This protease is specific for peptide bonds adjacent to aromatic amino acid residues (Trp, Phe, Tyr). Example= bovine pancreatic chymotrypsin The principle of transition state stabilization and also provides a classic example of general acid-base catalysis and covalent catalysis In addition to its action on polypeptides, chymotrypsin catalyzes the hydrolysis of small esters and amides. The Chymotrypsin Mechanism Involves Two Phases • Acylation phase: the peptide bond is cleaved, and an ester linkage is formed between the peptide carbonyl carbon and the enzyme • Deacylation phase: the ester linkage is hydrolyzed and the nonacylated enzyme is regenerated The pH Dependence of Chymotrypsin-Catalyzed Reactions • • • Optimal activity at pH 8 – His57 is unprotonated and Ile16 is protonated The transition just above pH 7 is due to changes in kcat – results from protonation of His57 The transition above pH 8.5 is due to changes in 1/Km – results from the ionization of the αamino group of Ile16 The rate of chymotrypsin-catalyzed cleavage generally, exhibits a bell-shaped pH-rate profile The Chymotrypsin Reaction • Serine proteases = proteases with a Ser residue that acts as the nucleophile – Acylation phase nucleophile is the oxygen of Ser195 – Chymotrypsin is serine proteases that utilize the catalytic triad to carry out the hydrolysis of peptide bonds. • Catalytic triad = hydrogen bonding network→a set of three coordinated amino acids that can be found in the active site of some enzymes. – In chymotrypsin, Ser195 is linked to His57 and Asp102 • These amino acids work together to carry out the catalytic function of breaking peptide bonds. HIV Protease Inhibitors • HIV protease inhibitors form noncovalent complexes with the enzyme – designed as transition-state analogs – can be considered irreversible inhibitors • The HIV protease is an aspartyl protease. Two active-site Asp residues facilitate the direct attack of a water molecule on the carbonyl group of the peptide bond to be cleaved. • The HIV protease is most efficient at cleaving peptide bonds between Phe and Pro residues. The active site has a pocket that binds an aromatic group next to the bond to be cleaved. Hexokinase Undergoes Induced Fit on Substrate Binding • Yeast hexokinase = bisubstrate enzyme that catalyzes the reversible reaction: Induced Fit in Hexokinase • When glucose (but not water) and Mg⋅ATP bind, the binding energy derived induces a conformational change in hexokinase to the catalytically active form The Catalytic Mechanism of Hexokinase • Active-site amino acid residues participate in general acid-base catalysis and transition-state stabilization The Enolase Reaction Mechanism Requires Metal Ions • Enolase = catalyzes the reversible dehydration of 2-phosphoglycerate to phosphoenolpyruvate: – the use of an enzymatic cofactor, in this case a metal ion An Understanding of Enzyme Mechanism Produces Useful Antibiotics • Peptidoglycan = major component of the bacterial cell wall – Consists of polysaccharides and peptides cross-linked in several steps that include a transpeptidase reaction Penicillin interferes with the synthesis of peptidoglycan, the major component of the rigid cell wall that protects bacteria from osmotic lysis. Peptidoglycan consists of polysaccharides and peptides crosslinked in several steps that include a transpeptidase reaction Transpeptidase Inhibition by β-Lactam Antibiotics • β-lactam antibiotics bind to the active site of transpeptidase • The covalent complex irreversibly inactivates the enzyme, blocking synthesis of the cell wall β-Lactamases • Β-lactamases = enzymes that cleave βlactam antibiotics • Bacteria that express β-lactamases become resistant to β-lactam antibiotics • Compounds that mimic the structure of a βlactam antibiotic can inactivate β-lactamases Regulatory enzymes Regulatory Enzymes • Regulatory enzymes = catalytic activity increases or decreases in response to certain signals – Allows the cell to meet changing needs for energy and biomolecules Both classes of regulatory enzymes tend to be multisubunit proteins, and in some cases the regulatory site(s) and the active site are on separate subunits. Modulation of Regulatory Enzymes • Activities of regulatory enzymes are modulated in a variety of ways: – Allosteric enzymes = function through reversible, noncovalent binding of regulatory compounds called allosteric modulators or allosteric effectors (small metabolites or cofactors) – Reversible covalent modification – Binding of separate regulatory proteins – Removal of peptide segments by proteolytic cleavage Some enzymes are stimulated or inhibited when they are bound by separate regulatory proteins. Others are activated when peptide segments are removed by proteolytic cleavage; unlike effectormediated regulation, regulation by proteolytic cleavage is irreversible. Allosteric Enzymes Undergo Conformational Changes in Response to Modulator Binding • Homotrophic = regulation in which the substrate and modulator are identical • Heterotropic = regulation in which the modulator is a molecule other than the substrate The modulators for allosteric enzymes may be inhibitory or stimulatory. Note that heterotropic modulators should not be confused with uncompetitive and mixed inhibitors. Enzymes with several modulators generally have different specific binding sites for each. The Regulatory Enzyme Aspartate Transcarbamoylase • Aspartate transcarbamoylase (ATCase) = catalyzes the formation of carbamoyl aspartate, an early step in pyrimidine biosynthesis: Allosteric enzymes are typically larger and more complex than nonallosteric enzymes, with two or more subunits. ATCase has 12 polypeptide chains organized into 6 catalytic subunits (organized as 2 trimeric complexes) and 6 regulatory subunits (organized as 3 dimeric complexes). The Quaternary Structure of Aspartate Transcarbamoylase ATP = Positive regulator CTP = Negative regulator ATCase effectively illustrates both homotropic and heterotropic allosteric kinetic behavior. The Kinetic Properties of Allosteric Enzymes Diverge from Michaelis-Menten Behavior • Plots of V0 versus [S] usually produce a sigmoid saturation curve, rather than a hyperbolic curve • [S]0.5 or K0.5 represents the [S] giving half-maximal velocity of the reaction Sigmoid kinetic behavior reflects cooperative interactions between multiple protein subunits. In other words, changes in the structure of one subunit are translated into structural changes in adjacent subunits, an effect mediated by noncovalent interactions at the interface between subunits. The Sigmoid Curve of a Homotropic Enzyme • A relatively small increase in [S] in the upright part of the curve causes a comparatively large increase in V0 Modulation in Which K0.5, but Not Vmax, Is Altered • For heterotropic allosteric enzymes: – Activators may cause the curve to become more hyperbolic – Inhibitor may cause the curve to become more sigmoidal Modulation in Which Vmax, but Not K0.5, Is Altered • Less common type of modulation for heterotropic allosteric enzymes Some Enzymes Are Regulated by Reversible Covalent Modification Phosphoryl Groups Affect the Structure and Catalytic Activity of Enzymes • Protein kinases = catalyze the attachment of phosphoryl groups to specific amino acid residues (Ser, Thr, Tyr, His) • Phosphoprotein phosphatases = protein phosphatases = remove phosphoryl groups from the same target proteins Regulation of Muscle Glycogen Phosphorylase Activity by Phosphorylation To serve as an effective regulatory mechanism, phosphorylation must be reversible. In general, phosphoryl groups are added and removed by different enzymes, and the processes can therefore be separately regulated. Phosphorylation in Glycogen Phosphorylase Causes a Conformational Change The regulation of glycogen phosphorylase by phosphorylation illustrates the effects on both structure and catalytic activity of adding a phosphoryl group. Multiple Phosphorylations Allow Exquisite Regulatory Control • Residues that are typically phosphorylated in regulated proteins occur within common structural motifs (consensus sequences) The Ser, Thr, or Tyr residues that are typically phosphorylated in regulated proteins occur within common structural motifs, called consensus sequences, that are recognized by specific protein kinases Multiple Regulatory Phosphorylations • Provide the potential for extremely subtle modulation of enzyme activity • Sequential phosphorylation processes can be hierarchical Some Enzymes and Other Proteins Are Regulated by Proteolytic Cleavage of an Enzyme Precursor • Zymogen = inactive precursor that is cleaved to form an active protease enzyme • Proteases are not the only proteins activated by proteolysis. • Proprotein or proenzyme = precursors that are cleaved to form other proteins A Cascade of Proteolytically Activated Zymogens Leads to Blood Coagulation • Regulatory cascade = a mechanism that allows a very sensitive response to—and amplification of—a molecular signal – Example = formation of a blood clot Blood Clots • Blood clot = aggregate of specialized cell fragments that lack nuclei (platelets) crosslinked and stabilized by proteinaceous fibers consisting mainly of the protein fibrin (derived from the soluble zymogen fibrinogen) Platelet Activation • Caused by collagen exposure to blood • Causes the release of signaling molecules such as thromboxanes to stimulate the activation of additional platelets The Cleavage of Fibrinogen to Fibrin • Fibrinogen is converted to fibrin by the proteolytic removal of amino acid residues • Thrombin = serine protease that catalyzes peptide removal • Factor XIIIa = transglutaminase enzyme that catalyzes the formation of covalent cross-links between fibrins Blood clotting is mediated by two interlinked regulatory cascades of proteolytically activated zymogens. Two Regulatory Cascades Lead to Fibrinogen Activation • Intrinsic pathway = involves components found in the blood plasma all • Extrinsic pathway = tissue factor pathway = involves the protein tissue factor (TF) which is not present in blood Genetic defects in the genes encoding the proteins required for blood clotting result in diseases referred to as hemophilias. Some Regulatory Enzymes Use Several Regulatory Mechanisms • when chemical resources are plentiful, cells synthesize and store glucose and other metabolites • when chemical resources are scarce, cells use these stores to fuel cellular metabolism • the availability of specific catalysts allows for the regulation of these reactions Cells catalyze only the reactions they need at a given moment.