Enzymes Binding and Catalysis Lecture

Enzymes More E Rate (!) = kcat D[S] Rate (!) = kcat[E] (µM•sec-1) (sec-1) (µM) More E Time (t) Rate (!) Time (t) [S] Concentration (M) [S] Concentration (M) 3) What is the relationship between reaction rate and [E]? Concentration (M) Catalysis • The turnover number (kcat) is the number of catalytic cycles/unit time per enzyme active site. • Range of values from <1 to 107 sec-1 [S] Time (t) No enzyme, “no rate” (really slow) Double the [enzyme], double the rate slope = 1.0 [E] Said to be “Diffusion Controlled” Enzymes Enzyme nomenclature a. Reaction Nomenclature b. Enzyme helpers (cofactors) c. Naming enzymes Reaction Nomenclature Enzyme•Substrate Complex Substrate (S) Binding Enzymes E + S ⇌ ES ⇌ EP ⇌ E + P (ES) Enzyme (E) Chemistry S P Enzyme•Product Complex (EP) Product (P) E E (ES ⇌ EP) - Reaction coordinate written as a horizontal line - Substrates binding with arrow down; substrates are denoted in order A, B, C Products released with arrow up; products are denoted in order P, Q, R Enzyme forms are denoted in order as E, F, G Define the number of substrates and/or products separately as uni=1, bi=2, ter=3 For multi-substrate enzymes: Define the order of binding and release separately as ordered or random Define the relationship of substrates-to-products as sequential or ping-pong A P What about more E E complicated reactions? (EA ⇌ EP) This is a uni-uni reaction. (No need to designate last two when there is only one substrate and one product) Reaction Nomenclature Enzymes A B E A E + P1 Q E (EAB ⇌ EPQ) Sequential ordered bi random bi EP2 + P1 P Q B E P (EAB ⇌ EPQ) B Sequential random bi ordered bi A P Q E Reaction Nomenclature Enzymes Two-substrate/Two-product reactions: bi bi Sequential ordered bi ordered bi Sequential ordered bi bi Ordered bi bi Sequential random bi random bi Sequential random bi bi Random bi bi Ping-pong ordered bi ordered bi Ping-pong bi bi Which is the bi bi reaction catalyzed by LDH? Enzyme Helpers Enzymes Cofactors are small molecules that some enzymes require for activity. The two main classes of cofactors are coenzymes (organic molecules derived from vitamins) and metals. Covalently bound coenzymes are called prosthetic groups. Cofactors Metal ions Coenzymes Coenzymes (co-substrate) Prosthetic (covalent) An enzyme with its cofactor is a holoenzyme. Without the cofactor, the enzyme is called an apoenzyme. Cofactors Enzyme Naming Enzymes Enzymes *Trivial: Name -dehydrogenase Reaction Catalyzed redox/hydride transfer • • • Nearly all enzymes end with the suffix of “-ase.” • Generally, the names are of the form “substrate or product – reaction catalyzed.” For example, lactate dehydrogenase is for an enzyme that removes a hydrogen (plus 2e–, i.e., a hydride) from lactate, yielding the carbonyl in pyruvate. • There are two ways of naming enzymes; 1) Trivial and 2) Systematic lactate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase -oxidase redox/O2 as oxidizer cytochrom e oxidase glucose oxidase • -oxygenase redox/O2 incorporated -hydroxylase redox/-OH incorporated cyclooxygenase Ribulose Bisphosphate Carboxylase Oxygenase tyrosine hydroxylase • phenylalanine hydroxylase -kinase transfer/Pi into substrate from ATP • hexose kinase protein kinase A -hydrolase • hydrolysis with H2O (esterase, deacylase) trypsin • phospholipase C - phosphorylase hydrolysis with Pi instead of H2O glycogen phosphorylase b Thym idine phosphorylase -mutase move Pi from one part of molecule to another phosphoglycerate m utase phosphoglucose m utase • - isomerase configuration change • • triosephosphate isom erase phosphogluco isom erase - synthase synthesis • * Not all possible types listed -synthetase • Bullets are those also given as systematic am inoacyl-tRNA synthetases acyl-CoA synthetase Reaction At alcohol -oxidoreductase redox Examples Type Sub-type Sub-class enzyme specific EC# Name 1.1.1.28 1.2.1.59 1.1.3.4 1.14.16.1 L-lactate:NADH oxidoreductase D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase β-D-glucose:oxygen 1-oxidoreductase L-phenylalanine,tetrahydrobiopterin:oxygen oxidoreductase (4-hydroxylating) 2.6.1.1 L-aspartate:2-oxoglutarate aminotransferase lactate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase glucose oxidase phenylalanine hydroxylase Primary amine transfer 2.3.1.85 Acyl-CoA:malonyl-CoA C-acyltransferase 2.7.1.1 ATP:D-hexose 6-phosphotransferase Peptide bonds Aspartate aminotransferase fatty acid synthase hexose kinase Serine mechanism hydrolysis 3.4.21.4 Serine endopeptidylamino acid hydrolase 3.1.4.3 Phosphatidylcholine:cholinephosphohydrolase trypsin phospholipase C C-C bond cleavage -lyase Aldehyde product bond cleavage 4.1.2.13 Fructose 1,6-bisphosphate:triosephosphate lyase 4.2.1.2 (S)-malate hydro-lyase 4.1.1.1 2-oxo-acid carboxy-lyase (aldehyde-forming) Intramolecular configuration change -isomerase aldolase fumarase pyruvate decarboxylase Aldose:ketose configuration change 5.3.1.9 Glucose-6-phophate isomerase phosphoglucoisomerase 5.3.1.1 D-glyceraldehyde-3-phosphate:aldose-ketose-isomerase 5.1.1.4 Proline racemase 5.4.2.11 D-phosphoglycerate 2,3-phosphomutase (2,3-diphosphoglycerate-dependent) Ester linkage -ligase * Trivial Name Uses NAD Nitrogen-group transfer -hydrolase 1.1.1.1 Systematic (https://www.enzyme-database.org/class.php) -transferase synthesis that requires ATP • Great website: EC numbers Types fatty acid synthase nitric oxide synthase triosephosphate isomerase proline racemase phosphoglycerate mutase Only 1 subclass synthesis 6.1.1.1 6.4.1.1 Tyrosine aminoacyl-tRNA ligase Pyruvate:carbon-dioxide ligase (ADP-forming) Tyr-aminoacyl-tRNA synthetases pyruvate carboxylase Naming Enzymes Enzymes Trivial Names Correlation of trivial and systematic: OTHLIL Naming Enzymes Enzymes Oxidoreductase: oxidation-reduction reaction 1.1.1.28 Transferase: transfer of functional group between molecules 2.7.1.1 OT OTHLIL Naming Enzymes Enzymes OTHLIL HL Hydrolase: hydrolysis (addition of water breaks bond) 3.2.1.20 Lyase: addition of group to a double bond or removal of group to form double bond without hydrolysis or oxidation 4.2.1.2 Naming Enzymes Enzymes Isomerase: transfer of functional group within a molecule (rearrangement) 5.4.2.11 Ligase: joining of two molecules (bond formation) coupled with ATP hydrolysis 6.4.1.1 ENZYMES (The WHAT and the How) What must ALL enzymes to to achieve these amazing rate enhancements? How do enzymes do what they do…….. Mechanistically? Enzymes How do enzymes achieve these HUGE rate enhancements? OK, what does a similar plot Let’s review what governs the rates of chemical reactions: the transition state energy Reaction rate ∝ DG‡ (activation energy) look like using an enzyme? Reaction Coordinate Diagram + DG‡ – Enzymes CAN change this! – DG – Enzymes CANNOT change this! The Equilibrium Constant will NEVER be changed by an enzyme – + Enzymes Catalytic Strategies – Position Effects: bind substrates where they need to be for reaction (rather than depending on random collisions) – Polarization of bonds: make substrates more reactive by polarizing bonds (make better nucleophiles, (Electrostatics) electrophile, or leaving groups) – Strain of bonds: bind substrates in such a way that they ”look” like products (put strain on bonds that are to be (Geometry) broken (sessile)) – De-solvation: assist in removal of water shell around substrates or adding to products upon release (S & P are usually in direct contact with residues at the active site (no water)) Enzymes Catalytic Strategies EXAMPLE: Carbonic Anhydrase CO2 + H2O ⇌ H2CO3 ⇌ H+ + HCO3– Carbonic Acid • Named for the reverse reaction, the dehydration of Carbonic Acid to get water and carbon dioxide • Mostly b-structure • It needs a Zn2+ metal cofactor • Held in the active site by 3 His residues • Fourth ligand is a water molecule Bicarbonate Enzymes Catalytic Strategies CO2 + H2O ⇌ H2CO3 ⇌ H+ + HCO3– • Transition State Binding •His-64 ⇌ H+•His-64 ⇌ His-64 H+ + 2a ‡•Thr-199 Enzymes Catalytic Strategies CO2 + H2O ⇌ H2CO3 ⇌ H+ + HCO3– •Thr-199 ⇌ What is another example of binding the TS? ⇌ • Transition State Binding ‡ Enzymes Illustration of TS Stabilization Idea: Imaginary Stickase Enzymes Mechanistic Strategies HOW do Enzymes lower Activation Energies? - enzymes use may use none, one, or more of these There are THREE major strategies used by enzymes: – acid-base catalysis: give and take protons – covalent catalysis: change reaction paths – metal ion catalysis: use redox cofactors, pKa shifters Enzymes Mechanistic Strategies General Acid-Base Catalysis EXAMPLES: Carbonic Anhydrase Proteases Phosphoglyceromutase Proline Racemase Enzymes Mechanistic Strategies General Acid-Base Catalysis Amino Acids Recall, pKa values can shift by orders of magnitude in the microenvironment of the active site Enzymes Mechanistic Strategies Covalent Catalysis • A transient covalent bond between the enzyme and the substrate • Changes the reaction pathway – un-catalyzed: H2O A—B ®A+B – catalyzed (X = catalyst): H2O A — B + X :® A — X + B ®A + X :+ B • Requires a nucleophile on the enzyme EXAMPLES: Which Amino Acids? Ser, Thr, Cys, Lys, Asp, Glu Carbonic Anhydrase Proteases Amino transferase Aldolase – can be a reactive hydroxyl, thiolate, amine, or carboxylate • May also be with metals (dative-covalent bond/coordinate) Enzymes Mechanistic Strategies Metal Ion Catalysis • Involves a metal ion bound to the enzyme • Interacts with substrate to facilitate binding – stabilizes negative charges • Participates in oxidation reactions EXAMPLES: Carbonic Anhydrase Carboxylpeptidase A Cytochrome oxidase Enzymes Mechanistic Strategies – acid-base catalysis: – covalent catalysis: – metal ion catalysis: H+ + •His-64 His-64 2a H+•His-64 What about our enzyme, carbonic anhydrase? Enzyme Kinetics What Is Enzyme Kinetics? • Kinetics is the study of the rate at which compounds react. • The rate of enzymatic reaction is affected by: – enzyme – substrate – effectors – Temperature – Reaction conditions (salts, buffers, pH) •Thr-199 Enzyme Kinetics Why Study Enzyme Kinetics? • Quantitative description of biocatalysis • Determine the order of binding of substrates • Elucidate acid-base catalysis • Understand catalytic mechanism • Find effective inhibitors (drugs) • Understand regulation of activity Enzyme Kinetics Review of Chemical Kinetics Consider a simple reaction: Keq = Thermodynamics tells us: [B] [A] and RA +L k1 k-1 BL R KR eq +=L making product k1 k-1 RL Kinetics tells us: The rate of a chemical reaction (!) is proportional to the concentration of the specie(s) participating in the ! ∝ [A] rate limiting step. making reactant Furthermore, the reaction rate (!) is determined by measuring how much A disappears as a function of time or how much B appears as a function of time. B ! Suppose that we can readily measure the disappearance of A. The velocity of the reaction is given by solving the differential equation above to get the formula below, where k1 is a proportionality constant. ! 1 When the velocity of a reaction is directly proportional to a single reactant concentration, the reaction is called a first-order reaction and the proportionality constant has the units s-1. Enzyme Kinetics Review of Chemical Kinetics When the velocity of a reaction is directly proportional to the concentrations of two reactants, the reaction is called a second-order reaction and the proportionality constant has the units M-1s-1. Many important biochemical reactions are biomolecular or second-order reactions. or The rate equations for these reactions are: ! 2 1 and ! 1 [B] During an enzyme catalyzed reaction when the enzyme binds to substrate, it’s a “pseudo” secondorder reaction….. Enzyme Kinetics Apply Chemical Kinetics to Enzymes Enzyme•Substrate Complex Substrate (S) (ES) Binding Enzyme (E) Chemistry Rate (!) Enzyme•Product Complex (EP) [E] Product (P) E + S ⇌ ES ⇌ EP ⇌ E + P • The rate that an individual enzyme -But, ONLY if athere is AMPLE substrate. Recall,this thisis has units of can go around this cycle is called -What st if [S] is low? 1 order rate constant the “turnover number,” or kcat (sec-1)velocity will now have a dependence on -The • And, the rate depends on the [E] [S], or (recall) " ∝ [S] Enzyme Kinetics How to Do Kinetic Measurements Experiment: 1. Mix enzyme + substrate. 2. Record rate of substrate disappearance and/or product formation as a function of time (the velocity of reaction). 3. Plot initial velocity (!0) at that substrate concentration [S]. 4. Change substrate or enzyme concentration and repeat to get the best slopes. rate The rate is getting slower and slower Why is this curving off? [S] As S goes to P, the [S] decreases. OK, back to the question: what is the equation that describes this behavior? Enzyme Kinetics “Initial” velocity How to Do Kinetic Measurements Experiment: 1. Mix enzyme + substrate. 2. Record rate of substrate disappearance and/or product formation as a function of time (the velocity of reaction). 3. Plot initial velocity (!0) at that substrate concentration [S]. 4. Change substrate or enzyme concentration and repeat to get the best slopes. rate The rate is getting slower and slower Why is this curving off? [S] As S goes to P, the [S] decreases. OK, back to the question: what is the equation that describes this behavior? Enzyme Kinetics This question was answered in 1913 by Michaelis & Menten They used these principles and derived the equation that describes the relationship between !0 and [S] for enzymecatalyzed reactions that show “hyperbolic” behavior…..... The famous Michaelis-Menten Equation Enzyme Kinetics Derivation of Michaelis-Menten Equation: 1. Start with a model mechanism. 2. Identify constraints and assumptions. 3. Carry out algebra 1. Simplest Model Mechanism: one reactant, one product, no inhibitors k1 k2 E+S⇌ ES ⇌ E+P k k -1 -2 Enzyme Kinetics Derivation of Michaelis-Menten Equation: 2. Identify constraints and assumptions. There are 4 important ASSUMPTIONS for this derivation. 1. Use only INITIAL rate (!0). This means [P] = 0, so there is no back reaction and you can ignore k-2. The mechanism simplifies further: k1 k2 E+S⇌ ES ⇀ E + P k -1 2. The slow step is AFTER binding. In other words, binding is a rapid equilibrium. This is the so-called RAPID EQUILIBRIUM ASSUMPTION. !0 = k2 [ES] 3. The substrate is in vast excess of the enzyme (a true catalyst). This means you can ignore the amount of substrate in the ES complex, and [S]free = [S]Total. We can get everything in easily measured quantities: [E]T, [S]T, and !0, but not [ES]. 4. The rate of formation of ES and the rate of breakdown are are equal. This is the so-called STEADY-STATE ASSUMPTION. Therefore, the [ES] does not change. Enzyme Kinetics Derivation of Michaelis-Menten Equation: k k E+S⇌ ES ⇀ E + P k 1 3. Carry out the algebra. 2 -1 • Starting with the Steady state assumption d [ ES ] = rateof formation of ES- rateof breakdown of ES = 0 dt Collect [ES] terms: k1 [E][S] = k2 [ES] + k-1 [ES] k1 [E][S] = (k2 + k-1) [ES] Substitute the expression [E] = [E]T – [ES], because free [E] is difficult to determine: k1 ([E]T – [ES])[S] = (k2 + k-1) [ES] Collect [ES] terms: k1 [E]T[S] – k1 [ES][S] = (k2 + k-1) [ES] k1 [E]T[S] = (k2 + k-1 + k1 [S] )[ES] Enzyme Kinetics k1 -1 • k2 E+S⇌ ES ⇌ E+P k k SUMMARY: -2 The final form in case of a single substrate is the Michaelis-Menten equation: !v0 = = k cat [E tot ][S] V max[S] = K m + [S] Km + [S] • kcat (turnover number): how many substrate molecules one enzyme molecule can convert per second • Km (Michaelis constant): an approximate measure of a substrate’s affinity for an enzyme; actually ratio of rate constants for formation and loss intermediate involved in rate-limiting step. • During steady state, the maximum velocity (Vmax) occurs when all of the enzyme is in the ES complex and is dependent on the breakdown of that complex (k[ES]). • The microscopic meaning of Km and kcat depends on the details of the mechanism. Enzyme Kinetics Two enzymes play a key role in the metabolism of alcohol. Some people respond to alcohol consumption with facial flushing and rapid heart beat, symptoms caused by excessive amounts of acetaldehyde in the blood. There are two different acetaldehyde dehydrogenases in most people, one with a low KM and one with a high KM. The low KM enzyme is genetically inactivated in some individuals. The enzyme with the high KM cannot process all of the acetaldehyde, and so some acetaldehyde appears in the blood. So, if knowing the values of the constants, Km and Vmax, for enzymes and their substrates is important, how are they determined?