Enzyme Catalysis and Enzyme

Kinetics

Student Edition 5/23/13 Version

Dr. Brad Chazotte
213 Maddox Hall
chazotte@campbell.edu
Pharm. 304
Web Site: Biochemistry
http://www.campbell.edu/faculty/chazotte Fall 2014
Original material only ©2005-14 B. Chazotte

Goals- Catalysis
• Understand the nomenclature of enzymes.
• Understand the roles of cofactors and coenzymes and be familiar
with examples of each.
• Understand the meaning of activation energy and the concepts of
transition state theory & catalytic rate enhancement.
• Be generally familiar with the types of enzyme catalytic
mechanisms.
• Learn the biologically important electrophiles & nucleophiles.

Enzymes
Protein molecules that utilize the chemical and physical properties of their
component amino acids to facilitate biochemical reactions that otherwise would
be difficult to accomplish under physiological conditions.

• An enzyme provides a specific environment that allows the biochemical reaction
to occur more rapidly.
• The distinguishing feature of an enzyme-catalyzed reaction is that it occurs within
a pocket on the protein.
• This pocket is termed the active site and the molecule(s) that binds to the active
site and is converted is called the substrate.
• The molecule(s) produced by the reaction is called the product.

Lehninger 2005 Figure p.191

• occur with a high degree of specificity with respect to both the reactants and the products. and ~pH 7. 1 Atm. • occur under relatively mild conditions under 100° C. Enzymes: General Properties Enzymatically catalyzed reactions: • are typically 106 to 1012 times more rapid than the corresponding uncatalyzed reactions. • can be controlled by non-substrate molecules. covalent enzyme modification. Voet. 282 . or the synthesis or degradation of the enzyme (amount of enzyme present). i.e.0. allosteric control. Voet & Pratt 2002 p.

Here are the 6 major classes Enzymes are commonly named by adding the suffix –ase to the substrate the enzyme converts or a phase that describes the reaction catalyzed by the enzyme.3 . Lehninger 2000 Table 8. International Classification on Enzymes (OTHLIL) To be systematic the IUMBMB developed a numerical coding and naming system.

Enzyme Commission Number An example: The four-part enzyme classification number is a series of 4 numbers separated by periods.9.3.ch/sprot/enzyme.3 Sub-subclass With oxygen as an acceptor See enzyme classification website @ http://expasy. 1.9. Complex IV 1. Subclass Acting on a heme group of donors 1.1 Cytochrome c oxidoreductase trivial (common) names : cytochrome oxidase.html .9. Class Oxidoreductases 1.

. Specificity of Enzymes Geometric complementarity – the enzyme’s binding site has a structure complementary to the substrate it needs to bind. Stereospecificity – binding of chiral substrates and the catalysis of their reactions is highly specific due in large part to the inherent chirality of the L-amino acids that comprise the enzyme. Electronic complementarity – amino acids that form the enzyme’s binding site are arranged to specifically interact and attract the substrate molecule.

1 . Enzyme- Substrate Complex Illustration Voet. Voet & Pratt 2013 Fig 11.

Cofactors and Coenzymes Some enzymes require some help. . Help can be in the form cofactors which are either one or more inorganic ions or a complex organic or metalloorganic molecules called a coenzyme.

Lehninger 2000 Table 8. Cofactors table Holoenzyme: catalytically active complete enzyme together with its bound coenzyme and/or metal ions. Prosthetic group: a coenzyme or metal ion that is covalently or very tightly bound to the protein.2 . Apoenzyme (aproprotein): protein part of such an enzyme.

Coenzymes as Transient Carriers Table Human Deficiency Disease Perncious anemia Pellagra Megaloblastic anemia Beriberi .

. What do enzymes do? Enzymes as catalysts change the rate of a chemical reaction but do not alter the equilibrium.

. & The Reaction Coordinate Thermodynamics Again? Transition state theory was developed to describe chemical reactions by applying thermodynamic equilibrium concepts. Activation Energy (Ea). Transition State Theory.

Transition State Diagram Consider the bimolecular Rx (a) : HA – HB + HC  HA + HB-.5 . Voet & Pratt 2013 Fig 11.HC Consider the generic bimolecular Rx (b) : A + B  X‡  P + Q rate = k ~ e-ΔG‡/RT . ΔG‡ = Ea Voet.

Transition State Diagram: Effect of Catalyst A catalyst functions by lowering the activation energy of a reaction. Voet & Pratt 2013 Fig 11. the energy barrier for the reactants to become products.7 . Voet.

Catalytic Rate Enhancement*: a ΔG‡ Calculation The enhancement of catalyzed vs uncatalyzed is given by: k= eΔΔG‡/RT ln k = ΔΔG‡/RT RT ln k = ΔΔG‡ for a 100-fold change in rate at 25 ºC (8.15 ºK) x ln(100) = ΔΔG‡ (2478.000.71 kJ mol -1 compare H-bond ~20 kJ mol-1 1.000-fold: 34.25 kJ mol -1 covalent bond ~300-500 kJ mol-1 * Enhancement meaning the absolute value – hence the omitted “-” sign Voet. Voet & Pratt 2013 Chap 11 .416 J mol-1 = 11.605) = ΔΔG‡ 11.314514 J ºK-1 mol-1 x 298.97 J mol-1 ) x (4.42 kJ mol-1 = ΔΔG‡ 10-fold: 5.

speeds up both reactions! ΔG effects the likelihood of reactant going to products OR products going to reactants based on the free energy difference between reactants and products. Effect of ΔG‡ versus ΔG Important Concepts! A+ B  P+ Q ΔG‡ effects the likelihood of reactants going to products AND products to reactants . thermodynamics ΔG < 0 forward reaction favored A+ B  P+ Q .

CATALYTIC MECHANISMS Enzymes are effective as catalysts due to: 1) their ability to rearrange covalent bonds using their various amino acid side chains. metal ions and coenzymes. . 2) their ability to specifically bind the substrate molecule in an enzyme-substrate complex and to use noncovalent interactions for binding to significantly lower the free energy.

Preferential Binding of the Transition State Complex . Acid-Base Catalysis 2. Covalent Catalysis 3. Electrostatic Catalysis 5. Types of Enzyme Catalytic Mechanisms 1. Proximity and Orientation Effects 6. Metal Ion Catalysis 4.

Voet & Pratt 2013 Fig 11.8 . Acid-Base Catalysis Mechanism: Keto-Enol Tautomerization ketone Uncatalyzed Hydroxyl “ol” “ene” acid General Acid Catalyzed General Base Catalyzed base δ ≡ partial charge Voet.

• General Acid Catalysis: A process in which partial proton transfer from an acid lowers ΔG‡ and accelerates the reaction.ions present in water. Types of Acid-Bases Catalysis • Specific Acid-Base Catalysis: Non-enzymatic reactions: Uses only the H+ on OH. . • General Base Catalysis: A process in which partial proton extraction by a base lowers ΔG‡ and accelerates the reaction. (No other molecules involved) Ions are transferred between water and the intermediate faster than the intermediate breaks down to reactants.

Amino Acids in General Acid-Base Catalysis Lehninger 2005 Figure 6.9 .

pH and Enzyme Activity Lehninger 2005 Figure 6.17 .

3’Cyclic ribonucleotide intermediate Acid catalysis Voet.10 . Voet & Pratt 2013 Fig 11.RNase A Mechanism: An Acid-Base Catalysis Base catalysis 2’.

Nucleophilic Rx between catalyst & substrate to form covalent bond.11 . 2. Covalent Catalysis: Decarboxylation of Acetoacetate uncatalyzed carbonyl elimination decarboxylation covalent bond electron withdrawal Transition state Typical Stages: 1. Elimination of the catalyst (reverse of first step) Voet. Voet & Pratt 2013Fig 11. 3. Withdrawal of electrons from reaction center by the (now) electrophilic catalyst.

the less easily it can degrade. Covalent Catalysis • Covalent Catalysis (nucleophilic catalysis) definition: accelerates the reaction via the transient formation of a catalyst-substrate covalent bond. . Therefore good candidates for covalent catalysis like imidazoles and thiols have high polarizability (mobile electrons) have high nucleophilicity and also can form good leaving groups. The typical way this happens is a nucleophile group on the enzyme forms a covalent bond with an electrophile group on the substrate. • Important – The more stable the covalent bond formed in the transition state.

12 . Voet & Pratt 2013 Fig 11. Biologically Important Electrophiles & Nucleophiles “The nucleophilicity of a substance is closely related to its basicity” Voet.

Metal Ion Catalysis: Carbonic Anhydrase Participate in catalytic process by: • Binding to substrates to properly orient them for Rx • Mediating redox Rx via reversible metal ion oxidation state changes. Voet. Active site opening charge shielding Nearly 1/3 of all enzymes utilize a metal ion for their catalytic function. • Electrostatic CO2 + H2O HCO3. Voet & Pratt 2008 Fig 11.+ H+ stabilization or neg.13 .

Electrostatic Catalysis The charge distribution around enzyme active sites appears to be arranged to stabilize the transition state of the catalyzed reactions. .

• They bring substrate into contact with catalytic groups & multiple substrates with each other. Proximity & Orientation Effects Enzymes tend to be catalytically efficient. Voet & Pratt 2006 p. 328 . ~ 5-fold boost • They bind their substrates in the proper orientation to promote the reaction ~100-fold boost • They freeze out relative translational and rotational motion of catalytic groups and substrates – in transition state little relative motion of catalytic groups ~107-fold boost Voet.

Preferential Transition State Binding S ES “An enzyme may bind the transition state with greater affinity than the substrates or products” This increases the molecules in the transition state.15 . thus proportionally increasing the reaction rate Lehninger 2013 Figure 11.

Kinetics The study of reaction rates .

be able to plot it and extract kinetic constants. • Know the nomenclature for enzyme kinetics. • Understand the concepts of Km and Vmax. how their L-B plots look. BiBi and ping-pong) and how that effects their kinetic equations. and how to get quantitative information from those plots. Goals -Kinetics • Know the difference between 1st and 2nd order reactions and their half-lives. • Understand how pH and temperature (Arrhenius eq. • Understand basic types of multisubstrate enzymes (sequential and random.) affect enzyme activity . • Understand the difference between rapid-equilibrium and steady-state approaches to enzyme kinetics. • Know the various types of enzyme inhibition. Be aware that there are other types of kinetic plots • Understand enzyme reaction reversibility and how that affects the kinetic equation. • Know the Michaelis-Menten equation and how to plot it. • Understand the Lineweaver-Burke Plot.

Write the velocity equations. . Substitute in the differential equations the expression for the enzyme species. Write differential equations for each enzyme species. 3. 2. Setting Up A Kinetic Analysis 1.

Internet Explorer Demonstration .

Reaction Velocity The instantaneous rate of product appearance or reactant disappearance. k is proportional to the frequency at which the reacting molecules come together. the molecularity .e. Chemical Kinetics – Elementary Rxs Reaction Stoichiometry A→ P substance A. Reaction Order The number of molecules that must simultaneously come together (collide) to generate a product. product P A → I1 → I2 → P intermediates In Rate Constant k The proportionality constant at constant temperature describing the rate of an elementary reaction. i.

Reaction Order – First Order .

i. First Order Reaction A → P First Order Equation: d[P] d[A] v= dt = . dt = k[A] The reaction at time t is proportional to the concentration of A.1 .e. it is a constant Voet. Voet & Pratt 2013 Fig 12. Ao is the initial reactant concentration k is in s-1 units For first order: The half-life is independent of [A0].

Determining order & rate constant for Irreversible 1st order Rx Matthews et al.1999 Figure 11.1 .

dt = .Second Order Reactions (Bimolecular) 2A → P Second Order Equation: d[A] v= dt = k[A]2 k is in M-1 S-1 units 1 1 [A] = [Ao] + kt Half-life : t½ = 1/k[Ao] A+B →P Second Order Equation: d[A] d[B] v = .dt = k[A][B] k is in M-1 S-1 units Pseudo first order (for B if [A] >>[B] & vice versa) .

k ⌡0 dt ln [A] = ln[Ao] – kt or [A] = [Ao] e-kt . Rate Equation Describes reaction progress as a function of time.k dt Integrate from [Ao] to [A] ⌠ [A] ⌠t ⌡ [ Ao] d ln [A] = . Derivation: (where t = time) d[A] [A] = d ln[A] = .

Half Life Calculation First Order k t½ = ln [A0]/2 [Ao] t½ = ln 2/k = 0.693 / k Half Life: t½ Second Order t½ = 1/(k [Ao]) .

Enzyme Kinetics The study of enzyme reaction rates Lehninger 205 Figure 8.6 .

[E] enzyme concentration: [E]T total enzyme ES enzyme substrate complex [ES] complex concentration P. Enzyme Kinetic Definitions: E Enzyme.… “products” A. v = reaction velocity vo = initial reaction velocity. when [P] ~ 0 . Q. R. C …. “substrates” I. J. B. K… “inhibitors” k rate constant k1 forward rate constant k-1 reverse rate constant kp the catalytic rate constant.

Therefore. [S].203 . & Ks. an equilibrium expression. one has to make certain assumptions for calculations Rapid Equilibrium (Henri-Michaelis-Menten) Early components of the reaction are at equilibrium. Lehninger 2005 Figure p.e. Two Approaches to Enzyme Kinetics Problem: One does not know the concentration of ES during a reaction. One still needs a velocity equation to insert expression for [ES]. Steady-State (Briggs-Haldane) {most used approach} Shortly after the reaction starts [ES] would reach a steady-state and this would be close to the equilibrium level. Permits one to express [ES] in terms of [E]. i.

Henri-Michaelis-Menten I
Unireactant Enzyme
E + S k1
k-1 ES k2
E + P
E, S, and ES equilibrate very rapidly compared to formation of E + P

Instantaneous velocity depends on [ES]  v = k2 [ES] (1)
The total enzyme is written: [E]T = [E] + [ES] (2)

Divide equation (1) by [E]T
v = k2 [ES]
[E]T [E] + [ES] (3)

Henri-Michaelis-Menten II
E + S k1
k-1 ES k2
E + P
With rapid equilibrium assumption [ES] can be expressed in terms of
[S], [E] and Ks (the dissociation constant of the ES complex)
Ks = [E] [S] = k-1  [ES] = ([S]/ Ks) [E] (4, 5)
[ES] k1
Substitute for [ES] into eq. 3 using eq 5.

v = k2 ([S]/Ks)[E]
[E]T [E] + ([S]/Ks)[E] (6)

Henri-Michaelis-Menten III
E + S k1
k-1 ES k2
E + P
Cross multiple by k2 and cancel out [E] (on right side)
v = ([S]/Ks)
k2 [E]T 1 + ([S]/Ks) (6)

If v = kp [ES] then kp [E]T = Vmax (enzyme is saturated) (7)

v = ([S]/Ks)
Vmax 1 + ([S]/Ks) (8)
v = [S]
Vmax Ks + [S] (9)

Voet & Pratt 2013 Fig 12.2 .A Simple Enzyme Reaction and the Steady-State (Progress Curves) Voet.

. and ES will not be in equilibrium. Steady-State (Briggs-Haldane) I Unireactant Enzyme E + S k1 k-1 ES k2 E + P If the rate ES forms E + P is >> the rate ES goes back to E + S (K2 > k-1) and [S] >> [E]T then E. i. S. Instantaneous velocity is v = k2 [ES] (1) v = k2 [ES] [E]T [E] + [ES] (from before) (2) If [ES] is constant. then the rate that ES forms is equal to the rate that ES decomposes. d[ES]/dt = 0 .e.

e. k1 ES formation: E + S → ES (3) kp k-1 ES decomposition: ES → E + P andES → E + S (4) rate of ES formation: = k1 [E][S] (5) rate of ES decomposition: = k-1 [ES] + k2 [ES] (6) = (k-1 + k2) [ES] (6a) . d[ES]/dt = 0 . then the rate that ES forms is equal to the rate that ES decomposes. Steady-State (Briggs-Haldane) II E + S k1 k-1 ES k2 E + P If [ES] is constant. i.

8 for [ES] k1[E][S] (9) [ES] = (k-1 + kp) define Michaelis constant k-1 + kp [S] (for above reaction) km = k1  [ES] = km [E] . (7) rate of ES formation = rate of ES decomposition k1 [E][S] = (k-1 + kp) [ES] (8) Solve eq. Steady-State (Briggs-Haldane) III E + S k1 k-1 ES kp E + P At steady-state. d[ES]/dt = 0 .

Steady-State (Briggs-Haldane) IV E + S k1 k-1 ES k2 E + P v = ([S]/Km) = v = [S] Vmax 1 + ([S]/Km) Vmax Km + [S] (10a. saturated.e. . i.b) Can rearrange ES expression k-1 + k2 [S] [E] Km = k1  = [ES] When [S] = Km Km v= Km + Km Vmax = ½Vmax Vmax = k2 [E]T the highest velocity when all of the enzyme is [ES].

k1 [E] [S] (13) d[P] dt = (k2) [ES] (14) Also know that: [E]T = [E] + [ES] (15) Segal Enzyme Kinetics 1975 p.k1 [E] [S] (11) d[ES] dt = k1 [E] [S] .(k-1 + k2) [ES] (12) d[S] dt = (k-1) [ES] . 28 . Steady-State (Briggs-Haldane) V Differential Equations for Unireactant Enzyme E + S k1 k-1 ES k2 E + P One can write 4 differential equations to describe the above reaction d[E] dt = (k-1 + k2) [ES] .

Steady-State Kinetic Plot .

12 Voet. Initial Velocity vs Substrate [ ] in a Simple Enzyme Reaction Michaelis-Menten equation o Lehninger 2000 Figure 8. Voet & Pratt 2013 Fig 12.3 .

HTML DISPLAY Michaelis-Menten Kinetics 12-1b_MichaelisMenten\MichaelisMenten.htm .

2. If Km is known the assay conditions can be altered so that [S] >> Km so that Vmax can be determined which is a measure of [E]T. Significance of Km Operational Definition: The substrate concentration at which the reaction velocity is half maximal. 218 . when Km = [S] then vo = ½Vmax 1. 3. i.e. The substrate with the lowest Km has the highest affinity for the enzyme. or from the same tissue at different developmental stages (Vmax is not a constant but depends on kp and [E]T. 5. The “best” substrate has the highest Vmax/Km ratio. Km can be altered by ligand binding – one mode of enzyme regulation. Since it is constant for a given enzyme/substrate.) Km will vary with temperature and pH. It establishes an approximate value for the intracellular level of a substrate. Segal Biochemical Calculations 1976 p. 4. its numerical value provides a means of comparing enzymes from different organisms or from different tissues of the same organism. It indicates the relative “suitability” of alternate substrates for an enzyme.

e. more rate constants. i. When [S] << Km very little ES is formed and then [E] ≈ [E]T Vo ≈ (k2/Km)[E]T [S] ≈ (kcat/Km) [E] [S] Under these conditions (kcat/Km) is a measure of the enzyme’s catalytic efficiency since this apparent second order rate constant (depends on BOTH [E] and [S]) the rate of the reaction depends on how often E and S encounter each other in solution. Most efficient enzymes have this ratio near the diffusion-controlled limit: 108 – 109 M-1s-1 kcat/Km ≤ k1 .turnovers) that each active site catalyzes per unit time (Note: some enzymes have more than one active site) However. Catalytic Efficiency & Turnover Number Define the catalytic constant: kcat = Vmax/[E]T This is also known as the turnover number – the number of reaction processes (number of moles of substrate transformed per minute per mole of catalytic site under optimal conditions . more complex enzymes have a more complicated expression for kcat.

Kinetic Data Analyses Michaelis-Menten Michaelis-Mentenequation equation o .

Voet & Pratt 2013 Fig 12. Lineweaver-Burke Plot y = (m) x + b 1 Km 1 + 1 vo Vmax [S] Vmax Voet.4 .

Eadie-Hofstee Plot y = m * x + b v Vmax Km* v + [S] Km Matthews et al.1999 Figure 11.17 .

Reversibility I Effect of Product on Forward Reaction k1 k2 k3 E +S ES EP E+P k-1 k-2 k-3 The direction of the reaction When [P] = 0 Vmaxf [S] will depend upon the ratio of vf = [P]/[S] relative to Keq (initial) Kms + [S] When [S] = 0 Vmaxr [P] vr = (final) Kmp + [P] .

vnet = k2 [ES] .k-2 [EP] vnet k2 [ES] .k-2 [EP] [E]t [E] + [ES] + [EP] Know [ES] = ([S]/Ks) [E] and [EP] = ([P]/Kp) [E]. substitute in vnet k2 ([S]/Ks) [E] .k-2 ([P]/Kp) [E] Factor [E] out [E]t [E] + ([S]/Ks) [E] + ([P]/Kp) [E]  . Reversibility II Effect of Product on Forward Reaction k1 k2 k3 E +S ES EP E+P k-1 k-2 k-3 From rapid equilibrium assumptions set up net velocity eqs.

Vmaxr ([P]/Kmp) vnet 1+ ([S]/Kms)] + ([P]/Kmp) Final equation in terms of steady-state . Reversibility III Effect of Product on Forward Reaction k1 k2 k3 E +S ES EP E+P k-1 k-2 k-3 Multiply k2 [E]t([S]/Ks) .k-2 [E]t ([P]/Kp) right side vnet by [E]t 1+ ([S]/Ks)] + ([P]/Kp) Vmaxf ([S]/Kms) .

Multisubstrate Enzymes Voet. Voet & Pratt 2013 Figure 12.5 .

Sequential Reaction Mechanisms “Reactions in which all substrates must combine with the enzyme before a reaction can occur and products are released” E A+ B P + Q .

367 . Voet & Pratt 2013 p. Sequential Mechanisms: Types Ordered Bi-Bi Random Bi-Bi Voet.

14a .Bisubstrate Rx Steady-State Kinetic Analysis: Random BiBi Lehninger 2000 Figure 8.

Ping-Pong Non-Sequential Reaction Mechanisms “Group transfer reactions in which one or more products are released before all susbtrates have been added.” .

Non-Sequential Mechanism
MgATP MgADP glucose Glucose-6-P
Ping-
Pong
Bi-Bi

V [A][B]
Vmax Kma[A] + Kmb[B] + [A][B]

Voet, Voet & Pratt 2013 p. 367

Bisubstrate Rx Steady-State Kinetic
Analysis: BiBi Ping Pong

Lehninger 2000 Figure 8.14b

King-Altman Diagrams

Enzyme Inhibition Inhibitor .Any substance that reduces the velocity of an enzyme-catalyzed reaction.Any substance that irreversibly binds to an enzyme.) . (appears similar to noncompetitive inhibition. Reversible Types: • Competitive • Uncompetitive • Mixed (Noncompetitive) Inactivator .

Voet & Pratt 20 Figure 12.8 k2[E]T [E] + ([S]/Km) [E] + ([I]/KI) [E] v ([S]/Km) [S] [S] Vmax 1 + ([S]/Km) + ([I]/KI) Km(1 + ([I]/KI)) +[S] αKm + [S] . Competitive Inhibition Kmapp = Km [1 + ([I]/Ki)] KI = [E][I] / [EI] v = k2 [ES] vmax = k2[E]T v k2 [ES] [E]T [E] + [ES] + [EI] v ([S]/Km) [E] Voet.

Voet & Pratt 2013 Figure 12.20 Voet. Competitive Inhibition Matthews et al.1999 Figure 11.7 .

Uncompetitive Inhibition: M-M enzyme in k1 k-1 k 2 L-B Plot KI = [ES][I] / [ESI] v = k2 [ES] vmax = k2[E]T v k2 [ES] [E]T [E] + [ES] + [ESI] Voet. Voet & Pratt 2013 Fig 12.9 v ([S]/Km) [E] vmaxI = vmax / /(1 + ([I]/KI)) k2[E]T [E] + ([S]/Km) [E] + ([S][I]/KmKI) [E] v ([S]/Km) v [S] [S] Vmax 1 + ([S]/Km) + ([S][I]/Km KI) VmaxI (Km/(1 + ([I]/KI)) +[S] αKm + [S] .

Mixed Inhibition: M-M enzyme in L-B Plot KI = [E][I]/[EI] and K’I = [ES][I]/[ESI] v k2 [ES] [E]T [E] + [ES] +[EI] + [ESI] v [S] v [S] Vmax Km (1+ ([I]/KI)) + [S] (1+ ([I]/K’I)) VmaxI αKm + α’ [S] Voet. Voet & Pratt 2013 Fig 12.10 .

2 .9 Voet. Voet & Pratt 2013 Table 12. Michaelis-Menten Equations: Effects of Inhibitors Table An example for competitive inhibitor the X intercept is: 1/Km app =1/αKm = 1 /(1+[I]/Ki)Km Lehninger 2005 Table 6.

Temperature Effects Enzyme Activity .

to free energy (thermodynamics) dP/dt = k’ e-ΔG‡/RT [A] [B] substituting into the first equation k’ =  define  vibra. Kinetics. energy of the vibration for the decomposition on X‡ . bond breaks as X‡ to products and  prob. and Transition State Theory I K‡ k’ A+ B X‡ P+Q dP/dt = k [A][B] = k’ [X‡] under rapid equilibrium assumption write: K‡ = [X‡] / [A][B] equilibrium constant expression ΔG‡ = -RT ln K‡ relate equil. (0 -1) ν = ε/h h is Planck’s const. X goes to products rather than reactant. Thermodynamics. freq. const. ε is the avg.

Mech. The energy of a classical oscillator at T. Thermodynamics.6261 x 10-34 J s k’ = kBT/h via substitution k = (kBT/h) e-ΔG‡/RT = A e -ΔG‡/RT . (the available thermal energy) where kB = Boltzman constant 6. and Transition State Theory II K‡ k’ A+ B X‡ P+Q ε = k BT from Stat. Kinetics.

303R log k = log A –Ea/2.303RT Matthews et al.1999 Figure 11.3 . Arrhenius Equation log A k = Ae-Ea/RT Slope ln k = ln A –Ea/RT or -Ea/2.

17 .pH Effects Enzyme Activity Lehninger 2004 Figure 6.

phosphorylation and dephosphorylation Voet.g. e. Voet & Pratt 2013 0. Enzyme Regulation Control of Enzyme availability Cells control the rate of enzyme synthesis and degradation and thus can control the amount of enzyme present. • via covalent modification of an enzyme. Control of Enzyme activity • via binding of small molecule “allosteric effectors” that alter catalytic activity.381 .

11 . Allosteric Effect 2.0 mM 0.4 mM Voet. Voet & Pratt 2013 Fig 12.

Kinetics and Mechanism The steady-state kinetic analysis of a reaction cannot unambiguously establish the reaction’s mechanism .

Bisubstrate Rx: Examples Transferase Redox Voet. Voet & Pratt 2008 Fig 12.5 .

age. e. • Cytochromes P450: superfamily of enzymes – occur in nearly all living organisms. and environmental factors” • Cytochromes P450: a major function is to detoxify xenobiotics – involved in metabolic clearance of a many of drugs in use today.389 . • Human genome has 57 isoforms -33% of isoforms are in liver • Nomenclature: P450 isozymes are named by the letters CYP followed by a number designating its family. and another number. Voet & Pratt 2012 p. Cytochrome P450 & Drug Metabolism • “Differences in reactions to drugs arise from genetic differences among individuals as well as differences in their disease states.g. sex. other drugs they are taking. an uppercase letter designating its subfamily. CYP2D6 Voet.

• PAH polycyclic aromatic hydrocarbons (carcinogenic – in smoke. glycine. Bad: Sometimes converts an innocuous compound to a toxic agent . Cytochrome P450 & Drug Metabolism II Typical reaction type: RH + O2 + 2 H+ + 2e. broiled meat) • PCB polycyclic biphenyls (electrical insulators. sulfate & acetate – further increases water solubility Good: Allow xenobiotics to be detoxified and/or cleared.g. acetaminophen metabolism at high dosages . e. RH. plasticizers) • steroids (P450s also involved in their biosynthesis) • drugs (many types) Convert Xenobiotics to a more water soluble form to facilitate excretion • New hydroxyl group can be enzymatically conjugated to glucuronic acid.→ ROH + H2O • Different Cyt P450 are SPECIFIC for particular types of a wide variety of typically lipophilic compounds.

Both drugs given together – Bioavailability of drug B increases. Case 1: Drug A metabolized by or inhibits Cyt P450 isozyme. Both drugs given together. What happens to rate of Drug B metabolism? Why? What is the resultant problem? . Why? Case 3: Additionally Drug B is metabolized to a toxic product. Same isozyme metabolizes drug B.Cytochrome P450 & Drug Metabolism III Cyt P450s often mediate drug-drug interactions. Why? What if drug B has a low therapeutic index? Case 2: Drug A induces increased expression of the particular isozyme of Cyt P450 Both drugs given together – Bioavailability of Drug B decreases. Text Example: Two drugs A & B.

End of Lectures .