Advanced Enzyme Kinetics and Metabolism BOC 324 Part A Dr. A. - PowerPoint PPT Presentation

Advanced Enzyme Kinetics and Metabolism BOC 324 Part A Dr. A. van Tonder (for 3 rd quarter; Part B in 4 th quarter with Dr. E. van Heerden)

BOC 324 Part A SOURCES Textbook: “Biochemistry” Mathews et al : Ch 11 pp. 360-413 Internet Resources: http://www-biol.paisley.ac.uk/kinetics/contents.html http://www.cf.ac.uk/biosi/staff/kille/dentals/dental5_99/ Articles: Articles 1-8 are in the study guide

Advanced Enzyme Kinetics TOPICS:  1. Enzyme kinetics: Basics  2. Determination of kinetic constants  3. Kinetics of enzyme inhibitors  4. Kinetics of multisubstrate reactions  5. Kinetics of allosteric enzymes

MIND MAP

1. Enzyme Kinetics: Basics  Contents  Revision : BOC226 work (Ch 11 in Mathews) plus Internet sources plus Wikipedia (article #8)  Steady state models for 1S, 1P when [S]>>[E]  The effect of [S] on v  The effects of [E]  The meaning of k cat and k cat /K m  The significance of K m , k cat and k cat /K m

REVISION What is an enzyme? It is a BIOLOGICAL CATALYST!!!  The reaction catalysed by an enzyme uses exactly the same reactants and produces exactly the same products as the uncatalysed reaction.  Like other catalysts, enzymes do not alter the position of equilibrium between substrates and products.  However, unlike uncatalysed chemical reactions, enzyme-catalysed reactions display saturation kinetics.

 For a given enzyme concentration and for relatively low substrate concentrations, the reaction rate increases linearly with substrate concentration; the enzyme molecules are largely free to catalyze the reaction, and increasing substrate concentration means an increasing rate at which the enzyme and substrate molecules encounter one another:

 What an enzyme does: e.g.:

 The reduction of activation energy (ΔG) increases the number of reactant molecules with enough energy to reach the activation energy and form the product.  By providing an alternative reaction route and by stabilizing intermediates the enzyme reduces the energy required to reach the highest energy transition state of the reaction.  Not so simple – may look like this:

 The favored model for the enzyme-substrate interaction is the induced fit model of Daniel Koshland (1958)....  This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding.

Catalysis by induced fit - Stabilising effect of strong enzyme binding. - Two different mechanisms of substrate binding: uniform binding: strong substrate binding, differential binding: strong transition state binding. - The stabilizing effect of uniform binding increases both substrate and transition state binding affinity, while differential binding increases only transition state binding affinity.

 These conformational changes also bring catalytic residues in the active site close to the chemical bonds in the substrate that will be altered in the reaction.  After binding takes place, one or more mechanisms of catalysis lowers the energy of the reaction's transition state, by providing an alternative chemical pathway for the reaction.  There are five possible mechanisms of "over the barrier" catalysis as well as a "through the barrier" mechanism (see Wikipedia article for detail): - Catalysis by bond strain - Catalysis by proximity and orientation - Catalysis involving proton donors/acceptors (Acid/Base Catalysis) - Electrostatic catalysis - Covalent catalysis - Quantum tunnelling

1. Catalysis by bond strain - The affinity of the enzyme to the transition state is greater than to the substrate itself. - Induces structural rearrangements which strain substrate bonds into a position closer to the conformation of the transition state, so lowering the energy difference between the substrate and transition state and helping catalyze the reaction. 2. Catalysis by proximity and orientation - Increases the rate of the reaction as enzyme-substrate interactions align reactive chemical groups and hold them close together. - This reduces the entropy of the reactants and thus makes reactions such as ligations or addition reactions more favourable - There is a reduction in the overall loss of entropy when two reactants become a single product.

3. Catalysis involving proton donors/acceptors (Acid / Base Catalysis) - Proton donors and acceptors, i.e. acids and bases, may donate and accept protons in order to stabilize developing charges in the transition state. - Typically has the effect of activating nucleophile and electrophile groups, or stabilizing leaving groups. 4. Electrostatic catalysis - Stabilization of charged transition states can also be by residues in the active site forming ionic bonds (or partial ionic charge interactions) with the intermediate. - These bonds can either come from acidic or basic side chains found on amino acids such as Lys, Arg, Asp or Glu or come from metal cofactors such as zinc.

5. Covalent catalysis - Involves the substrate forming a transient covalent bond with residues in the active site. - Adds additional covalent intermediate to the reaction, and helps to reduce the energy of later transition states of the reaction. - Covalent bond must, at a later stage in the reaction, be broken to regenerate the enzyme. - Found in enzymes such as proteases like chymotrypsin and trypsin, where an acyl-enzyme intermediate is formed. 6. Quantum tunnelling - Some enzymes operate with kinetics which are faster than predicted. - In "through the barrier" models, a proton or an electron can tunnel through activation barriers. - Quantum tunnelling for protons has been observed in tryptamine oxidation by aromatic amine dehydrogenase. - Does not appear to provide a major catalytic advantage.

 The two most important kinetic properties of an enzyme are: 1. how quickly the enzyme becomes saturated with a particular substrate, and 2. the maximum rate it can achieve.  Knowing these properties suggests what an enzyme might do in the cell and can show how the enzyme will respond to changes in these conditions. k 1 k cat v 0 = V max [S] [E] + [S] [ES] [E] + [P] K m + [S] k - 1

 Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes, with a focus on their reaction rates.  The study of an enzyme's kinetics reveals the catalytic mechanism of this enzyme, its role in metabolism, how its activity is controlled, and how a drug or a poison might inhibit the enzyme. Dihydrofolate reductase from E. coli with its two substrates, dihydrofolate (right) and NADPH (left), bound in the active site.

What is the most plentiful single enzyme on earth? Answer: ribulose bisphosphate carboxylase / oxygenase (or RUBISCO) - Catalyses the attachment of carbon dioxide to ribulose bisphosphate, a short sugar chain with five carbon atoms and then clips the lengthened chain into two identical phosphoglycerate pieces. - Why so abundant? It fixes only about three carbon dioxide molecules per second so plants make more of it – half of the protein in chloroplasts is Rubisco. 2 x 8 protein chains

Steady State Models for 1S, 1P Assumptions and Givens: [P] [S] Concentrations  d[ES]/dt = O (Steady state)  [P] = 0 at t = 0 [E] t Cannot  v = d[P]/dt = k cat [ES] [ES] measure [E]  [E] t = [E] + [ES] Time  V max = k cat [E] t Pre-steady Steady state  K m = {k -1 + k cat }/k 1 state ES almost constant ES forming = [S] ½ at V 0 = ½V max k 1 k cat [E] + [S] [ES] [E] + [P]  v 0 = V max [S] = k cat [E] t [S] k - 1 K m + [S] K m + [S] [S] (mM) >> [E] t ( 10 -8 - 10 -10 M) Michaelis-Menten [S] changes, [E] t constant

MICHEALIS-MENTEN KINETICS V [ S ] Leonor max 0 v Maud Menten 0 Michaelis K [ S ] (1879-1960) m 0 (1875-1949) 1913

M-M: The Effect of [S] on v  Low [S] : [S] << K m v 0 = V max [S]  V 0 = {V max /K m }[S] K m + [S]  V 0 [S]  First order reaction  [S] = Km  V 0 = {V max [S]}/2[S]  V 0 = ½V max  [S] > K m :  mixed order reaction  High [S] : [S] >> K m  V 0 = {k cat [E] t [S] }/[S] = k cat [E] t = V max  Zero order reaction  E is saturated with S

The Effects of [E] High [S] : [S] >> K m v 0 = k cat [E] t = V max V max [E] t K m independent of [E]

Deviations from Michaelis-Menten kinetics v o v o [S] o [S] o Substrate inhibition Positive co-operativity

Deviations from Michaelis-Menten kinetics v o v o [S] o [S] o Negative co-operativity Alternative pathways EA E EAB Products EB

Deviations from Michaelis-Menten kinetics v o v o [S] o [S] o Two or more molecules Failure to determine v o of the same substrate

Deviations from Michaelis-Menten kinetics v o tot V m a V m a b V [ S ] V [ S ] max 0 max 0 v b V m 0 a b K [ S ] K [ S ] m 0 m 0 [S] o a b K m K m More than one enzyme catalysing the same reaction

Deviations from Michaelis-Menten kinetics Enzyme reaction plus blank rate v o Blank rate [S] o Failure to subtract blank rate