Enzyme Kinetics… Virtually All Reactions in Cells Are Mediated by Enzymes • Enzymes catalyze thermodynamically favorable reactions, causing them to proceed at extraordinarily rapid rates • Enzymes provide cells with the ability to exert kinetic control over thermodynamic potentiality • Living systems use enzymes to accelerate and control the rates of vitally important biochemical reactions • Enzymes are the agents of metabolic function Enzymatic catalysis Role of enzymes • serve the same role as any other catalyst in chemistry • act with a higher specificity • acid and base catalyst possible due to proximal locations of amino acids • alter the structure location of quantity of the enzyme and you have regulated the formation of product - try that in organic chemistry ! • Don't lose sight of what enzymes do. catalyze reactions – nothing more reactants -> products. What do Enzyme ACTUALLY do? Naming Enzymes •Enzyme commission for each enzyme based on the type of reactions. Kind of like IUPAC •Yeah right - the mother of invention – he/she who finds/discovers/purifies/clones the enzyme, names if. Trivial names rule… • First recognition of an enzyme and naming – 1833: alcohol precipitate of malt extract converted starch into sugar – called diatase (first use of suffix –ase) • Proteases traditionaly are the only enzymes which don’t end in ‘ase’ and use ‘-in’ • Enzyme is coined after the Greek for “in yeast” • 1050s started need to use a standardized naming: Otto Hofmann-Ostenhof and Dixon & Webb – common, non-systematic names still rule. Enzyme Nomenclature Provides a Systematic Way of Naming Metabolic Reactions O T H L I L Enzyme reaction types: Enzymes are classified into six major classes based on the reaction they catalyze. 1) Oxidoreductases catalyze oxidation-reduction reactions. Most of these enzymes are known as dehydrogenases, but some are called oxidases, peroxidases, oxygenases or reductases. 2) Transferases catalyze group-transfer reactions (functional groups). Many require the presence of coenzymes. A portion of the substrate molecule usually binds covalently to these enzymes or their coenzymes. This group includes the kinases! 3) Hydrolases catalyze hydrolysis. They are a special class of transferases, with water serving as the acceptor of the group transferred 4) Lyases catalyze nonhydrolytic and nonoxidative elimination reactions, or lysis, of a substrate, generating a double bond. In other words these reactions catalyze group elimination to form double bonds. A lyase that catalyzes addition reaction in cells is often termed a synthase. 5) Isomerases catalyze the isomerase reactions. Because these reactions have only one substrate and one product, they are among the simplest enzymatic reactions. 6) Ligases catalyze ligation or joining, of two substrates. These reactions require the input of chemical potential energy of a nucleoside triphosphate such as ATP. i.e. bond formation coupled to ATP hydrolysis. Ligases are usually referred to as synthetases. Often involved in joining carbon atoms to N, S or O atoms. 1 Enzyme specificity - One of the most powerful actions of enzymes. – Group complementation - the ability to recognize specific regions of the substrate to align reactants with catalytic site. – Based on non-covalent molecular interactions. – Lock and key vs. induced fit - both occur. Induced fit takes place when binding of one part of the substrate to the enzyme alters the conformation of the enzyme to make a true "fit" – Binding does not mean activity Enzyme specificity An enzyme can bind and react stereo-specifically with chiral compounds This can happen due to a three point attachment – Binding can then only occur in one way and therefore the products are not a mixture. General Information on Enzymology Enzyme nomenclature • Active site • Substrate vs. reactant • Prosthetic groups, coenzyme and cofactors • Activity vs. reaction • Suffix -ase Another important reminder - not all activity is on or off. Many times the enzyme has a low or constitutive activity that can be increased many times. It is a common mistake to think of enzymes being on or off. Job of Enzyme Catalysts • Specifically bind substrate(s) (reactants), decrease transition state energy to increase the rate (NOT thermodynamics) of a favorable reaction: • High specificity • Low side reactions • Ability to be regulated: Active site – amino acids and cofactors binding and “doing” the reaction, are regulated by the rest of the protein How do enzymes work? • By reducing the energy of activation • This happens because the transition state is stabilized by a number of mechanisms involving the enzyme/protein • Without enzymes reactions occur by collisions between reactants or addition of various organic catalysts • The energy barrier between the reactants and product, is the free energy of activation. • The free energy (DG) of the reaction is what determines if a reaction is spontaneous. Reactions are often multistep Two intermediate transition states. • Two step reactions will have a slow and fast step – based on activation energy • The higher activation step is the rate-limiting step of the reaction Enzyme Kinetics • Kinetics is the branch of science concerned with the rates of reactions • Enzyme kinetics seeks to determine the maximum reaction velocity that enzymes can attain and the binding affinities for substrates and inhibitors • Analysis of enzyme rates yields insights into enzyme mechanisms and metabolic pathways • This information can be exploited to control and manipulate the course of metabolic events 2 Rates of a reaction… Molecularity – the number of molecules that must collide/interact for a reaction to occur. • For enzymes, molecules that are involved at the active site for a step of the reaction. • Unlike non-enzyme catalyzed reactions, the rate is too complex but often initially described as unimolecular or bimolecular • Remember, order – refers to rate equation, while molecularity defines the mechanism Gen Chem – Rates of Reaction… Quick Review – MOST enzymes catalyze reactions in a first or second order reaction. - Less about collisions of freely moving molecules than binding and reacting on a surface of a catalyst (enzyme active site): For single/uni-molecularity reactions – 1st order A->P The rate is proportional to the concentration of substrate Enzyme Kinetics Kinetic defn.: Of or relating to or produced by motion. Kinetics defn.: the branch of chemistry or biochemistry concerned with measuring and studying the rates of reactions. Enzyme kinetics are defined differently than basic chemistry kinetics because of the large catalyst… Measuring Rates of Enzyme Catalyzed Reactions Louis Michaelis and Maud Menten's theory • assumes the formation of an enzyme-substrate complex • assumes that the ES complex is in rapid equilibrium with free enzyme • assumes that the breakdown of ES to form products is slower than 1) formation of ES and 2) breakdown of ES to re-form E and S Enzyme Kinetics An enzyme-catalyzed reaction of substrate S to product P, can be written Actually, the enzyme and substrate must combine and E recycled after the reaction is finished, just like any catalyst. Because the enzyme actually binds the substrate the reaction can be written as: The simplest reaction is a single substrate going to a single product. Rate or velocity of the reaction depends on the formation of the ES •The P -> ES is ignored •The equilibrium constant Keq is based on the idea that the reaction is limited to the formation of the ES complex and that only K1 and K-1 are involved because the thermodynamics of the reversal of K2 cause it to be minimal How fast an enzyme catalyzes a reaction is it's rate. The rate of the reaction is in the number of moles of product produced per second [ES] Remains Constant Through Much of the Enzyme Reaction Time Course in Michaelis-Menten Kinetics 3 Time course for a typical enzyme-catalyzed reaction obeying the Michaelis-Menten, Briggs-Haldane models for enzyme kinetics. • The early state of the time course is shown in greater magnification in the bottom graph. Enzyme-Catalyzed Reactions • The relationship between the concentration of a substrate and the rate of an enzymatic reaction is described by looking at the concentration of S and v • At low concentrations of the enzyme substrate, the rate is proportional to S, as in a first-order reaction • At higher concentrations of substrate, the enzyme reaction approaches zero-order kinetics • This behavior is a saturation effect Psuedo First Order Kinetics For the most part enzyme reactions are treated as if there is only one substrate and one product. If there are two substrates, one of them is held at a high concentration (0 order) and the other substrate is studied at a lower concentration • so that for that substrate, it is a first order reaction. This leads us to the M and M equation. •Mathematical model of the representation of the M&M eq. - For the reaction: 1) The Michaelis constant Km is: Think of what this means in terms of the equilibrium. Large vs. a small Km 2) When investigating the initial rate (Vo) the Michaelis-Menten equation is: Graphical representation is a hyperbola. Think of the difference between O2 binding of myoglobin and hemoglobin. •When [S] << Km, the velocity is dependent on [S] •When [S] >> Km, the initial velocity is independent of [S] •When [S] = Km, then Vo = 1/2 V max Understanding Vmax The theoretical maximal velocity Vmax is a constant Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate Vmax is asymptotically approached as substrate is increased Understanding KmThe "kinetic activator constant" Km is a constant Km is a constant derived from rate constants Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S Small Km means tight binding; high Km means weak binding Conditions for Michaelis -Menten Two assumptions must be met for the Michaelis-Menten equation 1) Equilibrium -the association and dissociation of the substrate and enzyme 4 is assumed to be a rapid equilibrium and Ks is the enzyme:substrate dissociation constant.