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Tuesday, November 5th, 2013

Kinetics reaction rates (Eact) Thermodynamics - states under particular set of conditions which way the reaction is going will it go from left to right or from right to left? Takes into account bond energy, randomness, entropy, etc. Enzyme Kinetics The fact that a something is spontaneous in one direction or the other does not mean it is fast. Why do we study kinetics? - Can the reaction be run at a reasonable rate? How does kinetics differentiate between SN1 and SN2 reactions? - Is the rate dependent of one starting material or both? The kinetic behavior of a reaction leads us to certain conclusions of the reaction. Catalysis speeds up a reaction by lowering the energy of activation (Ea) - If Eact is lowered, the reaction is sped up. rate = k[S] k = Ae-Eact/RT k = rate constant Enzymes biological naturally occurring catalysts (biocatalysts) How do enzymes function? How do they lower Eact? - Proximity effect bind - Orientation effect the way it binds A and B - Induced fit In every case, the energy necessary to lower Eact is generated by the binding of the substrate to the enzyme (positive interaction).

First Order Kintetics (S P) First order kinetics is dependent only on one reactant the concentration of S (for example, SN1). S starting material (substrate) P product

Second Order Kinetics (A+B P+Q) Second order depends on two different starting materials..

How can the rate of the following reaction be increased? alkene alkane - Add heat (every 10 increase doubles de reaction) - Increase the concentration of the catalyst (nickel). Increasing the concentration of alkene or hydrogen pressure will not increase the rate of the reaction because the catalyst is the driving force of this reaction.. The catalyst is part of the reaction by bringing H2 and alkene together in some way. Catalyst saturation the surface of the catalyst has limited number of catalytic sites. Once sites are saturated, the reaction cannot go any faster. Enzyme Catalyzed Process Single Substrate Reactions

ES and EP are indistinguishable from one another usually shown as a single species. Single Substrate Catalyzed Reaction


Michaelis-Menten equation:

- Simplifying assumptions made in order to derive to Michaelis-Menten equation: 1st assumption: The contribution of the reverse reaction (product going back to ES) is negligible.. This gives us the following: 2nd assumption: The concentration of enzyme substrate complex after a short induction period is constant. The rate of formation of enzyme substrate complex and the rate of breakdown of enzyme substrate complex is the same. ( ) - Michaelis constant: A collection of rate constant. It is the ratio of rate constants of breakdown divided by the rate constant of formation.

- Cannot make the reaction go faster than Vmax (the maximum rate of the reaction). Vmax cannot be measured but it can be inferred by the limit of the reaction. Under the following conditions: - When [S] << KM, the reaction is first order with the rate directly proportional to the substrate concentration. When you double the substrate concentration, the velocity is doubled. - When [S] >> KM, Vo = Vmax, meaning that the rate is maximal. The reaction is zero order, independent of substrate concentration. - When [S] = KM, then Vo = Vmax/2. KM is equal to the substrate concentration at which the reaction rate is half of its maximal value.

In order to determine KM and Vmax values by hand, the doublereciprocal plot equation can be used:

- The Michaelis-Menten equation is transformed into a double-reciprocal plot by taking the reciprocal of both sides, thus resulting in a straight-line plot. A double-reciprocal plot of enzyme kinetics is generated by plotting as a function . Slope: Vertical axis intercept: Horizontal axis intercept:

The maximal rate, Vmax, reveals the turnover number of an enzyme, which is the number of substrate molecules converted into product by an enzyme molecule in a unit time when the enzyme is fully saturated with substrate. - This equation gives the ratio of molecules transformed per second (turnover number) Catalytic Efficiency Catalytic efficiency the efficiency of the enzymatic process

- Some catalytic efficiency values approach a diffusion maximum.

- The most efficient enzymes have catalytic efficiency values near the diffusioncontrolled limit of 108 to 109 M-1 s-1. Enzyme Inhibition Two types of inhibition: - Irreversible inhibitors Substances by their activity inactivate enzymesdrugs and nerve gases.. - Reversible inhibitors Inhibitor binds to enzyme (with two possible binding sites on the enzyme).

Thursday, November 7th, 2013

Reversible Inhibition Reversible inhibition can be addressed with the Michaelis-Menten model. MichaelisMenten kinetics is kinetics for reversible enzymatic reactions. Three types: two pure forms & one mixed form. - Pure forms: Competitive Inhibition

Inhibitor binds at the same site as substrate. Diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate. o The inhibitor and the substrate are in direct competition for the same site hence, the name. o Who wins is a function on the concentration of substrate and inhibitor & how tightly the binding to each of the two competing elements is which does the enzyme prefer? The substrate or the inhibitor?

As the concentration of a competitive inhibitor increases, higher concentrations of substrate are required to attain a particular reaction velocity. The reaction pathway suggests how sufficiently high concentrations of substrate can completely relieve competitive inhibition.

A double-reciprocal plot of enzyme kinetics in the presence and absence of a competitive inhibitor illustrates that the inhibitor has no effect on Vmax but increases KM. Competitive inhibitor has a steeper slope because the rate decreases with the addition of inhibitor. All the double-reciprocal plots will intersect at . o The point of intersection is Uncompetitive Inhibition

The binding site for the inhibitor is somewhere else than the binding site for the substrate. two binding sites. o May or may not be proximal to one other or vise versa. Free enzyme will not bind inhibitor; only enzyme substrate (ES) binds inhibitor. o Can either go to product or it can bind to an inhibitor to form a ES inhibitor complex (which is dead, no reaction). An uncompetitive inhibitor does not affect the slope of the double-reciprocal plot. Vmax and KM are reduced by equivalent amounts. The intersect in axis changes.

- Mixed (Noncompetitive) Inhibition

Inhibitor can bind to either E or ES. Has two different K1 since they are two different species

Intersection occurs to the left of the axis. Both Vmax and KM are changing

o Pure noncompetitive inhibition when the two K1 are equal. A double-reciprocal plot of enzyme kinetics in the presence and absence of a noncompetitive inhibitor shows that KM is unaltered and Vmax is decreased. KM is unaltered but Vmax changes Wrapping up single substrate reactions: - Single substrate reactions represent a relatively small subset of enzymatic reactions. - At least 60% of enzymatic processes are bimolecular.

Two Substrate Reactions Reversible process Enzyme with two binding sites which can be close or faraway. - Must be close enough from one another in order for the reaction to take place. Multiple substrate reactions divided into two classes: - Single-displacement (sequential) reaction Both A and B are on the enzyme at the same time. MUST be on enzyme in order for the reaction take place. Two subtypes: ordered and random. Ordered the order in which the enzyme binds the two substrates (A and B) is obligatory, it is fixed. It must bind A before it will bind B. o Since this is a reversible reaction, the product that corresponds to A is going to be last thing off the enzyme. The first on is the last off. Random Can go on in any order and can come off in any order. Cleland Notation (Ordered vs. Random) Ordered The coenzyme always binds first and the lactate is always released first. Leading substrate: NADH

Random Either creatine or ATP may bind first, and either phosphocreatine or ADP may be released first.

- Double-displacement (ping-pong) reaction The enzyme operates on the two substrates independent of one another: Takes one substrate, catalyzes a reaction with it, resulting in the enzyme being covalently modified, and finally releases the product that results from this reaction. Takes second substrate, binds to the displaced section of the first substrate, and finally releases the second product. Both A and B are NOT on the enzyme at the same time. Only one substrate product pair is on the enzyme at any point in time. There are two forms of the enzymes: for example, E & E-NH2 (amino transferase). Cleland Notation

1) Takes aspartate 2) Removes amino group from asparte and becomes an amine modified enzyme, resulting in oxaloacetate. 3) Takes -ketoglutarate and binds amine group to it resulting in glutamate.

Can use kinetics parameters to tell the difference between single- and doubledisplacement. - The double-reciprocal plot [Aconc.] vs. rate of reaction

- In these double-reciprocal plots, the concentration of substrate 1 is varied while the concentration of substrate 2 is held constant. This is repeated for several values of [S2], generating several separate lines. Intersecting lines indicate single-displacement reaction (mixed inhibition.). Parallel lines indicate a ping-pong (double-displacement) pathway. Get parallel lines because increasing [S2] does not change the ratio of Vmax to KM. Another way to distinguish between single- and double-displacement: A doubledisplacement enzyme will catalyze a half reaction.

Can use kinetics to distinguish between random and ordered single-displacement processes. - Product inhibition adding product will slow reaction down. - Example: NADH has to be on the enzyme before it will bind pyruvate. Adding NAD + (product of NADH) will act as an inhibitor. Plotting vs. at various concentrations of NAD+.


[NAD+]1 no NAD+

Results in a competitive inhibition plot


Why does NAD+ competitively inhibit this reaction? Both NAD+ and NADH are competing for the same site. What results in adding lactate? Lactate does not compete with NADH but in order for lactate to [lactate]1 bind, NADH has to be on the no lactate enzyme. Lactate is binding at the same cite that pyruvate does. this is an uncompetitive inhibition plot


2.5 4.0 10.0 20.0 40.0 100 2,000 20,000

V0 (M/min)
28 40 70 95 112 128 139 140

Vmax probably not higher than 140 because the last interval was a 10-fold increase which only increased V0 by almost nothing. KM is probably 10x10-6 since