Biochemistry I Fall Term, 2004

October 4, 2004

Lecture 14: Enzyme Kinetics

Links: (I) Review Quiz on Lecture 14 concepts (I) Substrate Saturation Kinetics shows various graphs used to analyze kinetic data (S) Graphing Quiz: Michaelis- Menten Kinetics: Determine Vmax and KM Campbell's text is excellent on all of the assigned sections for today's lecture. These notes develop the same concepts in a slightly different order and add some material in the Transition State Theory and Catalytic Efficiency and the Specificity sections below. For the simple enzyme-catalyzed reaction: S <==> P S is substrate; and P is product. The enzyme forms a complex with the substrate, in much the same way as a protein-ligand complex, and performs some chemical reaction/transformation of the bound substrate. The resultant product is released. Important Features of Enzyme Catalysis. 1. Enzymes increase the rate of reactions. 2. Enzymes do not change the equilibrium point of reactions. 3. Enzymes are specific for their substrates: Geometric complementarity Energetic complementarity. 4. Enzymes are regulated: 1

at genetic level (transcription, translation); by concentration of substrate and product; allosterically.

Background Kinetics 1. Order of Reactions: A -> P rate ~ [A]1 1st order 2nd order 2nd order

A + A -> P A + B -> P

rate ~ [A]2 rate ~ [A][B]

2. Velocity of a reaction: v = -d[A]/dt = d[P]/dt = k[A][B] (for a second order reaction) 3. Transition State Theory: Is there a physical limit to the rate of a chemical reaction? There is indeed such an upper limit; and transition state theory provides an estimate of the rate constant. At some point along the reaction progress curve, the chemical species reach a high energy configuration called the transition state. It is represented by the symbol, X (Campbell, Fig. 5.1). Transition state theory states that the rate of reaction is directly proportional to the concentration of the transition state: v = k'[X] where k' is the rate at which the transition state will decay to products. The key assumption made in transition state theory is that the substrates and products are in equilibrium with the transition state. For example, the equilibrium constant of a second order reaction in the forward reaction is: K = [X]/[A][B] Therefore the velocity of the reaction is: v = k'K[A][B]. Comparing this equation to that for a second order reaction gives: k = k' K. An expression for k' was derived by H. Eyring who postulated that a reaction velocity could not exceed the vibrational frequencty of the bond that is broken. This maximum frequency can be expressed using only fundamental physical constants: 2

 = kBT/h
where kB is Boltzmann's constant and h is Planck's constant. The vibrational frequency, = 6.3*1012 sec-1 at T = 300 K. Finally, we substitute the free energy equivalent of the equilibrium constant K, to get the expression for a rate constant according to transition state theory: k = (kBT/h)exp(-G/RT) The pre-exponetial portion is the maximum frequency possible; the exponential represents the fraction of reactants in the transition state (at temperature, T). From the above equation it is clear how enzymes increase the rate of the reaction - they must do so by lowering the free energy of the transition state in the enzyme substrate complex. This is shown by the free energy diagrams below:

Note that enzymes bind to both the substrates and products and stabilize the transition state. The transition state of the enzyme substrate complex is stabilized in two ways: First, the enzyme transition-state complex is stabilized by direct interactions between the enzyme and the transition state. This reduces the free energy of the transition state. Second, the enzyme may provide a number of functional groups to aid in catalysis. Since these groups are positioned in well defined locations, the entropy of bringing these groups into the catalytic site is substantially less than having these functional groups diffuse freely in solution.

Steady State Enzyme Kinetics: Steady-state reactions are easy to measure because the rate of the reaction is constant for relatively long periods of time. Steady-state rates are those which are most relevant to metabolic levels. The analysis of steady state kinetics can only provide limited information on the kinetic mechanism.

The simplest reaction scheme is:

where, k1 is the forward rate constant for substrate binding k-1 is the reverse rate constant for substrate binding. k2 is the catalytic rate constant (containing terms related to the transition state). The ES complex is also called the "Michaelis complex". The velocity of the reaction is: v = d[P]/dt = k2[ES] The change in [ES] as a function of time is: d[ES]/dt = k1[E][S] - k-1[ES] - k2[ES] During the steady state: d[ES]/dt = 0 0 = k1[E][S] - k-1[ES] - k2[ES] The goal is to relate this equation to readily measurable experimental parameters, such as: The total amount of enzyme: ET = [E] + [ES] The concentration of substrate: [S] The measured steady state velocity (v = k2 [ES])

We do not have a suitable way to measure [E], but since we know the total enzyme concentration, we can substitute as follows: [E] = ET - [ES] [ES](k-1 + k-2) = k1[S](ET - [ES]) 4

[ES](k-1 + k2) = k1 E [ES] - k1[ES][S] [ES](k-1 + k2 + k[S]) = k1 E [S] [ES] = k1E [S]/(k-1 + k2 + k1[S]) The velocity of the reaction: v = k2[ES] = k1k2E [S]/{k-1 + k2 + k1[S]} = k2E [S]/{(k-1 + k2)/ k1+ [S]} Defining a few terms: The Vmax or maximal velocity: Vmax = k2ET This is the highest reaction rate that can be attained because all (i.e. ET) of the enzyme is saturated with substrate.

The KM or Michaelis constant: KM = (k-1 + k2)/k1 This is the substrate concentration that gives a reaction velocity equal to 1/2 of Vmax. Note that in the case of slow reaction kinetics (k2<<k-1), the KM is also the dissociation constant for substrate binding. The use of these definitions gives the Michaelis-Menten Equation

The class handout, Substrate Saturation Kinetics shows a plot of reaction velocity versus [S]. Also shown is a graph of the data in a double reciprocal plot, otherwise known as a "LineweaverBurk Plot".

Catalytic Efficiency and the Specificity of Enzyme Reactions: kcat/KM The catalytic constant of an enzyme is defined as: kcat = Vmax/ET In the case of the simple example discussed above, kcat = k2. (The units of kcat are time-1; typically, sec-1. Thus, if kcat = 1000 sec-1, the enzyme can convert 1000 molecules of substrate into product each second at saturating [S].) Treating the enzyme catalyzed reaction as a simple second order reaction: v = k[E][S] Compare this equation to the Michaelis-Menten equation above. At very low substrate concentrations (ET =[E]), the Michaelis-Menten equation becomes: v = k2 [ET][S] = (kcat/KM)[E][S] kcat/KM is the second order rate constant for this reaction. It represents the selectivity of the enzyme at limiting substrate concentrations. Selectivity can also be expressed as the ratio, Vmax/KM for a series of measurements where [ET] is constant. Using the definitions of KM: kcat/KM = k1k2/(k-1 + k2) If the chemical reaction is rate-limiting (k-1>>k2) then: kcat/KM = k1k2/k-1 , and the observed reaction rate depends both on how well the substrate binds as well as the rate of catalysis. If the chemical reaction is not rate-limiting (k-1<<k2) then: kcat/KM = k1 and the rate is only limited by how fast the substrate can diffuse into the active site.
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