College aantekeningen
Lecture notes Enzymology, BIC20806, wur
- Instelling
- Wageningen University (WUR)
Lecture notes of all lectures given during the course Enzymology, BIC20806 at Wageningen University.
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Voorbeeld van de inhoud
Enzymology lecture notesLecture 1: introduction
Enzymes have some advantages: they are very efficient, environmentally acceptable (natural,
biodegradable, good food grade) and they operate under mild conditions (less waste, less energy
required). Also, enzymes are selective (stereoselective, regioselective and chemoselective), can be
made by fermentation (cheap), can be modified/optimised, catalyse a broad spectrum of reactions
and accept unnatural substrates.
Enzymes are biological catalysts: a compound which enhances the rate of a chemical reaction
without being destroyed or incorporated into the product. Therefore, the first lectures will be about
catalysis. Features of catalysts:
Make an alternative reaction path in which less activation energy (ΔG+) is
needed
Equally increase the rate of the backward and forward reactions, catalysis thus
has no effect on the equilibrium position.
There are organic, inorganic and biological catalysts. The other way of
subdivision is about the physical state, which can either be homogenous (freely
dissolved in a solution) or heterogenous (a solid in liquid or gaseous
environment).
Lecture 2: reaction kinetics
A reaction can be one-step (1), second-step (2) or multiple steps (3). The reaction
step with the slowest rate is rate-determining. The rate is determined by the
magnitude of the activation energy Ea.
Bimolecular reaction: two particles collide and react.
Monomolecular: 1 particle dissociates and reacts. A large reaction runs through
multiple bi- and monomolecular reactions. The concentration of a compound can be
determined as a function of time. Ideally, you measure both substrate and product because
sometimes, there are side reactions. Methods:
Chromatography (gas chromatography, GC; high pressure liquid chromatography, HPLC)
UV-spectroscopy (plot the absorbance over time and use Lamberts-Beer equation to
determine the concentration)
NMR spectroscopy (deuterated solvents are necessary and can be performed quantitively)
Titration (excellent for quantitative measures, the pH electrode and burette are coupled to a
computer, the slope is equal to the reaction time).
Rate equations:
First-order Second-order Reversible reactions
A -> B A + B -> P. Now, the reaction A -> B, but it can also go the other
The rate of the reaction rate depends on both A and way around. For this, take the
depends on the B. simplest possibility. This is difficult
concentration of reactant A, For simplification, you can to solve.
the more A there is, the assume pseudo-first order
faster the reaction. kinetics: A >> B. This means
that the second order rate
equation is simplified to a
, The half-life can be first order equation.
calculates as followed: t1/2 =
ln2/k1.
Here, k1’ is the pseudo first-
order constant.
The Michaelis Menten constant, Km, is equal to the ratio of the rate of
the formation and the breakdown of the enzyme-substrate complex
ES. If Km is low, the substrate is tightly bound.
The Michaelis Menten equation can be seen on the left. In industry,
you want to work at Vmax.
Every reaction has to overcome an energy barrier E a to reach the
transition state (TS). At higher temperatures, more particles are able
to overcome the energy barrier. Arrhenius came up with an equation to describe this event.
If A, a pre-exponential factor, is unknown, E a can be measured at different temperatures. k 1
can be divided by k2, so that A cancels out (see image). The activation energy can never be
negative.
Especially ΔS‡ is extremely useful to determine the number
of molecules involved in the rate limiting step of a reaction:
ΔS‡ ~ 0: rate limiting step is monomolecular
ΔS‡ << 0: rate limiting step is bimolecular
This information can be used to determine the reaction
mechanism.
In a bimolecular reaction, the solvent has a huge influence
on the reaction rate. Both A and B are surrounded by solvent molecules, which have to be pushed
aside to reach A and B. The stronger the bond between solvent and reactant, the more difficult this is
(slower reaction time).
Ground state destabilization is a thermodynamic concept suggesting that there is an increase in
energy in going from one state to another. Enzymes use ground state destabilization in 3 ways:
1. Removal of solvent (substrate is more “naked”)
2. Distortion of the conformation of the substrate
3. Catalysis by complexation, bringing the reactants together in the optimum orientation and
keeping them there for a short period of time. This leads to an increase of the effective
concentration of the reactants and therefore to an increase of the reaction rate.
Organic polymers like enzymes may have apolar pockets, which can lead to less solvation of the polar
species and therefore higher reaction rates and changes in the pK a.
Knowing the structure of the transition state is important for the determination of the rate-
determining step (reaction mechanisms) and for the design of enzyme inhibitors. Nowadays, with
computer mathematical models, we can make a model of the energy of the transition state.
Hammond postulate: the transition state closely resembles the species with the highest energy
content. An exothermic reaction has a low Ea and the TS resembles ground state (early TS). An
endothermic reaction has a high Ea and the TS resembles the product (late TS).
The kinetic isotope effect (KIE) is the change in the reaction rate of a chemical reaction when one of
the atoms in the reactants is replaced by one of its isotopes. It is defined as the ratio of rate
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