Enzymology
The enzyme substrate complex
What is an enzyme
How does the substrate interact productively with the enzyme
The enzyme's active site binds to the substrate.
Increasing the temperature generally increases the rate of a reaction, but dramatic changes in temperature and
pH can denature an enzyme, thereby abolishing its action as a catalyst.
The induced fit model states an substrate binds to an active site and both change shape slightly, creating an ideal
fit for catalysis.
When an enzyme binds its substrate it forms an enzyme-substrate complex.
Enzymes promote chemical reactions by bringing substrates together in an optimal orientation, thus creating an
ideal chemical environment for the reaction to occur.
The enzyme will always return to its original state at the completion of the reaction.
Understand what an enzyme is, how it works and how we can monitor it.
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What is an enzyme – a substance produced by a living organism which acts as a catalyst to bring about a
specific biochemical reaction. Enzymes can do 5 types of chemistry.
What are enzymes made from - Enzymes are large molecules that speed up the chemical reactions inside
cells. Each type of enzyme does on specific job. Enzymes are a type of protein, and like all proteins, they
are made from long chains of different amino acids. Protein - Amino acids - C, H, N, O, S, post-translational
modifications (P, sugar, acetyl groups, other proteins) - cofactors (metals e.g. Fe, Cu, Zn) H 2O - Protons,
neutrons and electrons.
What do enzymes look like – enzymes are proteins (primary, secondary, tertiary, quaternary, quinary
structure)
Where are enzymes found – in cells. Intracellular (vagaries of the intracellular millieu; membrane
associated, viscosity, salinity, pH, macromolecular crowding, solvent excluded). Extracellular (secreted in
ECM, tethered outside cell, true extracellular e.g. saliva). Psychrophilic to hyhperthermophilic
What do enzymes do - Enzymes are catalysts: a substance that increases the rate of a chemical reaction
but remains chemically unchanged after turnover. Catalyse reactions, Oxidoreductase, Transferase,
Hydrolase, Isomerase, Lyase, Ligase (synthetase). Broad range of substrates.
What sort of substrates do enzymes use – proteins (post-transitional modification, disulphide bond formation
reduction), RNA, DNA, small molecules/metabolites, ATP, NAD/P, sugars, lips, in/organic cofactors
How do enzymes catalyse chemical reactions – lowers the activation energy/lowers the transition state.
They optimise the position of atoms for interactions. Quantum mechanical tunneling
Enzymes are proteins: Primary amino acid sequence that folds to a given functionally active structure.
Enzyme are specific: Stereo-, regio- and chemically specific.
The Enzyme substrate complex
, The substrate binds with high affinity but reversibly to the enzyme. Tight substrate binding is anti-catalytic.
The substrate orientation and proximity is key to a productive ES complex (collision theory)
The Substrate-Enzyme interaction involves multiple interactions e.g. H-bonding, van der Walls, ionic, formal
covalent.
Substrate binding is anti-catalytic. Binding your substrate makes your enzyme a
worse enzyme. Low energy is more stable.
Forward rate constant – K1/K on
Backwards rate constant – K-1/K
off
Kd = k-1 / k1 v – velocity
Chemical Reactions: Transition state theory
The transition state, or activated complex, for a chemical reaction is a transient configuration of atoms or
molecules that lies at the point of maximum energy on the minimum energy pathway leading from
reactants to products.
By analogy, we can think of this maximum energy position as a saddle point, or the top of a mountain pass.
dG* is the free energy of activation and its magnitude indicates how likely a reaction is to occur. And so rate
dGo is the free energy of reaction and if it is negative we have an exothermic reaction and if positive an
endothermic reaction but it says nothing about the rate of the reaction, simply whether it is
thermodynamically favourable.
Rates of reactions can be described by a high energy intermediate at the lowest energy ‘saddle-point’, the transition-
state
— The TS is in equilibrium with the reactant
— The TS is transient (unstable)
— The TS decays to form a product.
Limitations:
— Only applies for systems that obey classical mechanics
— Requires the TS to be long lived enough to be describable using a Boltzmann
distribution
— Only applies to relatively low energy reactions.
The physical properties of amino acids give rise to stereo and regio specific
binding of the substrate
Residues that bind the substrate may also take part in the chemical
mechanism, e.g. by altering electron density to promote nucleophilic bond
cleavage as in serine proteases.
Bonding and catalytic residues can be one and the same. In some respects
that is because what allows you to stabilise the ES complex also you to
stabilise the t‘s complex.
ES vs TS stabilisation. Same residue can be used for both
Physical models for ES formation 1. Basic
Lock and key is wrong.
,Induced fit – the active site and the substrate are initially not perfect matches for each other. The
substrate induces a change of shape in the enzyme to be complementary.
Physical models for ES formation
In the induced fit model, the ligand binds to the inactive form. The weak interactions
between the ligand and the protein induces a conformational change in protein to the
active form.
In the conformational selection model, the active and inactive forms
exist in equilibrium, but the vast majority of protein is in the inactive conformation. When ligand is
added, it only binds to the active form. The net effect is that the equilibrium shifts towards the active
conformation.
The essential difference is whether conformational change happens before or after ligand binding.
Induced fit says that it happens after; conformational selection says it happens before.
The Free energy landscape of protein structure
Anfinsen’s principle. Unique, stable, kinetically accessible (no traps) and population shift.
The image shows how a molecule minimizes the free energy of a molecule. This could lead to
protein folding.
An energy landscape is a mapping of all possible conformations of a molecular entity, or the
spatial positions of interacting molecules in a system, and their corresponding energy levels,
typically Gibbs free energy.
The term is useful when examining protein folding; while a protein can theoretically exist in a nearly infinite number
of conformations along its energy landscape, in reality proteins fold (or "relax") into secondary and tertiary
structures that possess the lowest possible free energy. The key concept in the energy landscape approach to
protein folding is the folding funnel hypothesis.
In catalysis, when designing new catalysts or refining existing ones, energy landscapes are considered to avoid low-
energy or high-energy intermediates that could halt the reaction or demand excessive energy to reach the final
products.
Summary and Enzyme Functions
The ES complex is a specific molecular architecture that
allows a chemical reaction to take place.
The enzyme has a high affinity for the substrate and
dictates the proximity and orientation of the substrate to
promote chemical reactivity.
There are two major models for the formation of the ES
complex, induced fit and conformational selection
(population shift).
NB// These two concepts are important when we think
about allostery in subsequent lectures.
Ligand binding sites 1: detection and characterisation
Simple and complex binding interactions
Experimental techniques
, Fluorescence and ITC
Only if the E.S complex breaks down to E+S very much faster than its conversion to E.P complex (and eventually
product) is Km = Kd.
In cases where k-1 >> kcat you can assume that Km=Kd=k-1/k1.
Kinetics
Single binding site
Only if the E.S complex breaks down to E+S very much faster than its conversion to E.P complex (and
eventually product) is Km = Kd.
In cases where k-1 >> kcat you can assume that Km=Kd=k-1/k1.
Scatchard plots - is a plot of the ratio of concentrations of bound ligand to unbound ligand versus the bound ligand
concentration. It is a method for analyzing data for freely reversible ligand/receptor binding interactions. It is an
equation for calculating the affinity constant of a ligand with a protein .
(i) r/[L] = nKa-rKa L = ligand
(ii) r = [L]bound / [P]0 P=protein
(iii) Ka = [LP]/[L][P]
Scatchard plot: r/[L] vs r
It is the ratio of the concentration of bound ligand to total available binding sites, and n is the number of
binding sites per protein molecule. Ka is the association (affinity) constant from the equation
Scatchard plot has a slope of –Ka and a y-intercept of nKa
Binding vs catalytic site
two step consecutive reversible model in Enzyme assays 2
Complex binding interactions
Cooperativity
Experimental techniques Fluorescence (polarisation,
ITC (energetics) FRET)
Surface technologies (e.g. SPR) (conformational change) Absorption (e.g. charge transfer
complex absorbance)
Radio-labelled ligands
