Enzyme catalysis
Enzyme catalysis is the catalysis of chemical reactions by
specialized proteins known as enzymes. Catalysis of biochemical
reactions in the cell is vital due to the very low reaction rates of the
uncatalysed reactions.
The mechanism of enzyme catalysis is similar in principle to other
types of chemical catalysis.
By providing an alternative reaction route and by stabilizing
intermediates the enzyme reduces the energy required to reach
the highest energy transition state of the reaction.
The reduction of activation energy (Ea) increases the number of
reactant molecules with enough energy to reach the activation
energy and form the product.
Enzyme Catalysis Basic principle – an enzyme increases the rate of a chemical reac6on by binding and stabilizing the transi'on state of its specific substrate 'ghter than the ground state. Enzymes make reac'ons go faster The cataly6c proper6es of enzymes are reflected in Km and kcat values MICHAELIS – MENTON SCHEME Enzyme (E) and substrate (S) first reversibly combine to give an enzyme – substrate complex (ES). Chemical processes then occur in a second step with a rate constant called kcat, or the turnover number, which is the maximum number of substrate molecules converted to product per ac6ve site of the enzyme per unit of 6me. The kcat is a rate constant that refers to the proper6es and reac6ons of the ES complex. For simple reac6ons, kcat is the rate constant for the chemical conversion of the ES complex to free enzyme and products. Important:
These defini6ons are valid only when the concentra6on of the enzyme is very small compared with that of the substrate. They apply only to the ini6al rate of forma6on of products: the rate of forma6on of the first few percent of the product, before the substrate has been depleted and products that can interfere with the cataly6c reac6on have accumulated. The first source of limitations for the Michaelis–Menten kinetics is that it is
an approximation of the kinetics derived by the law of mass action. In
particular, Michaelis–Menten kinetics is based on the quasi-steady state
assumption that [ES] does not change,
d[ES]/dt = 0
which is only approximately true: the rate of change of the complex [ES] is
very small but non-zero.
The second limitation is that Michaelis–Menten kinetics relies upon the
law of mass action which is derived from the assumptions of free
(Fickian) diffusion and thermodynamically-driven random collision.
However, many biochemical or cellular processes deviate significantly
from such conditions.
For example, the cytoplasm inside a cell behaves more like a gel than a
freely flowable or watery liquid, due to the very high concentration of
protein (up to ~400 mg/mL) and other “solutes”, which can severely limit
molecular movements (e.g., diffusion or collision). This causes
macromolecular crowding, which can alter reaction rates and
dissociation constants.
The Michaelis – Menten scheme explains why a maximum rate of reac'on, Vmax, is always observed when the substrate concentra6on is much higher than the enzyme concentra6on. This is the fastest a reac,on can go. Vmax is obtained when the enzyme is saturated with substrate. There are then no free enzyme molecules available to turn over addi6onal substrate. Hence, the rate is constant, Vmax, and is independent of further increase in the substrate concentra,on. The substrate concentra6on when the half maximal rate, Vmax/2, is achieved is called the Km. For many simple reac6ons, it can easily be shown that the Km is equal to the dissocia6on constant, Kd, of the ES complex. The Km describes the affinity of the enzyme for the substrate. For more complex reac6ons, Km may be regarded as the overall dissocia6on constant of all enzyme – bound species. A plot of the reac6on rate as a func6on of the substrate concentra6on for an enzyme catalyzed reac6on. Vmax is the maximal velocity. The Michaelis constant, Km, is the substrate concentra6on at half Vmax. The rate ν is related to the substrate concentra6on, [S], by the Michaelis – Menten equa6on: Determination of constants - Lineweaver–Burk plot
To determine the maximum rate of an enzyme-mediated reaction, a series of experiments is carried
out with varying substrate concentrations ([S]) and the initial rate of product formation is measured.
'Initial' here is taken to mean that the reaction rate is measured after a relatively short time period,
during which complex builds up but the substrate concentration remains approximately constant and
the quasi-steady-state assumption will hold. The measurements can then be plotted in a
Lineweaver–Burk plot, plotting the inverse of substrate concentration against the inverse of
the initial velocity
1 = Km + [S] = Km x 1 + 1
V0 Vmax [S] Vmax [S] Vmax
The values of the desired
constants KM and Vmax can be
read directly off the plot. Accurate
values for KM and Vmax can only
be determined by non-linear
regression of Michaelis-Menten
data.
This inverse plot, while useful for visualization, should never be the
source of the actual value of the enzyme constant due to large
insensitivity to errors inherent in all inverse plots. Should one need to
derive a value from an inverse plot, the The inverse plot, while useful for
visualization, should never be the source of the actual value of the
enzyme constant due to large insensitivity to errors inherent in all
inverse plots. Should one need to derive a value from an inverse plot,
the Hanes-Woolf plot is the most accurate.
The quan6ty kcat/Km is a rate constant that refers to the overall conversion of substrate into product. Very useful and widely used. The ul6mate limit to this value is set by the rate constant for the ini6al forma6on of the ES complex. This rate cannot be faster than the diffusion-­‐
controlled encounter of an enzyme and its substrate, which is between 108 to 109 per mole per second. The quan6ty is some6mes called the specificity constant because it describes the specificity of an enzyme for compe6ng substrates. It is a useful quan6ty for kine'c comparison of mutant proteins. Measure these numbers to assess how a muta6on affects a par6cular process. It’s therefore a way to see how important that part of your protein is. Km – recogni6ons proper6es on surface of proteins kcat – internal catalysis of process within the protein Overall concept:
Induced fit
The favored model for the enzyme-substrate interaction is the induced fit model. This model
proposes that the initial interaction between enzyme and substrate is relatively weak, but that these
weak interactions rapidly induce conformational changes in the enzyme that strengthen binding.
The advantages of the induced fit mechanism arise due to the stabilizing effect of strong enzyme
binding. There are two different mechanisms of substrate binding: uniform binding, which has strong
substrate binding, and differential binding, which has strong transition state binding.
The stabilizing effect of uniform binding increases both substrate and transition state binding
affinity, while differential binding increases only transition state binding affinity. Most proteins
seem to use the differential binding mechanism to reduce the Ea, so most proteins have high affinity
of the enzyme to the transition state. The substrate first binds weakly, then the enzyme changes
conformation increasing the affinity to the transition state and stabilizing it, so reducing the
activation energy to reach it.
Mechanisms of transition state stabilization
These conformational changes also bring catalytic residues in the active site close to the chemical
bonds in the substrate that will be altered in the reaction. After binding takes place, one or more
mechanisms of catalysis lowers the energy of the reaction's transition state, by providing an
alternative chemical pathway for the reaction. There are six possible mechanisms of "over the
barrier" catalysis as well as a "through the barrier" mechanism:
1: Catalysis by bond strain - this is the principal effect of induced fit binding, where the affinity of the
enzyme to the transition state is greater than to the substrate itself. This induces structural
rearrangements which strain substrate bonds into a position closer to the conformation of
the transition state, so lowering the energy difference between the substrate and transition state
and helping catalyze the reaction.
2: Catalysis by proximity and orientation - this increases the rate of the reaction as enzymesubstrate interactions by aligning reactive chemical groups and holding them close together. This
reduces the entropy of the reactants and thus makes reactions such as ligations or addition
reactions more favorable, there is a reduction in the overall loss of entropy when two
reactants become a single product.
3: Catalysis involving proton donors or acceptors (Acid/Base Catalysis) - proton donors and
acceptors, i.e. acids and bases, may donate and accept protons in order to stabilize developing
charges in the transition state. This typically has the effect of activating nucleophile and
electrophile groups, or stabilizing leaving groups. Histidine is often the residue involved in these
acid/base reactions, since it has a pKa close to neutral pH and can therefore both accept
and donate protons.
Mechanisms of transition state stabilization
4: Electrostatic catalysis - stabilization of charged transition states can also be by residues in the
active site forming ionic bonds (or partial ionic charge interactions) with the intermediate.
These bonds can either come from acidic or basic side chains found on amino acids such as lysine,
arginine, aspartic acid or glutamic acid or come from metal cofactors such as zinc. Metal ions are
particularly effective and can reduce the pKa of water enough to make it an effective nucleophile.
5: Covalent catalysis - involves the substrate forming a transient covalent bond with residues in the
active site or with a cofactor. This adds an additional covalent intermediate to the reaction, and
helps to reduce the energy of later transition states of the reaction. The covalent bond must,
at a later stage in the reaction, be broken to regenerate the enzyme. This mechanism is found in
enzymes such as proteases like chymotrypsin and trypsin, where an acyl-enzyme intermediate is
formed.
6: Quantum tunneling – the "over the barrier" mechanisms above have been challenged in some
cases by models and observations of "through the barrier" mechanisms (quantum tunneling). Some
enzymes operate with kinetics which are faster than what would be predicted by the classical ΔG‡.
In "through the barrier" models, a proton or an electron can tunnel through activation barriers.
Quantum tunneling for protons has been observed in tryptamine oxidation by aromatic amine
dehydrogenase. Not super convincing.
General: Enzymes decrease the ac'va'on energy of chemical reac'ons The Michaelis complex, ES, undergoes rearrangement to one or several transi'on states before the product is formed. Energy is required for these rearrangements. The input energy required to bring free enzyme and substrate to the highest transi6on state of the ES complex is called the ac'va'on energy of the reac6on. In the absence of enzyme, spontaneous conversion of substrate to product also proceeds through transi'on states that require ac'va'on energy. The rate of chemical reac6on is strictly dependent on its ac6va6on energy, and the more than 1 million-­‐fold enhancement of rate achieved by enzyme catalysis results from the ability of the enzyme to decrease the ac6va6on energy of the reac6on. This decrease in ac6va6on energy is achieved by enzymes in several different ways like we’ve seen. Generally -­‐ the higher the affinity of the enzyme for the transi'on state makes the transi'on energe'cally favorable and thus decreases the ac'va'on energy. If, on the other hand, the enzyme were to bind the unaltered substrate more strongly than the transi6on state, the decrease in binding energy on the forma6on of the transi6on state would increase the ac6va6on energy and catalysis would not be achieved. It is therefore cataly'cally advantageous for the enzyme’s ac've site to be complementary to the transi'on state of the substrate rather than to the normal structure of the substrate. Summary: The enzyme likes substrates transi'on state more than its ground state This means that the ac6va6on energy required for forming the ES transi6on complex drops Catalysis occurs