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.
Lnzyme CaLalysls
8aslc prlnclple - an enzyme lncreases Lhe raLe of a chemlcal reacuon by
blndlng and sLablllzlng Lhe trans|non state of lLs speclc subsLraLe
nghter than the ground state.
Lnzymes make reacnons go faster
1he caLalyuc properues of enzymes are reecLed ln k
m
and k
cat
values
Lnzyme (L) and subsLraLe (S) rsL reverslbly comblne Lo glve an enzyme -
substrate comp|ex (LS).
Chemlcal processes Lhen occur ln a second sLep wlLh a raLe consLanL called k
cat
, or
Lhe turnover number, whlch ls Lhe maxlmum number of subsLraLe molecules
converLed Lo producL per acuve slLe of Lhe enzyme per unlL of ume.
1he k
caL
ls a raLe consLanL LhaL refers Lo Lhe properues and reacuons of Lhe LS
complex. lor slmple reacuons, k
cat
|s the rate constant for the chem|ca|
convers|on of the LS comp|ex to free enzyme and products.
MICnALLIS - MLN1CN SCnLML
1hese denluons are valld only when Lhe concenLrauon of Lhe enzyme ls very
small compared wlLh LhaL of Lhe subsLraLe. 1hey apply only Lo Lhe lnlual raLe of
formauon of producLs: Lhe raLe of formauon of Lhe rsL few percenL of Lhe
producL, before Lhe subsLraLe has been depleLed and producLs LhaL can lnLerfere
wlLh Lhe caLalyuc reacuon have accumulaLed.
Important:
The first source of limitations for the MichaelisMenten kinetics is that it is
an approximation of the kinetics derived by the law of mass action. In
particular, MichaelisMenten kinetics is based on the quasi-steady state
assumption that [ES] does not change,
which is only approximately true: the rate of change of the complex [ES] is
very small but non-zero.
d[ES]/dt = 0
The second limitation is that MichaelisMenten 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.
1he M|chae||s - Menten scheme explalns why a max|mum rate of reacnon,
V
max
, ls always observed when Lhe subsLraLe concenLrauon ls much hlgher Lhan
Lhe enzyme concenLrauon. !"#$ #$ &"' ()$&'$& ) *')+,-. +). /-.
V
max
ls obLalned when Lhe enzyme |s saturated w|th substrate. 1here are Lhen
no free enzyme molecules avallable Lo Lurn over addluonal subsLraLe. Pence,
Lhe raLe ls consLanL, V
max
, and ls #.0'1'.0'.& -( (2*&"'* #.+*')$' #. &"' $23$&*)&'
+-.+'.&*),-.4
1he subsLraLe concenLrauon when Lhe half maxlmal raLe, Vmax]2, ls achleved ls
called Lhe k
m
. lor many slmple reacuons, lL can easlly be shown LhaL Lhe k
m
ls
equal Lo Lhe dlssoclauon consLanL, k
d
, of Lhe LS complex.
1he k
m
descrlbes Lhe amnlLy of Lhe enzyme for Lhe subsLraLe. lor more complex
reacuons, k
m
may be regarded as Lhe overall dlssoclauon consLanL of all enzyme
- bound specles.
A ploL of Lhe reacuon raLe as a funcuon of Lhe subsLraLe concenLrauon for an
enzyme caLalyzed reacuon. v
max
ls Lhe maxlmal veloclLy. 1he Mlchaells
consLanL, k
m
, ls Lhe subsLraLe concenLrauon aL half v
max
. 1he raLe v ls relaLed Lo
Lhe subsLraLe concenLrauon, [S], by Lhe Mlchaells - MenLen equauon:
Determination of constants - LineweaverBurk 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
LineweaverBurk plot, plotting the inverse of substrate concentration against the inverse of
the initial velocity
1
=
Km + [S] = Km x 1 + 1
V
0
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.
1he quanuLy k
cat
]k
m
ls a rate constant that refers to the overa|| convers|on of
substrate |nto product. very useful and wldely used.
1he ulumaLe llmlL Lo Lhls value ls seL by Lhe raLe consLanL for Lhe lnlual
formauon of Lhe LS complex. 1hls raLe cannoL be fasLer Lhan Lhe dluslon-
conLrolled encounLer of an enzyme and lLs subsLraLe, whlch ls beLween 10
8
Lo
10
9
per mole per second. 1he quanuLy ls someumes called Lhe spec|hc|ty
constant because lL descrlbes Lhe speclclLy of an enzyme for compeung
subsLraLes. lL ls a useful quanuLy for k|nenc compar|son of mutant prote|ns.
Measure Lhese numbers Lo assess how a muLauon aecLs a parucular process.
lL's Lherefore a way Lo see how lmporLanL LhaL parL of your proLeln ls.
k
m
- recognluons properues on surface of proLelns
k
caL
- lnLernal caLalysls of process wlLhln Lhe proLeln
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 enzyme-
substrate 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 !G

.
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.
Genera|: Lnzymes decrease the acnvanon energy of chem|ca|
reacnons
1he Mlchaells complex, LS, undergoes rearrangemenL Lo one or several trans|non states before
Lhe producL ls formed. Lnergy ls requlred for Lhese rearrangemenLs. 1he lnpuL energy requlred
Lo brlng free enzyme and subsLraLe Lo Lhe hlghesL Lransluon sLaLe of Lhe LS complex ls called Lhe
acnvanon energy of Lhe reacuon. In the absence of enzyme, spontaneous convers|on of
substrate to product a|so proceeds through trans|non states that requ|re acnvanon energy. 1he
raLe of chemlcal reacuon ls sLrlcLly dependenL on lLs acuvauon energy, and Lhe more Lhan 1
mllllon-fold enhancemenL of raLe achleved by enzyme caLalysls resulLs from Lhe ablllLy of Lhe
enzyme Lo decrease Lhe acuvauon energy of Lhe reacuon.
1hls decrease ln acuvauon energy ls achleved by enzymes ln several dlerenL
ways llke we've seen. Genera||y - the h|gher the amn|ty of the enzyme for the
trans|non state makes the trans|non energenca||y favorab|e and thus decreases
the acnvanon energy.
lf, on Lhe oLher hand, Lhe enzyme were Lo blnd Lhe unalLered subsLraLe more
sLrongly Lhan Lhe Lransluon sLaLe, Lhe decrease ln blndlng energy on Lhe
formauon of Lhe Lransluon sLaLe would lncrease Lhe acuvauon energy and
caLalysls would noL be achleved.
It |s therefore cata|ynca||y advantageous for the enzyme's acnve s|te to be
comp|ementary to the trans|non state of the substrate rather than to the
norma| structure of the substrate.
Summary:
1he enzyme ||kes substrates trans|non state more than |ts ground state
1hls means LhaL Lhe acuvauon energy requlred for formlng Lhe LS
Lransluon complex drops
CaLalysls occurs