Transcript Enzymes

Enzymes:
Basic Concepts
and
Kinetics
Enzymes: catalysts of biological systems, speed up selective chemical
reactions needed for life.
They have remarkable catalytic power and specificity.
Nature selected some protein molecules to be enzymes.
There are very specific cases when other biomacromolecules can also carry
catalytic functions, like RNA (ribozymes) or antibodies (abzymes).
Catalysis takes place on a distinct part of the protein molecule called the
active site.
Enzymes use the full repertoire of intermolecular forces to attract, optimally orient and VERY specifically bind “the substrates” (the molecules
ought to be converted faster), then in the favored spatial positions the
chemical reaction between the two molecules will be highly facilitated.
Catalysis happen because enzymes stabilize the transition state (TS, the
highest energy species in the reaction). Selective stabilization of TS discriminates amongst potential reaction pathways.
Acceleration factor can be up to a million or more. Most reactions without
the enzyme practically does not occur at all.
carbonic anhydrase
1 million CO2/s
An enzyme generally catalyses only one reaction or very similar reactions.
Side reactions resulting in by-products are rare unlike in case of regular
chemical reactions.
Substrate specificity, however, may sometimes be kind of “loose” for some
enzymes (like Papain – very undiscriminating protease).
Some rather specific enzymes:
Trypsin: digestive enzyme, cleaves peptide bond on the –COOH side at Lys or Arg.
Thrombin: participates in blood clotting, hydrolyzes Arg-Gly bonds in specific peptide
sequences only.
Specificity and such a high precision in molecular action is possible because
of the intricately accurate design of the 3D structure of the enzyme
specializing in that particular reaction and substrate structures. This
function to develop needed millions of years of evolution.
Catalytic action sometimes needs some small extra molecular entities
attached covalently or non-covalently to the enzyme (cofactors).
Enzyme w/o its cofactor: apoenzyme, with it: holoenzyme.
Cofactors: 1. metals 2. coenzymes (small organic molecules, often derived
from vitamins; the tightly bound coenzymes are the prosthetic groups;
loosely bound coenzymes are rather just called cosubstrates)
Some enzymes use the same coenzymes showing similar mechanisms.
Enzymes may also transfer energy from one form to another, e.g. photosynthesis: light energy to chemical energy or in mitochondria: energy from
food to an ion gradient then to ATP. Other enzymes then can use the
energy of these molecules to convert energy or molecules even further
(e.g. myosin converts ATP energy to mechanical energy of contracting
muscles, or membrane transport proteins to move molecules/ions across
membranes).
Classification of enzymes:
There are common names (trypsin – no info on what it does), but also many
Es are named after the substrates and the reactions + the “ase” suffix. E.g.
peptide hydrolase – hydrolyzes peptide bonds, ATP synthase – synthesizes
ATP.
1964:
EC (Enzyme Commission) numbers: a 4 digit number to unambiguously identify enzymes.
Example:
nucleoside monophosphate (NMP) kinase (transfers a phosphoryl group –
a transferase (group 2)
Since it transfers a phosphoryl group: 2.7
Since the acceptor in the reaction is a phosphate: 2.7.4.
More precisely the acceptor is a nucleoside monophosphate: 2.7.4.4.
What drives (enzymatic) reactions? Which reaction will be spontaneous?
All answers are told by the thermodynamics considerations learned in
Physical Chemistry (please consult your previous studies and the Bioenergetics lectures).
Enzymes are “only” catalysts which enhance the rate of formation of the
dynamic reaction equilibria and the process gives the catalyst back
“unaltered” (enzymes also have a half-life though and then new molecules
of them are generated [expressed]).
TS is a transitory structure that is no longer the substrate but also not
yet the product, either. It is the least stable and most-seldom-occupied
species along the reaction pathway. It has the highest free energy.
DG‡ is (one expression of) the activation energy. Enzymes lower the activation energy facilitating the formation (stabilization) of the transition state.
S
‡
K
X
V
‡
v
P
‡
α [X ]
‡
DG α 1/[X‡]
V
(overall rate of reaction)
~ DG‡
I think that enzymes are molecules that are
complementary in structure to the activated
complexes of the reactions that they catalyze, that is, to the molecular configuration
that is intermediate between the reacting
substances and the products of reaction for
these catalyzed processes. The attraction
of the enzyme molecule for the activated
complex would thus lead to a decrease in its
energy and hence to a decrease in the energy of activation of the reaction and to an
increase in the rate of reaction.
Linus Pauling, Nature 161 (1948): 707
Remark: I think Stryer: Biochemistry, 6th ed. p.212, last eq. is not correct.
An enzyme-substrate complex (ES) forms to facilitate the stabilization of
the TS of the reaction.
first verification:
X-ray showed substrates and analogs bound to active sites.
cyt P450 – camphor complex
all catalytic
sites are
occupied.
Uncatalyzed reactions
do not saturate.
Time-resolved crystallography uses a light-sensitive
substrate analog. Photons are shed on resulting substrate and
then ES complex that is captured in a fraction of a second
by polychromatic synchrotron radiation X-ray beams.
Also spectroscopic characteristics of enzymes and/or substrates sometimes change upon the formation of the ES complex. It is particularly
obvious if there is a colored prosthetic group bound to an enzyme.
Trp synthetase uses a pyridoxal phosphate (PLP)
prosthetic group to make L-Trp from Ser and
indole. Addition of Ser changes drastically the
fluorescence of PLP. Addition of the 2nd
substrate, indole, decreases fluorescence to
lower than the free enzyme. This proves the
existence of the E-Ser and the E-Ser-indole
complexes. NMR and ESR can also prove ES
complex formations.
Lysozyme
Active site (AS):
-binds substrates
-binds cofactors
-contains the residues that brake
and make bonds (catalytic groups)
-a 3D cleft or crevice formed from
amino acids (aa) that might be far in
primary sequence
-H2O is excluded unless a substrate
-non-polar microenvironments with often
polar (“active”) residues in special positions
Why to have a big protein for those few “active” amino acids?
The “extra” aa are making the scaffold that position the “active” aa into
the exact 3D configuration required for action.
But why does not a protein use neighboring aa to form the AS?
Those aa are often sterically constrained from adopting the necessary
configuration.
The “extra” amino acids often constitute regulatory sites, sites of interaction with other proteins or ligands, or channels to bring the substrates
to the AS. Not to mention enzymes with multiple (but sometimes also
coupled) enzymatic functions.
Substrates are bound to enzymes by multiple weak interactions as
discussed earlier. The directional character of H-bonding among specific
sites on E and S makes the interaction often very specific.
Emil Fischer (1890): “lock and key”
analogy
(seems
now assume
an oversight).
The close contacts that hold
the ES
together
a matching shape of
Ribonuclease
S to E.
Daniel E. Koshland, Jr. (1958): E is flexible and dynamic and the shape of
the AS can considerably change upon substrate binding. The AS of most E
gets complementary in shape to S only after S is bound (induced fit).
Very current and still unsolved question: Does protein dynamics have
anything to do with the catalytic action? So far we believe the answer is
(suprisingly) NO!! /Protein Society Meeting, Stockholm, 2011/
Binding energy (BE) is released upon S binding to E. Only the correct substrate can participate in the maximal number of possible interactions between E and S, thus release the highest BE. But all possible interactions
can only be formed in the TS!! The release of BE can be
thought of as lowering the activation energy (see Linus Pauling earlier).
Paradox: the highest BE is released (the most stable interaction is) in the
TS where the least stable reaction intermediate exists.
The TS is too unstable to exist for long, it will collapse to either substrate
or product. Which accumulates in excess over time is decided by the DG of
the reaction.
The MichaelisMenten (M-M)
kinetics
(true for most
enzymes)
E+S
k1
k-1
ES
k2
k-2
E + P
If t ~ 0, k-2[P][E] ~ 0, so
E+S
k1
k-1
ES
k2
E + P
Vo should be measured early in time before P accumulates!
V0=k2[ES]
Rate of formation of ES: k1[E][S]
Rate of breakdown of ES: (k-1 + k2)[ES]
in steady state these are equal
According to the steady-state (Bodenstein`s) principle the concentrations of the
reaction intermediates under particular conditions do not change as a function of
time and remain approximately constant (d[ES]/dt ~ 0)
[E][S]/[ES]=(k-1 + k2)/k1=KM (Michaelis constant)
[ES]=[E][S]/KM
KM has a unit of concentration and it is independent of [S] or [E]
[E]=[E]T-[ES]
[ES]=([E]T-[ES])[S]/KM
[ES]=([E]T[S]/KM)/(1+[S]/KM)
[ES]=[E]T[S]/([S]+KM)
V0=k2[ES]
V0=k2[E]T[S]/([S]+KM)
Vmax is reached when [ES]=[E]T (all catalytic sites are occupied by substrate)
Vmax=k2[E]T
V0=Vmax [S]/([S]+KM)
M-M equation
V0=Vmax [S]/([S]+KM)
When [S] << KM
V0=(Vmax/KM)[S] (1st order; linear with [S])
When [S] >> KM
V0=Vmax (zero order, independent of [S])
KM and Vmax is determined from kinetic experiments by curve-fitting
analyses in computers. Before computers it was easier to linearize the
equation (error prone, it weighs data points differently at low and high [S]).
1/V0=(KM/Vmax)(1/[S]) + 1/Vmax
double-reciprocal or Lineweaver-Burk plot
KM depends on: substrate, ionic strength, pH, temperature
Significance/meaning of KM:
- KM is the substrate concentration when half of the AS are occupied (sig1.7 ms/
nificant catalysis takes place)
reaction
- A good approximation of the substrate concentration present in vivo
- ONLY when k-1 >> k2 (this is though very rarely the case for most
enzymes) KM approximates the Kd of ES (high KM then means weak binding
and vica versa) – frequent misinterpretation of KM (as Kd) even in today’s
scientific literature
Vmax reveals the turnover number (TN) of an enzyme. TN is the # of S
converted to P by a single E in unit time when E is fully saturated with S.
TN = k2 = kcat = Vmax/[E]T
e.g. a 10-6 M carbonic anhydrase solution catalyzes the formation of 0.6 M
H2CO3 in each second as long as the enzyme is fully saturated with CO2.
Fraction of active sites filled fES = V0/Vmax = [S]/([S] + KM)
In a “normal” situation an enzyme is not always fully saturated with substrates in vivo; rather the [S]/KM ~ 0.01 – 1.0 typically.
V0=k2[ES]
[ES]=[E][S]/KM
V0=(kcat/KM)[E][S]
When [S] << KM [E] ~ [E]T
V0=(kcat/KM)[E]T[S]
rate constant for the
interaction of S and E
determines the substrate
preference of an enzyme
Is there any physical limit for kcat/KM?
kcat/KM=(kcatk1)/(k-1 + kcat)=(kcat/(k-1 + kcat))k1 < k1
If kcat >> k-1, then kcat/KM ~ k1, so the ultimate limit on kcat/KM is k1
(the rate of formation for the ES). But this rate cannot be faster than
the diffusion-controlled encounter of an enyzme and its substrate!!
Diffusion controls k1, so k1 as well as kcat/KM cannot realistically be higher
than ~108-109 s-1M-1.
These enzymes attained “kinetic perfection”. Their catalytic velocities
practically are restricted only by how fast they can get to the substrate.
Diffusion in solution can also be partly overcome by confining S and P in the
limited volume of a multienzyme complex.
How can every encounter between E and S be productive, for the “kinetically
perfect enzymes”, when S can only effectively bind to the AS (which is a small part
/portion of E)? They suspect there are attractive electrostatic forces originated
from E which lure substrates to the AS. This is called the Circe effect.
If an enzyme is well characterized and known (and also in clinical practice)
then they use the so-called “activity” of the enzyme, as a unit of effectiveness. This actually means reaction velocity, under precisely known and
controlled conditions. It tells us how much S can be converted (or P be
generated) by the actual amount of E present in solution, (generally) at
the saturating concentration of S in unit time.
SI unit is: catal (mol/s)
In practice we rather use mmol/min (or Unit (U))
Especially during protein (enzyme) purification it is a good practice to
measure the specific activity of the enzyme, which means enzyme activity
per unit mass of protein (e.g. mmol/min/mg). If the specific activity is
raised along purification then the protein preparation gets more pure.
We have to consider that there are optimal conditions for E activity
(pH, I, T, cofactor) and comparison is only valid if all values have been
measured under the same experimental conditions.
Most (bio)chemical reactions include two or more compounds.
For enzymes this means multiple (and not only one!) substrates.
A + B
P + Q
Many such reactions transfer a functional group, a phosphoryl or an amino
group from one substrate to another. It is also possible that an electron or
two are transferred in oxido-reduction reactions.
There are two typical forms of multiple substrate reactions:
sequential or double-displacement reactions
Sequential reactions: all substrates get bound before any product is
released. Consequently, a ternary complex must be formed during catalysis
(E-S1-S2). There is an ordered (binding of Ss are in a defined seq.) and a
random sequential mechanism.
Good examples for ordered sequential mechanism are reactions that use
NAD+ or NADH as substrates, e.g. lactate dehydrogenase
Notation by W. Wallace Cleland
Example for random sequential mechanism:
(PC)
PC is an important energy source in muscle
Double-displacement (ping-pong) mechanisms
One or more products are released before all substrates are bound.
There is a substituted E-intermediate where E is temporarily modified.
Typical reactions are the transaminations, e.g.:
S1
P1
S2
substrates bounce on&off E: ping-pong analogy
P2
Allosteric enzymes may not always obey M-M kinetics; they might have
multiple subunits, multiple AS, sigmoidal V0 – [S] plots, regulatory ligand
(reversible) binding sites
cooperativity
Binding of one S to the 1st AS can alter the properties of the other ASs
, generally enhancing the binding probability of the 2nd S, in the same E
molecule (cooperativity, see Hb/Mb; positive homotropic effect).
Due to the versatile sorts of possibilities for regulation, allosteric enzymes
can satisfy the immediate needs of the cell, thus they are the key regulators of metabolic pathways (generally by the action of a heterotropic allosteric activator or inhibitor ligand on the same polypeptide chain, see later).
Inhibition
It is a major control mechanism in biological systems, where allosteric
regulation (inhibition) is a major player through binding small molecules
(metabolites) into allosteric sites. Also many drugs and toxic agents act
as inhibitors (inhibitor=I from now on).
Knowing the inhibitory mechanism tells us much about the mechanism of
action of an enzyme and vica versa.
TS analogs are especially potent Is.
There is reversible and irreversible inhibition.
Irreversible inhibition: I dissociates very slowly from E, bound strongly
covalently or non-covalently (Kd is small; several drugs are like this, e.g.
penicillin covalently modifies transpeptidase preventing bacterial cell wall
synthesis or aspirin covalently modifies cyclooxygenase blocking the synthesis of signal molecules involved in inflammation).
Reversible inhibition: more transient binding of I to E. Three types exist.
ES complex
EI complex
ESI complex
does not exist,
S and I are
mutually exclusive.
S and I often
resemble one
another.
I binds only
to ES.
A competitive I reduces the proportion of E that binds S. Competitive
inhibition can be relieved by increasing [S].
Examples:
- methotrexate is a potent competitive inhibitor (structural analog)
of dihydrofolate reductase (dTMP synthesis blocker, anticancer drug,
binds 1000x better than S to E)
- Ibuprofen competitively inhibits enzymes in signaling of inflammation.
- Statins lower cholesterol by competitively inhibit a key enzyme in
cholesterol biosynthesis (HMG-CoA reductase).
Uncompetitive inhibitory site is created only after forming the ES complex.
m=KM[S].
/Vmax
It cannot be overcome by increasing
In noncompetitive inhibition S and I can bind simultaneously to E but to
different sites. It cannot be overcome by increasing [S]. Here the kcat
decreases.
There is a so-called mixed inhibition: substrate binding is hindered and
kcat is reduced by I in the same time.
Kinetic pictures of reversible inhibitory mechanisms differ:
We need to measure the reaction rates at different S and I concentrations.
1. Competitive inhibition:
1/Vmax
apparent KM increases
Vmax does not change
KMapp=KM(1 + [I]/Ki)
2. Uncompetitive inhibition:
Example: glycophosphate (herbicide;
Roundup) is an uncompetitive inhibitor
of an enzyme for aromatic amino acid
synthesis.
1/Vmax
-1/KM
Vmax and KMapp
decrease
3. Noncompetitive inhibition:
Vmaxapp=Vmax/(1 + [I]/Ki)
Examples:
- deoxycycline (antibiotic)
noncompetitively inhibits collagenase (a protease). Used in the
treatment of periodontal disease.
Vmax is decreased (Vmaxapp)
KM does not change
The inhibitor simply lowers the
concentration of functional E.
- Lead (Pb) poisoning is due to
that Pb behaves as noncompetitive
inhibitor to lots of enzymes (Pb
can react with –SHs).
-1/KM
To determine the mechanism of action of an enzyme first we need to know
which residues (what functional groups) take part in the catalysis from E.
X-ray structure of the ES or an EI (esp. a covalently bound irreversible I)
can map the AS. Three types of irreversible Is there are: group-specific
reagents (1), reactive substrate analogs (affinity labels, 2) and suicide inhibitors (3).
Group-specific reagents:
diisopropylphosphofluoridate (DIPF, nerve gas): reacts only with highly
reactive Ser (e.g. ones in chymotrypsin’s and acetylcholinesterase’s ASs)
Iodoacetamide:
Reactive substrate analogs:
Structurally similar to the S and can covalently bind to AS residues.
substrate analog reacting
irreversibly with AS His
Suicide (mechanism-based) inhibitors: most specific way to modify the AS.
These are generally some versions of modified substrates which initially get
converted by the normal mechanism by E, but then are trapped in a specific
step during the mechanism in forms of intermediates which cannot further
be processed by E (due to a specific covalent modification of E, generally).
The covalently modified group on E is essential for catalysis.
Example: monoamine oxidase (MAO) inhibitors: N,N-dimethylpropargylamine
and Deprenyl. MAO deaminates neurotransmitters serotonin, dopamine
lowering their brain levels). Low level of dopamineDeprenyl
is implicated in Parkinson‘s
Disease, while low serotonin level in depression.
Transition-state analogs are potent inhibitors
First proposed by Linus Pauling (1948).
Example: Pro-racemase, in TS the tetrahedral a-C becomes trigonal.
Reprotonation can go towards either direction.
trigonal
binds 160x tighter to E than Pro
An analog holding – charge on a-C would be
even more potent.
Highly potent and specific Is can be designed if
I resembles the TS rather than S itself.
Antibodies (Ab) that recognise TS have catalytic activities.
Example: An Ab can catalyze the insertion of a metal ion into a porphyrin.
Ferrochelatase, last enzyme in heme synthesis, puts Fe2+ into protoporphyrin IX. During catalysis the planar ring must be bent for Fe2+ to enter.
A hard-to-find TS analog turned out to be methylmesoporphyrin (see below).
The idea came from the finding that N-Me-protoporphyrin is a potent inhibitor of ferrochelatase (Nalkylation forces the ring to bend). N-alkylated
porphyrins also chelate metals 10,000x better because
bending exposes the lone pairs of electrons of pyrrole
Ns that binds Fe2+.
They used this compound as antigen to produce a catalytic Ab (abzyme) that
also catalyzes the insertion Fe2+. The Ab was only 10x less potent than the
enzyme. In general, an abzyme can be generated by a TS analog. They generated abzymes for many more reactions already. These experiments prove
that conformation of the AS is indeed complementary to the TS.