Lecture 13
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Monday Exam 1
Today’s material will be on Exam 2
Intro to enzymes
Next Friday 3PM-Bonus seminar for Dr.
Howard Salis from Penn State University
Enzymes As Biological Catalysts
• Late 1700s, Early 1800s - digestion of meat protein by
gastric fluids.
• Starch degraded by animal saliva, plant extracts.
• mid 1800s - Louis Pasteur defined term “ferments” to
describe agents in yeast cells that converted sugars into
ethanol (fermentation). -Only believed live cells were
capable of fermentation.
• 1897 - Eduard and Hans Büchner used extracts of yeast
to ferment sugar.
• 1926 - James Sumner (Cornell Univ.) isolated and
crystallized urease enzyme from jack bean plant…
• Current search of Protein Data Bank (PDB) Sept 11,
2004 indicates 27112 protein structures
http://www.rcsb.org/pdb/
Enzymes
• Enzymes are classified and named according to the nature of the
chemical reactions they catalyze.
• Divided into 6 classes by the International Union of Biochemistry
and Molecular Biology (IUBMB).
• The six classes of enzymes:
1. Oxidoreductases - oxidation-reduction reactions
2. Transferases - Transfer of functional groups
3. Hydrolases - Hydrolysis reactions
4. Lyases - group elimination to form double bonds
5. Isomerases - Isomerizations (bond rearrangements)
6. Ligases - bond formation coupled with ATP hydrolysis
Enzymes
• All enzymes are assigned a number (EC number) which defines
exactly which reaction is catalyzed by the enzyme.
• Example trypsin is EC 3.4.21.4
EC 3.4.21.4
3 describes
the enzyme
class
(hydrolase)hydrolysis
reaction
4 because it is
21 denotes the 4th entry in
its subthis subclass
subclass
as a serine
peptidase
4 is for the
subclass
(acts on
peptide
bonds, so it
is a peptide
hydrolase)
• Enzyme Nomenclature Database - http://expasy.org/enzyme
Enzymes
•
The 6 enzyme classes can be illustrated by the general reactions catalyzed
1. Oxidoreductases:
A- + B  A + B-
2. Transferases:
A-B + C  A + B-C
3. Hydrolase:
A-B + H2O  A-H + B-OH
XY
4. Lyases:
A-B  A=B + X-Y
XY
YX
5. Isomerases:
A-B  A-B
6. Ligases (synthases)
A + B  A-B
Examples of enzyme classes
1.
2.
3.
4.
5.
6.
Alcohol dehydrogenase (EC 1.1.1.1.)
Hexokinase (EC 2.7.1.1)
Trypsin (EC 3.4.21.4)
Ribulose-bisphophate carboxylase (EC 4.1.1.39)
Triose phosphate isomerase (EC 5.3.1.1)
Tyrosine tRNA ligase (EC 6.1.1.1)
Cofactors
•
•
•
Enzymes are often composed of only protein. In this case only the aa
side chains are used for catalysis.
Some enzymes require additives (cofactors) for assisting with
catalysis for catalyzing oxidation-reduction reactions and many types of
group-transfer processes.
Types of cofactors
– Metal ions (Zn2+, Fe, Cu, Co, Mo, Mg)
– Coenzymes
– Organic molecules (NADH sometimes only shortly associated with the
enzyme so they are cosubstrates)
– Prosthetic groups (permanently associated with their protein by covalent
bonds (heme in hemoglobin)
– nucleotides
Coenzymes
• Chemically changed by the enzymatic reactions in which they
participate so they must be regenerated.
• Sometimes regenerated by a different enzyme (NAD+ 
NADH).
• Holoenzyme - a catalytically active enzyme-cofactor complex
• Apoenzyme - enzymatically inactive resulting from the removal
of the holoenzyme's cofactor.
Coenzymes
•
•
•
•
•
•
•
•
•
•
Common cofactors and uses (Table 13-1):
Biotin aids in carboxylation reactions (CO2 fixation)
Cobalamine (B12) coenzymes-aids in alkylation reactions (methylation)
Coenzyme A - acyl transfer (TCA cycle)
Flavin (vitamin B2) aids in oxidation reduction reactions (nitrate reductase)
Lipoic acid - acyl transfers via oxidation reduction processes
Nicotinamide coenzymes - NAD+ independent co-substrates for redox reactions
Pyridoxal (B6) aids in amino group transfers (provides aldehyde functional group)
Tetrahydrofolate- one carbon transfers
Thiamine pyrophosphate (B1)- aids in aldehyde transfers and alpha-keto acids
decarboxylations.
Coenzymes
Summary and review from last lecture
•
•
•
•
Supersecondary structures: be able to identify them.
Know the differences and be able to give examples of
primary, secondary, tertiary, and quaternary structure.
6 classes of enzymes.
Types of coenzymes and what types of reactions they
assist in.
Progression of enzyme-substrate interaction models
1. 1890 - Emil Fisher model of ES complex: “lock and key”
•
Enzyme is rigid, inflexible.
•
Only recognizes one substrate.
2. 1958 - Daniel Koshland: “induced fit model”
•
Assumes continuous changes in enzyme structure due
to substrate binding.
•
Verified by X-ray crystallographic experiments.
3. Present day, transition-state analog model
•
Active site both recognizes and orients substrate to
activate it for reaction.
•
Bound, distorted substrate takes on characteristics of
transition state.
The lock and key model to describe the formation of an ES
complex
The substrate has a
shape that is
complementary of
fits into a preformed
site on the enzyme.
Note: a, b and c
refer to specific
types of interactions
formed between
substrate and
enzyme.
The induced-fit model to explain binding of a substrate to
an enzyme active site.
Initially the enzyme
does not have a
preformed site for
substrate binding.
Initial binding of
the substrate
induces specific
conformational
changes in the
enzyme structure
to make it more
compatible to the
substrate’s size,
shape and polarity.
Transition-state analog model of enzyme action.
•
Assume the reaction
involves the cleavage of
the bond between atoms
A and B.
•
When the substrate is
bound in the ES complex
the bond to be cleaved is
already strained and
partially broken.
Enzyme-substrate interaction models
1. Understand the differences and similarities between “lock
and key”, “induced fit”, and transition-state analog
models
Stereospecificity
•
Enzymes are highly specific both in binding chiral
substrates and in catalyzing their reactions
•
Due to the inherent chirality of the primary structure
(L-amino acids) and asymmetric binding sites.
•
Example: Yeast alcohol dehydrogenase (YADH)
YADH
CH3CH2OH + NAD+
Ethanol
O
CH3CH + NADH + H+
Acetaldehyde
Page 461
Stereospecificity
•
Ethanol is a prochiral molecule
•
2 methylene H atoms may be distinguished if the
molecule is held in an asymmetric binding site.
OH
Hpro-S
C
CH3
Hpro-R
Page 461
Figure 13-3
Prochiral differentiation.
Stereospecificity
•
YADH reaction was carried out with deuterated ethanol
•
Shows direct hydrogen (D) transfer.
CH3CD2OH
+
H
O
YADH
CH3CD + H+
O
C
NH2
D
H
O
C
+
N
NH2
R
N
NAD+
R
NADD
Stereospecificity
•
NADD was isolated and used for the reverse reaction.
•
Shows the stereospecificity of the enzyme.
OH
O
YADH
CH3CH +
D
H
H
O
C
C
NH2
CH3
N
R
NADD
+ NAD+
D
Stereospecificity
•
The enantiomer can be made as follows
O
CH3CD + NADH + H+
OH
YADH
D
C
H
+ NAD+
CH3
•
In this reaction none of the deuterium can be
transferred from the product to NAD+ in the reverse
reaction.
Stereospecificity
•
If the ethanol is converted to the tosylate form and converted by
SN2 hydrolysis, the enantiomeric ethanol will be made that can be
converted.
CH3
CH3
CH
3
HCl
O S
O S
O
Cl
C
CH3
O
+
D
H
C
O
O S
+
O
OH
D
OH-
OH
OH
H
D
C
CH3
CH3
H
Geometric specificity
• In addition to stereospecificity, most enzymes are also
selective about the identities of the chemical groups on
their substrates (Geometric specificity).
•
Enzymes vary considerably in degree of geometric
specificity
•
Few enzymes are specific for only one compound (can
catalyze a small range of related compounds).
•
YADH catalyzes oxidation of small primary and secondary
alcohols to aldehydes and ketones but less efficiently than
for ethanol.
•
Methanol is catalyzed 25-fold slower than ethanol,
isopropanol 2.5-fold slower.
Regulation of enzymatic activity
• Catalytic activities need to be regulated. Two main
ways to do this.
1. Control the amount of enzyme available. This is
dependent on rate of synthesis of the enzyme and rate of
degradation of the enzyme.
2. Control of enzyme activity. Enzyme activity may be
directly regulated through conformational or structural
alterations.
Allosteric interactions
Control of enzyme activity through allosteric effectors
• Cooperative binding-as ligands or substrates are bound
by the enzyme, they increase the binding for the next
ligand or substrate through slight changes in the protein
structure.
•
The substrate-binding affinity may also change with the
binding of small molecules (in hemoglobin’s case, H+)
Control of enzyme activity through allosteric effectors
• Another example of allosteric control of an enzyme activity
is aspartate transcarbamoylse (ATCase).
NH2
O-
O
O
OPO32-
Carbamoyl
phosphate
+
NH2
CH2
H3
N+
COO-
Aspartate
•
C
ATCase
C
C
O-
O
CH2
C
O
N
H
COO-
N-carbamoylaspartate
Catalyzes the formation of N-carbamoylaspartate, first step
for the biosynthesis of pyrimidines (nucleic acids)
Page 465
Figure 13-5 The rate of the reaction catalyzed by
ATCase as a function of aspartate concentration.
Page 466
Feedback inhibition of ATCase
Control of enzyme activity through allosteric effectors
• ATCase is regulated by feedback inhibition-the
concentration of the product controls the activity of an
enzyme at the beginning of a pathway.
•
ATCase is inhibited by excess CTP, a pyrimidine
•
If the pyrimidines are all used up, CTP dissociates from
ATCase, allowing the enzyme to resume its normal activity.
•
ATP activates ATCase. ATP is a purine basd compound,
that is normally found in high amounts in the cell, so it
requires higher amounts of ATP to activate ATCase than
the amount of CTP to inactivate the enzyme.
Control of enzyme activity through allosteric effectors
•
ATCase is composed of 12 subunits (c6r6). c represent the
catalytic subunits and r represents the regulatory units.
•
The catalytic subunits are arranged in two sets of trimers
(c3).
•
Regulatory units are in three sets of dimers (r2).
•
If we separate the catalytic subunits (c3) from the
regulatory subunits, the catalytic subunits will have a
noncooperative (hyperbolic like myoglobin) substrate
saturation curve. This means that the maximum rate of the
enzyme is higher than that of the intact enzyme and it is
unaffected by the presence of ATP or CTP (no allosteric
effect).
•
The isolated regulatory dimers (r2) can bind to ATP or CTP
but have no enzymatic activity.
Control of enzyme activity through allosteric effectors
•
Allosteric theory predicts that the activator (ATP) will bind
to preferentially to the ATCase active state (R or high
substrate affinity).
•
Also predicts that the inhibitor (CTP) will bind
preferentially to ATCase inactive state (T or low substrate
affinity).
Page 467
Figure 13-7a X-Ray structure of ATCase. (a) (left) Tstate ATCase along the protein’s molecular threefold
axis of symmetry.
Page 467
Figure 13-7a X-Ray structure of ATCase. (a) (right) Rstate ATCase along the protein’s molecular threefold
axis of symmetry.
Page 467
Figure 13-7bX-Ray structure of ATCase. (b) (left) Tstate ATCase along the protein’s molecular twofold axis
of symmetry.
Page 467
Figure 13-7bX-Ray structure of ATCase. (b) (right) Rstate ATCase along the protein’s molecular twofold axis
of symmetry.
Page 468
Figure 13-8 Comparison of the polypeptide backbones
of the ATCase catalytic subunit in the T state (orange)
and the R state (blue).
Page 469
Figure 13-9 Schematic diagram indicating the tertiary
and quaternary conformational changes in two vertically
interacting catalytic ATCase subunits.
Summary of allosterism
•
Binding of substrates causes changes in quaternary
structure that facilitate the enzymatic reaction.
•
Binding of inhibitors can hold the enzyme in an inactive
state (unfavorable for the reaction to occur or for
substrates to bind).
•
Binding of activators can hold the enzyme in an active
state (favorable for the reaction to occur and for substrate
binding).
Review from last lecture
•
•
Understand differences between lock and key, induced fit,
and transition state analog models
Understand allosterism.
Thermodynamics
•
•
•
Determines if the reaction is spontaneous (does it occur).
Does not tell us how fast a reaction will proceed.
Catalysts (enzymes) can lower the activation barrier to get
from products to reactants.
Thermodynamics of Enzyme Function
• Catalysts lower the energy barrier
between reactants and products.
Free energy diagrams of simple chemical catalysis
Figure 2-21 Energetics of catalysis
The energy course of a
hypothetical reaction from
substrate S to product P
can be described in terms
of transition states and
intermediates.
For the uncatalyzed
reaction (blue curve) a
single transition-state
barrier determines the
rate at which product is
formed.
Multiple hills and valleys
are present for enzyme
pathway.
Figure 2-21 Energetics of catalysis (cont.)
In the presence of a catalyst (red curve), which in this
case is acting by changing the pathway of the
reaction and introducing additional smaller activationenergy barriers, intermediate I1, formed by crossing
transition-state barrier TSc1, leads to transition-state
barrier TSc2.
Its free energy (ΔGc), although the highest point in the
reaction, is considerably lower than the free energy
(ΔGu) of the uncatalyzed transition state, TSu.
After formation of a second intermediate, I2, a third
transition state, TSc3, leads to product.
Because TSc2 is the highest transition state in the
catalyzed reaction, the rate at which the reactants
pass over this barrier determines the overall rate and
thus it is said to be the rate-determining transition
state of the catalyzed reaction.
The rate-determining step of this reaction is thus the
conversion of I1 to I2.
Figure 2-21
Energetics of
catalysis
Figure 2-21 Energetics of catalysis (cont.)
The transition state is the highest point in free energy
on the reaction pathway from substrate to product.
It is the top of the activation-energy barrier (see TSu in
Fig. 2-21).
Chemically, it is a species that exists for about the time
required for a single atomic vibration to occur (about 10-15
s).
In the transition state, the making or breaking of chemical
bonds in the reaction is not yet complete: the atoms are
"in flight".
The stereochemistry and charge configuration of the
transition state is thus likely to be quite different from that
of either the substrate or the product, although it may
resemble one more than it does the other.
Figure 2-21
Energetics of
catalysis
Figure U2-3.2 The rate-determining step
•
•
The rate-determining step of a
reaction is the step with the
largest energy barrier.
In this example, the height of the
barrier between intermediates I1
and I2 is greater than between S
and I1 or I2 and P, and the rate
determining step is therefore I1
to I2.
U2-1 Enzyme Kinetics: General Principles
•
•
•
•
•
•
•
Enzymes function as biological catalysts to increase the rate
(speed) of chemical catalysis.
Reaction rates reflect key properties of enzymes and the
reactions they catalyze
Kinetics is the study of how fast chemical reactions occur.
The free energy change can tell us in which direction the reaction will
spontaneously occur.
The free energy change does not tell us how rapidly.
Some spontaneous reactions occur quickly, e.g. sec., others occur
almost imperceptibly over many years.
The rate of a chemical reaction or process, or the reaction rate, is the
change in the concentration of reacting species (or of the products of
their reaction) as a function of time (Fig. U2-1.1).
Figure U2-1.1 Reaction rates measure how fast processes
occur
(a) In this example, two reacting
species A and B (red and blue)
combine to form a product C
(purple).
(b) The concentration of product
molecules increases as the reaction
proceeds, and within two seconds
of the reacting species being mixed
together, the concentration of C
becomes 10 mM.
(c) The rate of the reaction during these
two seconds is therefore 5 mM s-1,
which is the slope of the line when
we plot the concentration of the
product against time.
Figure 6.6 A plot of initial reaction velocity versus the
concentration of enzyme [E].
Note that velocity
increases in a
linear fashion with
an increase in
enzyme
concentration.
Add more catalyst,
get faster reaction
rate.
-Not surprising!
Figure U2-1.2 Rate constants are measured
from reaction rates at different reactant
concentrations
• Follow the progress of the
reaction between molecules A
and B, as shown in Figure U21.1:
• Rate at which product C is
produced decreases as the
reaction proceeds (Fig. U21.2a).
• The rate decreases because
the concentration of reactants
decreases.
• The possibility also exists that
the reverse reaction (C → A +
B) becomes significant as the
concentration of product C
increases.
Figure U2-1.2 Rate constants are measured
from reaction rates at different reactant
concentrations
• To avoid these complications, we
can measure the initial rate of the
reaction (i.e, 5-10% of total reaction
time):
The rate before the concentration
of reactants decreases significantly
and before the accumulation of
product is able to interfere with the
reaction (Fig. U2-1.2b).