plantbiochemlecture5-enzymesb.ppt

Enzymes: Structure, Properties
and Mechanisms of Activity

Factors Affecting Enzyme Activity
• In addition to enzyme itself with its cofactors and coenzymes,
environmental conditions and enzyme inhibitors may influence on an
enzyme activity.
• Temperature (best be optimum, i.e. the temperature at which
enzymatic reaction occur fastest; then high temperatures could be
lethal, e.g. denaturation), pH (most like 6 - 8 pH near neutral) and
substrate concentration are the main environmental factors.
• The rate of reaction increases as substrate concentration increases (at
constant enzyme concentration).
• Maximum activity occurs when the enzyme is saturated (when all
enzymes are binding substrate).

Kinetic properties of enzymes
Study of the effect of substrate concentration on the rate of reaction

Effect of enzyme concentration [E]
on velocity (v) or reaction rate
In fixed, saturating [S], the
higher the concentration of
enzyme, the greater the initial
reaction rate.
This relationship will hold as long
as there is enough substrate
present.

Enzyme Kinetics
• Enzyme kinetics deal with determining the rate of a reaction and how it
changes in response to changes in experimental parameters.
• A key factor affecting the rate of a reaction catalyzed by an E is the
concentration of S. Studying the effects of S concentration is
complicated by the fact that S changes during the course of an in vitro
reaction as S is converted to product. One simplifying approach in
kinetic experiments is to measure the initial rate (initial velocity),
designated V0. In a typical reaction, the E may be present in nanomolar
quantities, whereas S may be five or six orders of magnitude higher. If
only the beginning of the reaction is monitored (often the first 60
seconds or less), changes in S therefore can be limited to a few
percent, and S can be regarded as constant.
• At relatively low concentrations of S, V0 increases almost linearly with an
increase in S. At higher S concentrations, V0 increases by smaller and
smaller amounts in response to increases in S. Finally, a point is reached
beyond which increases in V0 are vanishingly small as S increases. This
plateau-like V0 region is close to the maximum velocity, Vmax.

Michaelis and Menten Equation
• In 1913, Leonor Michaelis and Maud Menten, developed a kinetic
equation to explain the behavior of many simple enzymes. Key to the
development of their equation, is the assumption that the E first
combines with its S to form an ES complex in a relatively fast
reversible step:
• k1
• E + S ⇄ ES
• k-1
• The ES complex then breaks down in a slower second step to yield the
free E and the reaction product P:
• k2
• ES ⇄ E + P
• k-2
• If the slower second reaction limits the rate of the overall reaction,
the overall rate must be proportional to the concentration of the
species that reacts in the second step, i.e., ES.
• At any given instant in an enzyme-catalyzed reaction, the E exists in
two forms, the free or uncombined form E and the combined form ES.
At low S, most of the E is in the uncombined E form. Here, the rate is
proportional to S because the direction of the first equation above is
pushed toward formation of more ES as S increases.

• The maximum initial rate of the catalyzed reaction (Vmax) is observed
when virtually all of the E is present in the ES complex and E is
vanishingly small. Under these conditions, the E is saturated with its S,
so that further increases in S have no effect on rate. This condition
exists when S is sufficiently high that essentially all the free E has
been converted to the ES form. The saturation effect is a
distinguishing characteristic of enzymatic catalysts and is responsible
for the plateau observed in figure. The pattern seen in the figure is
sometimes referred to as saturation kinetics.
• When the E is first mixed with a large excess of S, there is an initial
period, the pre-steady state, during which the concentration of ES
builds up. This period is usually too short to be easily observed, lasting
just microseconds, and is not evident in the figure. The reaction quickly
achieves a steady state in which ES remains approximately constant
over time. The measured V0 generally reflects the steady state, even
though V0 is limited to the early part of the reaction. The analysis of
these initial rates is referred to as steady-state kinetics.

• The kinetic curves expressing the relationship between V0 and S have
the same general shape (a rectangular hyperbola) for most enzymes,
which can be expressed algebraically by the MM equation. Michaelis and
Menten derived this equation starting from their basic hypothesis that
the rate-limiting step in enzymatic reactions is the breakdown of the
ES complex to product and free enzyme. The MM equation is:
V0 = Vmax[S]/(Km + [S]).
• All these terms, [S], V0, Vmax, as well as the constant called the
Michaelis constant, Km, can be readily measured experimentally.
• The MM equation describes the kinetic behavior of a great many
enzymes, and all enzymes that exhibit a hyperbolic dependence of V0 on
S are said to follow Michaelis-Menten kinetics. However the MM
equation does not depend on the relatively simple two-step reaction
mechanism discussed above. Many enzymes that follow MM kinetics
have quite different mechanisms, and enzymes that catalyze reactions
with six or eight identifiable steps often exhibit the same steady-state
kinetic behavior. Even though the MM equation holds true for many
enzymes, both the magnitude and the real meaning of Vmax and Km can
differ from one enzyme to another. This is an important limitation of
the steady-state approach to enzyme kinetics.

• An important numerical relationship emerges from the MM equation in
the special case when V0 is exactly one-half Vmax. Km is equivalent to the
substrate concentration at which V0 is one-half Vmax.
• The Km can vary greatly from enzyme to enzyme, and even for different
substrates of the same enzyme. The Km is sometimes used (often
inappropriately) as an indicator of the affinity of an enzyme for its substrate.
Thus Km cannot always be considered a simple measure of the affinity of an
enzyme for its substrate.
• The meaning of the quantity
Vmax also varies greatly from
one enzyme to the next.

Enzyme Regulation
• Biochemical pathways in the living organisms need sophisticated
mechanisms for their regulation, due to several reasons:
• 1- Maintenance of an ordered state (i.e. timely production of
substances without wasting substances).
• 2- Conservation of energy (regulating the level of energy-generating
reactions just enough to meet the energy requirements).
• 3- Responsiveness to environmental changes (regulating the rate of
specific reactions to enable cells make relatively rapid adjustments to
variations in Temp, pH, ions, etc).
• Adjustment of the concentration and activities of certain enzymes is
key to the regulation of biochemical pathways. Control of enzymes
concentration and activities is accomplished by one of the followings:
• 1- Genetic control. Enzyme induction (i.e. the synthesis of enzymes in
response to changing metabolic need) is an efficient way of response of
cell to changes in environment. Enzyme repression (inhibition of
synthesis of certain key enzymes) may be accomplished by the end
product of a biochemical pathway.

Enzyme Regulation
• 2- Covalent modification. It is the regulation by reversible
interconversion between an active and inactive forms of the enzyme
molecule due to covalent modifications of enzymes structure. Covalent
attachment of a molecule to an amino acid side chain of a protein can
modify activity of enzyme.
• Many such enzymes have specific residues that may be phosphorylated
and dephosphorylated, methylated and demethylated, acetylated and
deacetylated or adenylated (the covalent addition of the nucleotide
adenosine monophosphate) and deadenylated.
3- Allosteric regulation. In each biochemical pathway at least one enzyme
sets the rate for the entire pathway (i.e. pacemaker or regulatory
enzyme). This enzyme usually catalyzes the first unique or committed
step in the pathway. Another typical control point is the first step of a
branch in a pathway that leads to an alternate product. Both covalent
modification and allosteric regulation are capable of regulating
pacemaker enzymes. Cells use allosteric regulation to respond
effectively to changes in intercellular conditions.

Enzyme Regulation
Allosteric enzymes are usually composed of several promoters whose
properties are affected by effector molecules. Allosteric enzymes
have a second regulatory site (allosteric site) distinct from the active
site.
Allosteric enzymes contain more than one polypeptide chain (have
quaternary structure).
Allosteric modulators bind noncovalently to allosteric site and regulate
enzyme activity via conformational changes.
• The binding of an effector (ligands) to an allosteric enzyme can affect
the binding of substrate to that enzyme. Allosteric effects may be
positive or negative. The binding of an effector shifts the curve
(enzyme activity in response to S concentration) to a higher (i.e. left or
decrease in Km: activator) or lower (i.e. right or increase in Km:
inhibitor) activity. Positive modulator binds to the allosteric site and
stimulates activity. Positive modulator of an enzyme usually is the
substrate of the reaction.
Negative feedback inhibition is a process in which the product of a
pathway inhibits the activity of the pacemaker enzyme.
Negative modulator (inhibitor) binds to the allosteric site and inhibits the
action of the enzyme. Usually it is the end product of a biosynthetic
pathway (i.e. end-product inhibition).

Enzyme Regulation
• Example: Phosphofructokinase (catalyzes the transfer of a phosphate
group from ATP to the OH group on C-1 of fructose-6-phosphate) is
the main regulatory control point in glycolysis. The enzyme is stimulated
by ADP, AMP and other metabolites and inhibited by PEP, citrate and
ATP. ATP is a S if binds to active site but is an inhibitor if binds to the
allosteric site of the enzyme.
• 4- Compartmentation. In eukaryotic cells biochemical pathways are
segregated into different organelles. Main purpose of this physical
separartion is that opposing processes are easier to control in this way.
E.g. FA biosynthesis occurs in the cytoplasm but FA oxidation during
energy generation occur in mitochondria. Another purpose of the
compartmentation is that each organelle can concentrate specific
substances such as substrates and coenzymes. The third purpose is
that special microenvironments are often created within organelles. E.g.
lysosomes contain hydrolytic enzymes mainly because these enzymes
require a high concentration of hydrogen ions for optimum activity
(lysosome pH = 5 vs cytoplasm pH = 7.2).

• PFK-1 catalyzes an early step in glycolysis
• Phosphoenol pyruvate (PEP), an
intermediate near the end of the pathway
is an allosteric inhibitor of PFK-1
Example of allosteric enzyme - phosphofructokinase-1
(PFK-1)
PEP

Dephosphorylation reaction
Usually phosphorylated enzymes are active.
Enzymes taking part in phospho-rylation are called
protein kinases
Enzymes taking part in dephosphorylation are called
phosphatases

Reversible and Irreversible Inhibitors
Reversible inhibitors – after combining with enzyme (EI complex is formed)
can rapidly dissociate. EI complex is held together by weak, noncovalent
interaction. Enzyme is inactive only when bound to inhibitor.
Reversible inhibition could be competitive or non-competitive.
Competitive inhibitor has a structure similar to the substrate thus can bind
to the same active site. The enzyme cannot differentiate between the two
compounds. When inhibitor binds, prevents the substrate from binding.
Inhibitor can be released by increasing substrate concentration.
Non-competitive inhibitor binds to an enzyme site different from the
active site. Inhibitor and substrate can bind enzyme at the same time.
Cannot be overcome by increasing the substrate concentration.
Suicide inhibitor. Inhibitor binds as a substrate and is initially processed by
the normal catalytic mechanism. It then generates a chemically reactive
intermediate that inactivates the enzyme through covalent modification.
It is called suicide because enzyme participates in its own irreversible
inhibition.

• Multienzyme complexes: different enzymes that catalyze sequential reactions
in the same pathway are bound together.
• Multifunctional enzymes: different activities may be found on a single,
multifunctional polypeptide chain.
• Metabolite channeling: is “channeling” of reactants between active sites. It
occurs when the product of one reaction is transferred directly to the next
active site without entering the bulk solvent. It can greatly increase rate of a
reaction.
• Channeling is possible in multienzyme complexes and multifunctional enzymes.
• Metabolism: is the entire network of chemical reactions carried out by living
cells. Metabolism also includes coordination, regulation and energy
requirement.
• Metabolites: are small molecule intermediates in the degradation and
synthesis of polymers.
• Most organism use the same general pathway for extraction and utilization of
energy. All living organisms are divided into two major classes of autotrophs
Multienzyme Complexes and
Multifunctional Enzymes

(a) Linear (b) Cyclic
(c) Spiral pathway
(fatty acid
biosynthesis)
A sequence of reactions that has a specific purpose (for instance:
degradation of glucose, synthesis of fatty acids) is called a metabolic
pathway.
Metabolic pathway may be:
Metabolic Pathways

Catabolic reactions - degrade molecules to create smaller molecules and
energy.
Anabolic reactions - synthesize molecules for cell maintenance, growth and
reproduction.
Metabolic pathways can be grouped into two paths: catabolism and
anabolism.
Catabolism is characterized by oxidation reactions and by release of free
energy which is transformed to ATP. Anabolism is characterized by
reduction reactions and by utilization of energy accumulated in ATP
molecules.
Catabolism and anabolism are tightly linked together by their
coordinated energy requirements: catabolic processes release the
energy from food and collect it in the ATP; anabolic processes use the
free energy stored in ATP to perform work.
Metabolism is highly regulated to permit organisms to respond to
changing conditions. Most pathways are irreversible.
•Flux - flow of material through a metabolic pathway which depends upon:
(1) Supply of substrates
(2) Removal of products
Catabolism and Anabolism

Anabolism and catabolism are coupled by energy

• Product of a pathway controls the rate of its own
synthesis by inhibiting an early step (usually the first
“committed” step (unique to the pathway)
Feedback inhibition
• Metabolite early in the pathway activates an enzyme
further down the pathway
Feed-forward activation

Stages of metabolism
Catabolism
Stage I. Breakdown of macromolecules (proteins,
carbohydrates and lipids to respective building
blocks.
Stage II. Amino acids, fatty acids and glucose
are oxidized to common metabolite (acetyl CoA)
Stage III. Acetyl CoA is oxidized in citric acid
cycle to CO2 and water. As result reduced
cofactor, NADH2 and FADH2, are formed which
give up their electrons. Electrons are transported
via the tissue respiration chain and released
energy is coupled directly to ATP synthesis.

Catabolism is characterized by convergence of three
major routs toward a final common pathway.
Different proteins, fats and carbohydrates enter the
same pathway – tricarboxylic acid cycle.
Anabolism can also be divided into stages, however the
anabolic pathways are characterized by divergence.
Monosaccharide synthesis begin with CO2,
oxaloacetate, pyruvate or lactate.
Amino acids are synthesized from acetyl CoA, pyruvate
or keto acids of Krebs cycle.
Fatty acids are constructed from acetyl CoA.
On the next stage monosaccharides, amino acids and
fatty acids are used for the synthesis of
polysaccharides, proteins and fats.

• Compartmentation of metabolic processes
permits:
- separate pools of metabolites within a cell
- simultaneous operation of opposing metabolic
paths
- high local concentrations of metabolites
• Example: fatty acid synthesis enzymes (cytosol),
fatty acid breakdown enzymes
(mitochondria)
Compartmentation of Metabolic
Processes in Cell

Compartmentation of metabolic processes

Pyruvate formed in the aerobic conditions undergoes
conversion to acetyl CoA by pyruvate dehydrogenase
complex.
Pyruvate dehydrogenase complex is a bridge between
glycolysis and aerobic metabolism – citric acid cycle.
Pyruvate dehydrogenase complex and enzymes of
cytric acid cycle are located in the matrix of
mitochondria.
OXIDATIVE DECARBOXYLATION OF PYRUVATE

Pyruvate translocase, protein embedded into the inner
membrane, transports pyruvate from the intermembrane space
into the matrix in symport with H+ and exchange (antiport) for
OH-.
Entry of Pyruvate into the Mitochondrion
Pyruvate freely diffuses through the outer membrane of mitochon-
dria through the channels formed by transmembrane proteins porins.

• Pyruvate dehydrogenase complex (PDH complex) is
a multienzyme complex containing 3 enzymes, 5
coenzymes and other proteins.
Conversion of Pyruvate to Acetyl CoA
Pyruvate
dehydrogenase
complex is giant,
with molecular
mass ranging
from 4 to 10
million daltons.
Electron micrograph of the
pyruvate dehydrogenase
complex from E. coli.

Enzymes:
E1 = pyruvate dehydrogenase
E2 = dihydrolipoyl acetyltransferase
E3 = dihydrolipoyl dehydrogenase
Coenzymes: TPP (thiamine pyrophosphate),
lipoamide, HS-CoA, FAD+, NAD+.
TPP is a prosthetic group of E1;
lipoamide is a prosthetic group of E2; and
FAD is a prosthetic group of E3.
The building block of
TPP is vitamin B1 (thiamin);
NAD – vitamin B5 (nicotinamide);
FAD – vitamin B2 (riboflavin),
HS-CoA – vitamin B3 (pantothenic acid),
lipoamide – lipoic acid

Overall reaction of pyruvate dehydrogenase complex
Pyruvate dehydrogenase complex is a classic example of
multienzyme complex
The oxidative decarboxylation of pyruvate catalized by
pyruvate dehydrogenase complex occurs in five steps.

Glucose
Glucose-6-
phosphate
Pyruvate
Glycogen Ribose, NADPH
Pentose phosphate
pathway
Synthesis of
glycogen
Degradation of
glycogen
Glycolysis Gluconeogenesis
Lactate
Ethanol
Acetyl Co A
Fatty Acids Amino Acids
The citric acid
cycle is the
final common
pathway for the
oxidation of fuel
molecules —
amino acids,
fatty acids, and
carbohydrates.
Most fuel
molecules
enter the
cycle as
acetyl
coenzyme A.

1. Citrate Synthase
• Citrate formed from acetyl CoA and oxaloacetate
• Only cycle reaction with C-C bond formation
• Addition of C2 unit (acetyl) to the keto double bond
of C4 acid, oxaloacetate, to produce C6 compound,
citrate
citrate synthase

2. Aconitase
• Elimination of H2O from citrate to form C=C bond
of cis-aconitate
• Stereospecific addition of H2O to cis-aconitate to
form isocitrate
aconitase aconitase

3. Isocitrate Dehydrogenase
• Oxidative decarboxylation of isocitrate to
a-ketoglutarate (a metabolically irreversible reaction)
• One of four oxidation-reduction reactions of the cycle
• Hydride ion from the C-2 of isocitrate is transferred to
NAD+ to form NADH
• Oxalosuccinate is decarboxylated to a-ketoglutarate
isocitrate dehydrogenase
isocitrate dehydrogenase

4. The -Ketoglutarate Dehydrogenase Complex
• Similar to pyruvate dehydrogenase complex
• Same coenzymes, identical mechanisms
E1 - a-ketoglutarate dehydrogenase (with TPP)
E2 – dihydrolipoyl succinyltransferase (with flexible
lipoamide prosthetic group)
E3 - dihydrolipoyl dehydrogenase (with FAD)
-ketoglutarate
dehydrogenase

5. Succinyl-CoA Synthetase
• Free energy in thioester bond of succinyl CoA is
conserved as GTP or ATP in higher animals (or ATP
in plants, some bacteria)
• Substrate level phosphorylation reaction
HS-
+
GTP + ADP GDP + ATP
Succinyl-CoA
Synthetase

• Complex of several polypeptides, an FAD prosthetic group and
iron-sulfur clusters
• Embedded in the inner mitochondrial membrane
• Electrons are transferred from succinate to FAD and then to
ubiquinone (Q) in electron transport chain
• Dehydrogenation is stereospecific; only the trans isomer is
formed
6. The Succinate Dehydrogenase Complex
Succinate
Dehydrogenase

7. Fumarase
• Stereospecific trans addition of water to the
double bond of fumarate to form L-malate
• Only the L isomer of malate is formed
Fumarase

8. Malate Dehydrogenase
Malate
Dehydrogenase
Malate is oxidized to form oxaloacetate.

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