The Control of Enzyme-Mediated

Reactions and Bioenergetics

References:
1. Widmaier, E. P., Raff, H., & Strang, K. T. (2006). Vander’s human physiology: the
mechanisms of body function. McGraw-Hill, New York, 10, 454-455.
2. Fox, S. I. (2006). Human Physiology 9th Editon (pp. 117-118). McGraw-Hill press, New York,
USA.
3. Murray, R. K. (2009). Harper's illustrated biochemistry.
4. Nelson, D. L., Lehninger, A. L., & Cox, M. M. (2008). Lehninger principles of biochemistry.
Macmillan.
5. Denniston, K., Topping, J., Woodrum, K., & Caret, R. (2013). General, organic, and
biochemistry. McGraw-Hill Higher Education.

Shakila Nargis Khan, PhD

Professor
Department of Microbiology
University of Dhaka
Dhaka 1000, Bangladesh
 General Pattern of a Metabolic Pathway

 The many thousands of different types of

enzymatic reactions within a cell do not occur
independently of each other. They are,
rather, all linked together by intricate webs
of interrelationships, the total pattern of
which constitutes cellular metabolism.
 A sequence of enzymatic reactions that
begins with an initial substrate, progresses
through a number of intermediates, and ends
with a final product is known as a metabolic
pathway.
 General Pattern of a Metabolic Pathway

 The enzymes in a metabolic pathway cooperate in a

manner analogous to workers on an assembly line,
where each contributes a small part to the final
product. In this process, the product of one enzyme
in the line becomes the substrate of the next
enzyme, and so on.

Figure: The general pattern of a metabolic pathway. In

metabolic pathways, the product of one enzyme
becomes the substrate of the next.
 General Pattern of a Metabolic Pathway

 Few metabolic pathways are completely linear. Most

are branched so that one intermediate at the branch
point can serve as a substrate for two different
enzymes. Two different products can thus be
formed that serve as intermediates of two pathways

Figure: A branched metabolic pathway. Two or more

different enzymes can work on the same substrate at the
branch point of the pathway, catalyzing two or more
different reactions.
 End-Product Inhibition
 The activities of enzymes at the branch points
of metabolic pathways are often regulated by a
process called end-product inhibition, which is a
form of negative feedback inhibition.
 In this process, one of the final products of a
divergent pathway inhibits the activity of the
branch-point enzyme that began the path
toward the production of this inhibitor.
 This inhibition prevents that final product from
accumulating excessively and results in a shift
toward the final product of the alternate
pathway.
 Inborn Errors of Metabolism
 Since each different polypeptide in the body is coded by a
different gene, each enzyme protein that participates in a
metabolic pathway is coded by a different gene.
 An inherited defect in one of these genes may result in a
disease known as an inborn error of metabolism.
 In this type of disease, the quantity of intermediates formed
prior to the defective enzymatic step increases, and the
quantity of intermediates and final products formed after the
defective step decreases.
 Diseases may result from deficiencies of the normal end
product or from excessive accumulation of intermediates
formed prior to the defective step.
 If the defective enzyme is active at a step that follows a
branch point in a pathway, the intermediates and final products
of the alternate pathway will increase. An abnormal increase in
the production of these products can be the cause of some
metabolic diseases.
 Inborn Errors of Metabolism

Figure: The effects of an inborn error of metabolism

on a branched metabolic pathway. The defective gene
produces a defective enzyme, indicated here by a line
through its symbol.
 Inborn Errors of Metabolism
 One of the conversion products of phenylalanine is a
molecule called DOPA, an acronym for
dihydroxyphenylalanine. DOPA is a precursor of the
pigment molecule melanin, which gives skin, eyes, and hair
their normal coloration. The condition of albinism results
from an inherited defect in the enzyme that catalyzes
the formation of melanin from DOPA

Figure: Metabolic pathways for the degradation of the amino acid

phenylalanine. Defective enzyme1 produces phenylketonuria (PKU),
defective enzyme5 produces alcaptonuria (not a clinically
significant condition), and defective enzyme6 produces albinism.
 Alteration of Enzyme’s Active Site by
Allosteric or Covalent Modulation
 Enzyme activity is a measure of the ability of
a given enzyme to convert its substrate(s)
into its product(s).
 Depending on the enzyme it is typically
assayed by measuring either the amount of
substrate that's disappearing, or the amount
of product that's appearing over a specified
period of time, such that the final result is
expressed in terms of moles of conversion
per unit mass of protein per unit time, for
example, nmol/mg protein/sec.
 Alteration of Enzyme’s Active Site by
Allosteric or Covalent Modulation
 Officially, enzyme activity is defined in terms of
"Units", i.e., the amount of enzyme that converts 1
µmol substrate to product in 1 minute, but this is
based on an assumption of purified enzyme, assayed
under conditions where the substrate is not limiting.
 However, in most real-world cases, one is not
working with a totally purified enzyme, but rather a
relatively crude mixture, e.g., an extract of tissue or
cells or body fluids.
 Thus, in the literature, enzyme activities are most
often reported as extent of conversion per unit time
and most kinetic calculations are based on this latter
method of expressing activity.
 Alteration of Enzyme’s Active Site by
Allosteric or Covalent Modulation
 If you measure the activity of a crude
extract you will get a fairly high total
activity, but it's specific activity, i.e.,
activity per unit protein will be low, because
your enzyme will be a very small part of the
extract.
 If you repeat measurements at each step
during a purification, the total activity will
decrease, but specific activity will go up,
because your enzyme is constituting an
increasing proportion of the total protein in
the sample.
• Specific Activity (SA) The concentration of enzyme in an impure preparation is
expressed in terms of units/ml.
• The Specific Activity (SA) of the preparation is reported as units/mg protein. As the
enzyme is purified, the specific activity will increase to a limit (that of pure enzyme).
• Since v varies with [S], pH, ionic strength, temperature, and like, a given preparation
can have an infinite number of specific activities. Consequently, specific activities are
usually reported for optimal assay conditions at a fixed temperature (usually 25, 30,
or 37oC), with all substrates present at saturating concentrations.
•
• International System (SI) of Units
• A new unit of enzyme activity, the katal, has been proposed. The katal (symbol: kat)
is the SI unit of catalytic activity.
• One katal is that amount of enzyme that catalyzes the conversion of one mole of
substrate per second under defined conditions. Thus,
• one International Unit = 1/60 katal = 16.67 nkatal.
• One katal = 6 x 107 Units.
• Specific activity can be expressed as katals/kg protein or katal/mg protein. The
molar activity of an enzyme is the katals/mole protein with units of per sec.
Turnover Number
• The “turnover number” of an enzyme is the number of substrate molecules
transformed per unit time by a single enzyme molecule (or by a single catalytic site)
when the enzyme concentration is the rate limiting factor.
• The enzyme carbonic anhydrase, an important enzyme found in high conc. In the
RBC, is among the most active of all enzymes with a turnover number of 36,000,000
per minute per enzyme molecule.
 Alteration of Enzyme’s Active Site by
Allosteric or Covalent Modulation
 In addition to changing the rate of enzyme-
mediated reactions by changing the
concentration of either substrate or enzyme,
the rate can be altered by changing enzyme
activity.
 A change in enzyme activity occurs when the
properties of the enzyme’s active site are
altered by either allosteric or covalent
modulation.
 Such modulation alters the rate at which the
binding site converts substrate to product, the
affinity of the binding site for substrate, or
both.
 Alteration of Enzyme’s Active Site by
Allosteric or Covalent Modulation
 The following figure illustrates the effect of increasing the
affinity of an enzyme’s active site without changing the
substrate or enzyme concentration.

Figure: At a constant substrate concentration, increasing the affinity of

an enzyme for its substrate by allosteric or covalent modulation
increases the rate of the enzyme-mediated reaction. Note that
increasing the enzyme’s affinity does not increase the maximal rate of
the enzyme-mediated reaction.
 Alteration of Enzyme’s Active Site by
Allosteric or Covalent Modulation
 Provided the substrate concentration is less than the
saturating concentration, the increased affinity of the
enzyme’s binding site results in an increased number of active
sites bound to substrate, and thus an increase in the reaction
rate.
 The regulation of metabolism through the control of enzyme
activity is an extremely complex process since, in many cases,
the activity of an enzyme can be altered by more than one
agent.
 The modulator molecules that allosterically alter enzyme
activities are product molecules of other cellular reactions.
The result is that the overall rates of metabolism can be
adjusted to meet various metabolic demands.
 In contrast, covalent modulation of enzyme activity is mediated
by protein kinase enzymes that are themselves activated by
various chemical signals received by the cell, for example, from
a hormone.
 Alteration of Enzyme’s Active Site by
Allosteric or Covalent Modulation

Figure: On a single enzyme, multiple sites can modulate

enzyme activity and hence the reaction rate by
allosteric and covalent activation or inhibition.
 Regulatory Enzymes
 In cellular metabolism, groups of enzymes work together in
sequential pathways to carry out a given metabolic process,
such as the multi-reaction breakdown of glucose to lactate
or the multi-reaction synthesis of an amino acid from
simpler precursors. In such enzyme systems, the reaction
product of one enzyme becomes the substrate of the next.
 Each pathway, however, includes one or more enzymes that
have a greater effect on the rate of the overall sequence.
 These regulatory enzymes exhibit increased or decreased
catalytic activity in response to certain signals.
 Adjustments in the rate of reactions catalyzed by
regulatory enzymes, and therefore in the rate of entire
metabolic sequences, allow the cell to meet changing needs
for energy and for biomolecules required in growth and
repair.
 Regulatory Enzymes
 In most multi-enzyme systems, the first enzyme of
the sequence is a regulatory enzyme.
 This is an excellent place to regulate a pathway,
because catalysis of even the first few reactions of a
sequence that leads to an unneeded product diverts
energy and metabolites from more important
processes.
 Other enzymes in the sequence are usually present at
levels that provide an excess of catalytic activity;
they can generally promote their reactions as fast as
their substrates are made available from preceding
reactions.
 The activities of regulatory enzymes are modulated in
a variety of ways.
 Regulatory Enzymes
 Allosteric enzymes function through
reversible, noncovalent binding of regulatory
compounds called allosteric modulators or
allosteric effectors, which are generally
small metabolites or cofactors.
 Other enzymes are regulated by reversible
covalent modification.
 Both classes of regulatory enzymes tend to
be multi-subunit proteins, and in some cases
the regulatory site(s) and the active site are
on separate subunits.
 Regulatory Enzymes
 Metabolic systems have at least two other
mechanisms of enzyme regulation.
 Some enzymes are stimulated or inhibited
when they are bound by separate regulatory
proteins.
 Others are activated when peptide segments
are removed by proteolytic cleavage; unlike
effector-mediated regulation, regulation by
proteolytic cleavage is irreversible.
 Important examples of both mechanisms are
found in physiological processes such as
digestion, blood clotting, hormone action, and
vision.
 Regulatory Enzymes
 Cell growth and survival depend on efficient use of
resources, and this efficiency is made possible by
regulatory enzymes.
 No single rule governs the occurrence of different
types of regulation in different systems. To a
degree, allosteric (noncovalent) regulation may
permit fine-tuning of metabolic pathways that are
required continuously but at different levels of
activity as cellular conditions change.
 Regulation by covalent modification may be all or
none—usually the case with proteolytic cleavage—or
it may allow for subtle changes in activity. Several
types of regulation may occur in a single regulatory
enzyme.
 Regulatory Enzymes
Enzymatic activity is regulated in five principle ways:
1. Allosteric control. Allosteric proteins contain distinct
regulatory sites and multiple functional sites.
cooperativity
2. Multiple forms of enzymes. Isozymes, or isoenzymes,
provide an avenue for varying regulation of the same
reaction at distinct locations or times.
3. Reversible covalent modification. The catalytic
properties of enzymes are altered by the covalent
attachment of a modifying group. Kinase - phosphatase
4. Proteolytic activation : Irreversible convert an inactive
enzyme into an active one.
5. Controlling the amount of enzyme present.
Transcription level
 Allosteric Enzymes
 Allosteric enzymes undergo conformational
changes in response to modulator binding.
 Allosteric proteins are those having “other
shapes” or conformations induced by the
binding of modulators.
 The same concept applies to certain
regulatory enzymes, as conformational
changes induced by one or more modulators
interconvert more-active and less-active
forms of the enzyme.
 The modulators for allosteric enzymes may
be inhibitory or stimulatory.
 Allosteric Enzymes
 Often the modulator is the substrate itself;
regulatory enzymes for which substrate and
modulator are identical are called
homotropic. The effect is similar to that of
O2 binding to hemoglobin: binding of the
ligand—or substrate, in the case of enzymes
—causes conformational changes that affect
the subsequent activity of other sites on the
protein.
 When the modulator is a molecule other than
the substrate, the enzyme is said to be
heterotropic.
 Allosteric Enzymes
 Note that allosteric modulators should not be
confused with uncompetitive and mixed inhibitors.
 Although the latter bind at a second site on the
enzyme, they do not necessarily mediate
conformational changes between active and inactive
forms, and the kinetic effects are distinct.
 The properties of allosteric enzymes are
significantly different from those of simple
nonregulatory enzymes. Some of the differences are
structural.
 In addition to active sites, allosteric enzymes
generally have one or more regulatory, or allosteric,
sites for binding the modulator.
 Allosteric Enzymes

Figure: Subunit interactions in an allosteric enzyme, and interactions with inhibitors and
activators. In many allosteric enzymes the substrate binding site and the modulator binding
site(s) are on different subunits, the catalytic (C) and regulatory (R) subunits, respectively.
Binding of the positive (stimulatory) modulator (M) to its specific site on the regulatory
subunit is communicated to the catalytic subunit through a conformational change. This change
renders the catalytic subunit active and capable of binding the substrate (S) with higher
affinity. On dissociation of the modulator from the regulatory subunit, the enzyme reverts to
its inactive or less active form.
 Allosteric Enzymes
 Just as an enzyme’s active site is specific for its
substrate, each regulatory site is specific for its
modulator.
 Enzymes with several modulators generally have
different specific binding sites for each.
 In homotropic enzymes, the active site and
regulatory site are the same.
 Allosteric enzymes are generally larger and more
complex than nonallosteric enzymes. Most have two
or more subunits.
 Aspartate transcarbamoylase, which catalyzes an
early reaction in the biosynthesis of pyrimidine
nucleotides, has 12 polypeptide chains organized into
catalytic and regulatory subunits.
 Allosteric Enzymes
 Just as an enzyme’s active site is specific for its
substrate, each regulatory site is specific for its
modulator.
 Enzymes with several modulators generally have
different specific binding sites for each.
 In homotropic enzymes, the active site and
regulatory site are the same.
 Allosteric enzymes are generally larger and more
complex than nonallosteric enzymes. Most have two
or more subunits.
 Aspartate transcarbamoylase, which catalyzes an
early reaction in the biosynthesis of pyrimidine
nucleotides, has 12 polypeptide chains organized into
catalytic and regulatory subunits.
 Feedback Inhibition
 Feedback inhibition refers to inhibition of an enzyme in a
biosynthetic pathway by an end product of that pathway.
 For example, for the biosynthesis of D from A catalyzed
by enzymes Enz1 through Enz3, high concentrations of D
inhibit conversion of A to B. Inhibition results not from
the “backing up” of intermediates but from the ability of
D to bind to and inhibit Enz1. Typically, D binds at an
allosteric site spatially distinct from the catalytic site of
the target enzyme. Feedback inhibitors thus are
allosteric effectors and typically bear little or no
structural similarity to the substrates of the enzymes
they inhibit. In this example, the feedback inhibitor D
acts as a negative allosteric effector of Enz1.
 Feedback Inhibition
 In a branched biosynthetic pathway, the initial reactions participate in
the synthesis of several products. The following figure shows a
hypothetical branched biosynthetic pathway in which curved arrows
lead from feedback inhibitors to the enzymes whose activity they
inhibit. The sequences:

each represent linear reaction sequences that are feedback-inhibited

by their end products. The pathways of nucleotide biosynthesis
provide specific examples.

Figure: Sites of feedback inhibition in a branched biosynthetic pathway. S1–S5 are

intermediates in the biosynthesis of end products A–D. Straight arrows represent
enzymes catalyzing the indicated conversions. Curved arrows represent feedback loops and
indicate sites of feedback inhibition by specific end products.
 Feedback Inhibition
 The kinetics of feedback inhibition may be
competitive, noncompetitive, partially
competitive, or mixed.
 Feedback inhibitors, which frequently are
the small molecule building blocks of
macromolecules (e.g., amino acids for
proteins, nucleotides for nucleic acids),
typically inhibit the first committed step in a
particular biosynthetic sequence.
 A much-studied example is inhibition of
bacterial aspartate transcarbamoylase by
CTP.
 Feedback Inhibition

Aspartate transcarbamoylase catalyzes the first step in

the biosynthesis of pyrimidines (such as cytidine
triphosphate CTP).
 Feedback Inhibition
ATCase is inhibited by CTP,
the final product of the
ATCase-initiated pathway.

-The inhibition of ATCase by CTP is an example of

feedback inhibition, the inhibition of an enzyme by the
end product of the pathway.
-CTP is structurally quite different from the
substrate and product of the reaction.
-Thus CTP must bind to a site distinct from the active
site (allosteric or regulatory site).
 Feedback Inhibition
 The enzyme exists in an equilibrium
between the T and R state.
 In the absence of substrate, almost all
the enzyme molecules are in the T
state(low affinity for substrates and
low catalytic activity).
 Occasional substrate binding to active
site → entire enzyme shifts to the R
state(higher binding affinity).
 Feedback Inhibition
Mechanism for allosteric regulation
1) Concerted mechanism
 the change in the enzyme is ‘all or none’
 explains the behavior of ATCase well

2) Sequential model
 the binding of ligand to one site on the
complex can affect neighboring sites without
T to R transition
 Most other allosteric enzymes have features
of both models.
Feedback Inhibition

- The enzyme is in the T state

when bound to CTP.
-Binding sites for CTP exist in
regulatory chains which are far
away from each active sites.
-How can CTP inhibit the
catalytic activity of the
enzyme?
 Feedback Inhibition

 CTP stabilize the T state.

 The binding of CTP shifts the equilibrium toward
the T state and makes it more difficult for
substrate binding to convert the enzyme into the R
state.
 Feedback Inhibition

In presence of CTP, more substrate In presence of ATP, the reaction

is required to attain a given reaction rate is increased.
rate.
High levels of ATP prevent CTP from inhibiting the
enzyme. Nonsubstrate effects on allosteric enzyme
Heterotropic effects
 Feedback Inhibition
 Physiological roles of the increase in ATCase
activity in response to increased ATP
concentration
 High ATP concentration signals a high
concentration of purine nucleotides in the
cell. (to balance between purine and
pyrimidine)
 A high concentration of ATP indicates that
energy is available for mRNA synthesis and
DNA replication and lead to the synthesis of
pyrimidines needed for these processes.
 Feedback Inhibition
 Multiple feedback loops can provide additional
fine control

Figure: Multiple feedback inhibition in a branched biosynthetic

pathway. Superimposed on simple feedback loops (dashed, curved
arrows) are multiple feedback loops (solid, curved arrows) that
regulate enzymes common to biosynthesis of several end products.
 Feedback Inhibition
 In the previous figure, the presence of excess
product B decreases the requirement for
substrate S2.
 However, S2 is also required for synthesis of A,
C, and D. Excess B should therefore also curtail
synthesis of all four end products.
 To circumvent this potential difficulty, each end
product typically only partially inhibits catalytic
activity.
 The effect of an excess of two or more end
products may be strictly additive or,
alternatively, may be greater than their
individual effect (cooperative feedback
inhibition).
 Feedback Inhibition
 Allosteric and catalytic sites are spatially distinct.
 The lack of structural similarity between a feedback
inhibitor and the substrate for the enzyme whose activity
it regulates suggests that these effectors are not
isosteric with a substrate but allosteric (“occupy another
space”).
 Jacques Monod therefore proposed the existence of
allosteric sites that are physically distinct from the
catalytic site.
 Allosteric enzymes thus are those whose activity at the
active site may be modulated by the presence of effectors
at an allosteric site.
 This hypothesis has been confirmed by many lines of
evidence, including x-ray crystallography and site-directed
mutagenesis, demonstrating the existence of spatially
distinct active and allosteric sites on a variety of enzymes.
 Feedback Regulation is Not Synonymous with Feedback
Inhibition
 In both mammalian and bacterial cells, end products
“feedback” and control their own synthesis, in many instances
by feedback inhibition of an early biosynthetic enzyme.
 We must, however, distinguish between feedback regulation,
a phenomenologic term devoid of mechanistic implications,
and feedback inhibition, a mechanism for regulation of
enzyme activity.
 For example, while dietary cholesterol decreases hepatic
synthesis of cholesterol, this feedback regulation does not
involve feedback inhibition.
 HMG-CoA reductase, the rate-limiting enzyme of
cholesterologenesis, is affected, but cholesterol does not
feedback-inhibit its activity. Regulation in response to
dietary cholesterol involves curtailment by cholesterol or a
cholesterol metabolite of the expression of the gene that
encodes HMG-CoA reductase (enzyme repression).
 The Kinetic Properties of Allosteric Enzymes Diverge
from Michaelis-Menten Behavior

Figure: Substrate-activity curves for representative allosteric enzymes. Three examples of

complex responses of allosteric enzymes to their modulators. (a) The sigmoid curve of a
homotropic enzyme, in which the substrate also serves as a positive (stimulatory) modulator, or
activator. Note the resemblance to the oxygen-saturation curve of hemoglobin. (b) The effects
of a positive modulator (+) and a negative modulator () on an allosteric enzyme in which K0.5 is
altered without a change in Vmax. The central curve shows the substrate-activity relationship
without a modulator. (c) A less common type of modulation, in which Vmax is altered and K0.5 is
nearly constant.
 Feedback Regulation is Not Synonymous with Feedback
Inhibition
 Allosteric enzymes show relationships between V 0
and [S] that differ from Michaelis-Menten
kinetics.
 They do exhibit saturation with the substrate
when [S] is sufficiently high, but for some
allosteric enzymes, plots of V0 versus [S] produce
a sigmoid saturation curve, rather than the
hyperbolic curve typical of nonregulatory
enzymes.
 On the sigmoid saturation curve we can find a
value of [S] at which v0 is half-maximal, but we
cannot refer to it with the designation Km,
because the enzyme does not follow the
hyperbolic Michaelis-Menten relationship.
 Feedback Regulation is Not Synonymous with Feedback
Inhibition
 Instead, the symbol [S]0.5 or K0.5 is often used to
represent the substrate concentration giving half-
maximal velocity of the reaction catalyzed by an
allosteric enzyme
 Sigmoid kinetic behavior generally reflects
cooperative interactions between protein subunits. In
other words, changes in the structure of one subunit
are translated into structural changes in adjacent
subunits, an effect mediated by noncovalent
interactions at the interface between subunits. The
principles are particularly well illustrated by a
nonenzyme: O2 binding to hemoglobin. Sigmoid kinetic
behavior is explained by the concerted and sequential
models for subunit interactions.
 Feedback Regulation is Not Synonymous with Feedback
Inhibition
 Homotropic allosteric enzymes generally are multi-subunit
proteins and, as noted earlier, the same binding site on
each subunit functions as both the active site and the
regulatory site.
 Most commonly, the substrate acts as a positive
modulator (an activator), because the subunits act
cooperatively: the binding of one molecule of substrate to
one binding site alters the enzyme’s conformation and
enhances the binding of subsequent substrate molecules.
 This accounts for the sigmoid rather than hyperbolic
change in v0 with increasing [S]. One characteristic of
sigmoid kinetics is that small changes in the concentration
of a modulator can be associated with large changes in
activity. As is evident from previous figure (a), a relatively
small increase in [S] in the steep part of the curve causes
a comparatively large increase in v0.
 Feedback Regulation is Not Synonymous with Feedback
Inhibition
 For heterotropic allosteric enzymes, those whose
modulators are metabolites other than the normal
substrate, it is difficult to generalize about the
shape of the substrate-saturation curve.
 An activator may cause the curve to become more
nearly hyperbolic, with a decrease in K 0.5 but no
change in Vmax, resulting in an increased reaction
velocity at a fixed substrate concentration (v 0 is
higher for any value of [S]; previous figure (b),
upper curve). Other heterotropic allosteric
enzymes respond to an activator by an increase in
Vmax with little change in K0.5 (previous figure
(c)).
 Feedback Regulation is Not Synonymous with Feedback
Inhibition

 A negative modulator (an inhibitor) may

produce a more sigmoid substrate-
saturation curve, with an increase in K0.5
(previous figure (b), lower curve).
 Heterotropic allosteric enzymes
therefore show different kinds of
responses in their substrate-activity
curves, because some have inhibitory
modulators, some have activating
modulators, and some have both.
 Isozymes
 Isozymes provide a means of regulation
specific to distinct tissues and
developmental states
 Isozymes (or isoenzymes) are enzymes
that differ in amino acid sequence yet
catalyze the same reaction.
 Have different Km, different regulatory
molecules
 Isozymes
-LDH(lactate dehydrogenase): catalyzes a step in anaerobic glucose
metabolism and glucose synthesis (pyruvate  lactate).
-H isozyme : expressed in heart muscle.
-M isozyme : expressed in skeletal muscle.
amino acid sequences are 75% identical each other.
H isozyme (square) has higher affinity for substrate than M (circle)

The rat heart LDH isozyme profile changes in the course

of development. Functional enzyme is tetrameric.
 Covalent Modification
 Covalent modification is a mean of regulating enzyme
activity.
 Some modifications are reversible.
 Covalent Modification
 Phosphorylation is a highly effective mean of regulating the
activities of target proteins.
 30% of eukaryotic proteins are phosphorylated.
 The enzymes catalyzing phosphorylation reactions are called
protein kinases.
 ATP is the most common donor of phosphoryl group.
Covalent Modification
 Covalent Modification
 Protein phosphatases reverse the effects of kinases by
catalyzing the removal of phosphoryl groups attacked to
proteins.
 Hydroxyl-containing side chain is regenerated and Pi is
produced.
 Vital role in cells because the enzymes turn off the signals
 One class of highly conserved phosphatase called PP2A
suppresses the cancer-promoting activity of certain
kinases.
 Covalent Modification
 The phosphorylation and
dephosphorylation are not the
reverse of one another;
irreversible under physiological
conditions without enzymes
 With only the help of kinases and
phosphatase, take place
 The rate of cycling between the
phosphorylated and the
dephosphorylated states depends
on the relative activities of
kinases and phosphatases
 Proteolytic Cleavage
 There are a number of important cases in
which enzymes are produced as inactive
forms.
 In the cells of the pancreas, for example,
many digestive enzymes are produced as
inactive zymogens, which are activated after
they are secreted into the intestine.
 Activation of zymogens in the intestinal
lumen (cavity) protects the pancreatic cells
from self-digestion.
 Proteolytic Cleavage
 Certain proteins are synthesized and secreted as inactive
precursor proteins known as proproteins.
 The proproteins of enzymes are termed proenzymes or
zymogens.
 Selective proteolysis converts a proprotein by one or
more successive proteolytic “clips” to a form that
exhibits the characteristic activity of the mature
protein, eg, its enzymatic activity.
 Proteins synthesized as proproteins include the hormone
insulin (proprotein = proinsulin), the digestive enzymes
pepsin, trypsin, and chymotrypsin (proproteins =
pepsinogen, trypsinogen, and chymotrypsinogen,
respectively), several factors of the blood clotting and
blood clot dissolution cascades, and the connective tissue
protein collagen (proprotein = procollagen).
Proteolytic Cleavage
Proteolytic Cleavage
Proteolytic Cleavage
 Proteolytic Cleavage

Figure: Activation of zymogens by proteolytic cleavage. Shown here is the formation of

chymotrypsin and trypsin from their zymogens, chymotrypsinogen and trypsinogen. The bars
represent the amino acid sequences of the polypeptide chains, with numbers indicating the
relative positions of the residues (the amino-terminal residue is number 1). Residues at the
termini of the polypeptide fragments generated by cleavage are indicated below the bars.
Note that in the final active forms, some numbered residues are missing. Recall that the
three polypeptide chains (A, B, and C) of chymotrypsin are linked by disulfide bonds.
 Inborn Errors of Metabolism
 One of the conversion products of phenylalanine is a
molecule called DOPA, an acronym for
dihydroxyphenylalanine. DOPA is a precursor of the
pigment molecule melanin, which gives skin, eyes, and hair
their normal coloration. The condition of albinism results
from an inherited defect in the enzyme that catalyzes
the formation of melanin from DOPA

Figure: Metabolic pathways for the degradation of the amino acid

phenylalanine. Defective enzyme1 produces phenylketonuria (PKU),
defective enzyme5 produces alcaptonuria (not a clinically
significant condition), and defective enzyme6 produces albinism.
 Section Summary: Metabolic Pathways
 Metabolic pathways involve a number of enzyme-catalyzed
reactions.
 A number of enzymes usually cooperate to convert an
initial substrate to a final product by way of several
intermediates.
 Metabolic pathways are produced by multi-enzyme systems
in which the product of one enzyme becomes the substrate
of the next.
 If an enzyme is defective due to an abnormal gene, the
intermediates that are formed following the step
catalyzed by the defective enzyme will decrease, and the
intermediates that are formed prior to the defective step
will accumulate. (1) Diseases that result from defective
enzymes are called inborn errors of metabolism. (2)
Accumulation of intermediates often results in damage to
the organ in which the defective enzyme is found.
 Section Summary: Metabolic Pathways

 Many metabolic pathways are branched, so

that one intermediate can serve as the
substrate for two different enzymes.
 The activity of a particular pathway can be
regulated by end-product inhibition. (1) In
end-product inhibition, one of the products
of the pathway inhibits the activity of a key
enzyme. (2) This is an example of allosteric
inhibition, in which the product combines
with its specific site on the enzyme, changing
the conformation of the active site.
 Bioenergetics
 Living organisms require the constant expenditure of
energy to maintain their complex structures and
processes. Central to life processes are chemical
reactions that are coupled, so that the energy
released by one reaction is incorporated into the
products of another reaction.
 Bioenergetics refers to the flow of energy in living
systems.
 Organisms maintain their highly ordered structure
and life-sustaining activities through the constant
expenditure of energy obtained ultimately from the
environment.
 The energy flow in living systems obeys the first and
second laws of a branch of physics known as
thermodynamics.
 Laws of Thermodynamics
 According to the first law of thermodynamics,
energy can be transformed (changed from one
form to another), but it can neither be created
nor destroyed. This is sometimes called the law
of conservation of energy.
 As a result of energy transformations, according
to the second law of thermodynamics, the
universe and its parts (including living systems)
become increasingly disorganized.
 The term entropy is used to describe the degree
of disorganization of a system.
 Laws of Thermodynamics
 Energy transformations thus increase the
amount of entropy of a system. Only energy that
is in an organized state—called free energy—can
be used to do work.
 Since entropy increases in every energy
transformation, the amount of free energy
available to do work decreases.
 As a result of the increased entropy described
by the second law, systems tend to go from
states of higher free energy to states of lower
free energy.
 Laws of Thermodynamics
 The chemical bonding of atoms into molecules
obeys the laws of thermodynamics.
 A complex organic molecule such as glucose
has more free energy (less entropy) than six
separate molecules each of carbon dioxide
and water.
 Therefore, in order to convert carbon
dioxide and water to glucose, energy must be
added. Plants perform this feat using energy
from the sun in the process of
photosynthesis
 Laws of Thermodynamics

Figure: A simplified diagram of photosynthesis. Some of the sun’s radiant energy is

captured by plants and used to produce glucose from carbon dioxide and water. As
the product of this endergonic reaction, glucose has more free energy than the
initial reactants.
 Endergonic and Exergonic Reactions

 Chemical reactions that require an input of energy

are known as endergonic reactions.
 Since energy is added to make these reactions “go,”
the products of endergonic reactions must contain
more free energy than the reactants.
 A portion of the energy added, in other words, is
contained within the product molecules.
 This follows from the fact that energy cannot be
created or destroyed (first law of thermodynamics)
and from the fact that a more-organized state of
matter contains more free energy, or less entropy,
than a less-organized state (second law of
thermodynamics).
 Endergonic and Exergonic Reactions

 The fact that glucose contains more free

energy than carbon dioxide and water can
easily be proven by combusting glucose to
CO2 and H2O.
 This reaction releases energy in the form of
heat.
 Reactions that convert molecules with more
free energy to molecules with less- and,
therefore, that release energy as they
proceed—are called exergonic reactions.
 Endergonic and Exergonic Reactions
 The total amount of energy released by a molecule in a
combustion reaction can be released in smaller portions, by
enzymatically controlled exergonic reactions within cells.
 This allows the cells to use the energy to “drive” other
processes, as described in the next section.
 Since the energy obtained by the body from the cellular
oxidation of a molecule is the same as the amount released
when the molecule is combusted, the energy in food molecules
can conveniently be measured by the heat released when the
molecules are combusted.
 Heat is measured in units called calories. One calorie is defined
as the amount of heat required to raise the temperature of one
cubic centimeter of water one degree on the Celsius scale. The
caloric value of food is usually indicated in kilocalories (one
kilocalorie = 1,000 calories), which are often called large
calories and spelled with a capital C.
 Endergonic and Exergonic Reactions

Figure: A comparison of combustion and cell respiration. Since

glucose contains more energy than six separate molecules each of
carbon dioxide and water, the combustion of glucose is an
exergonic reaction. The same amount of energy is released when
glucose is broken down stepwise within the cell.
 Coupled Reactions: ATP
 In order to remain alive, a cell must maintain its highly
organized, low-entropy state at the expense of free energy in
its environment.
 Accordingly, the cell contains many enzymes that catalyze
exergonic reactions using substrates that come ultimately from
the environment.
 The energy released by these exergonic reactions is used to
drive the energy-requiring processes (endergonic reactions) in
the cell. Since cells cannot use heat energy to drive energy-
requiring processes, the chemical-bond energy that is released
in exergonic reactions must be directly transferred to
chemical-bond energy in the products of endergonic reactions.
 Energy-liberating reactions are thus coupled to energy-
requiring reactions. This relationship is like that of two meshed
gears; the turning of one (the energy-releasing exergonic gear)
causes turning of the other (the energy-requiring endergonic
gear).
 Coupled Reactions: ATP

Figure: A model of the coupling of exergonic and endergonic

reactions. The reactants of the exergonic reaction (represented
by the larger gear) have more free energy than the products of
the endergonic reaction because the coupling is not 100%
efficient—some energy is lost as heat.
 Coupled Reactions: ATP
 The energy released by most exergonic reactions in the cell is
used, either directly or indirectly, to drive one particular
endergonic reaction: the formation of adenosine triphosphate
(ATP) from adenosine diphosphate (ADP) and inorganic
phosphate (abbreviated Pi).

Figure: The formation and structure of adenosine triphosphate (ATP). ATP is the
universal energy carrier of the cell. High-energy bonds are indicated by a squiggle
(~).
 Coupled Reactions: ATP
 The formation of ATP requires the input of a fairly
large amount of energy.
 Since this energy must be conserved (first law of
thermodynamics), the bond produced by joining Pi to
ADP must contain a part of this energy. Thus, when
enzymes reverse this reaction and convert ATP to
ADP and Pi, a large amount of energy is released.
 Energy released from the breakdown of ATP is used
to power the energy-requiring processes in all cells.
As the universal energy carrier, ATP serves to more
efficiently couple the energy released by the
breakdown of food molecules to the energy required
by the diverse endergonic processes in the cell.
 Coupled Reactions: ATP

Figure: A model of ATP as the universal energy carrier of the

cell. Exergonic reactions are shown as gears with arrows going
down (these reactions produce a decrease in free energy);
endergonic reactions are shown as gears with arrows going up
(these reactions produce an increase in free energy).
 Coupled Reactions: Oxidation- Reduction
 When an atom or a molecule gains electrons, it is said to
become reduced; when it loses electrons, it is said to become
oxidized.
 Reduction and oxidation are always coupled reactions: an atom
or a molecule cannot become oxidized unless it donates
electrons to another, which therefore becomes reduced.
 The atom or molecule that donates electrons to another is a
reducing agent, and the one that accepts electrons from
another is an oxidizing agent.
 It is important to understand that a particular atom (or
molecule) can play both roles; it may function as an oxidizing
agent in one reaction and as a reducing agent in another
reaction.
 When atoms or molecules play both roles, they gain electrons in
one reaction and pass them on in another reaction to produce a
series of coupled oxidation-reduction reactions—like a bucket
brigade, with electrons in the buckets.
 Coupled Reactions: Oxidation- Reduction
 Notice that the term oxidation does not imply that oxygen
participates in the reaction. This term is derived from the
fact that oxygen has a great tendency to accept electrons;
that is, to act as a strong oxidizing agent. This property of
oxygen is exploited by cells; oxygen acts as the final
electron acceptor in a chain of oxidation-reduction
reactions that provides energy for ATP production.
 Oxidation-reduction reactions in cells often involve the
transfer of hydrogen atoms rather than free electrons.
Since a hydrogen atom contains one electron (and one
proton in the nucleus), a molecule that loses hydrogen
becomes oxidized, and one that gains hydrogen becomes
reduced. In many oxidationreduction reactions, pairs of
electrons—either as free electrons or as a pair of
hydrogen atoms—are transferred from the reducing agent
to the oxidizing agent.
 Coupled Reactions: Oxidation- Reduction

 Two molecules that serve important roles in the

transfer of hydrogens are nicotinamide adenine
dinucleotide (NAD), which is derived from the
vitamin niacin (vitamin B3), and flavin adenine
dinucleotide (FAD), which is derived from the
vitamin riboflavin (vitamin B2).
 These molecules are coenzymes that function as
hydrogen carriers because they accept hydrogens
(becoming reduced) in one enzyme reaction and
donate hydrogens (becoming oxidized) in a
different enzyme reaction. The oxidized forms of
these molecules are written simply as NAD (or
NAD+) and FAD.
 Coupled Reactions: Oxidation- Reduction

Figure: Structural formulas for NAD+, NADH, FAD, and FADH2. (a)
When NAD+ reacts with two hydrogen atoms, it binds to one of them and
accepts the electron from the other. This is shown by two dots above the
nitrogen ( ) in the formula for NADH. (b) When FAD reacts with two
hydrogen atoms to form FADH2, it binds each of them to a nitrogen atom
at the reaction sites.
 Coupled Reactions: Oxidation- Reduction

Figure: The action of NAD. NAD is a coenzyme that transfers

pairs of hydrogen atoms from one molecule to another. In the
first reaction, NAD is reduced (acts as an oxidizing agent); in
the second reaction, NADH is oxidized (acts as a reducing
agent). Oxidation reactions are shown by red arrows, reduction
reactions by blue arrows.
 Coupled Reactions: Oxidation- Reduction

 Each FAD can accept two electrons and can bind

two protons. Therefore, the reduced form of
FAD is combined with the equivalent of two
hydrogen atoms and may be written as FADH2.
 Each NAD can also accept two electrons but can
bind only one proton. The reduced form of NAD is
therefore indicated by NADH + H + (the H+
represents a free proton).
 When the reduced forms of these two coenzymes
participate in an oxidationreduction reaction,
they transfer two hydrogen atoms to the
oxidizing agent
 Section Summary: Bioenergetics

 The flow of energy in the cell is called

bioenergetics.
 According to the first law of thermodynamics,
energy can neither be created nor destroyed but
only transformed from one form to another.
 According to the second law of thermodynamics,
all energy transformation reactions result in an
increase in entropy (disorder). (1) As a result of
the increase in entropy, there is a decrease in
free (usable) energy. (2) Atoms that are
organized into large organic molecules thus
contain more free energy than more-
disorganized, smaller molecules.
 Section Summary: Bioenergetics
 In order to produce glucose from carbon dioxide and
water, energy must be added. (1) Plants use energy
from the sun for this conversion, in a process called
photosynthesis. (2) Reactions that require the input
of energy to produce molecules with higher free
energy than the reactants are called endergonic
reactions.
 The combustion of glucose to carbon dioxide and
water releases energy in the form of heat. (1) A
reaction that releases energy, thus forming products
that contain less free energy than the reactants, is
called an exergonic reaction. (2) The same total
amount of energy is released when glucose is
converted into carbon dioxide and water within cells,
even though this process occurs in many small steps.
 Section Summary: Bioenergetics
 The exergonic reactions that convert food molecules
into carbon dioxide and water in cells are coupled to
endergonic reactions that form adenosine
triphosphate (ATP). (1) Some of the chemical-bond
energy in glucose is therefore transferred to the
“high energy” bonds of ATP. (2) The breakdown of
ATP into adenosine diphosphate (ADP) and inorganic
phosphate results in the liberation of energy. (3)
The energy liberated by the breakdown of ATP is
used to power all of the energy-requiring processes
of the cell. ATP is thus the “universal energy
carrier” of the cell.
 Oxidation-reduction reactions are coupled and
usually involve the transfer of hydrogen atoms.
 Section Summary: Bioenergetics

 A molecule is said to be oxidized when it loses electrons;

it is said to be reduced when it gains electrons.
 A reducing agent is thus an electron donor; an oxidizing
agent is an electron acceptor.
 Although oxygen is the final electron acceptor in the cell,
other molecules can act as oxidizing agents.
 A single molecule can be an electron acceptor in one
reaction and an electron donor in another. (1) NAD and
FAD can become reduced by accepting electrons from
hydrogen atoms removed from other molecules. (2) NADH
+ H+, and FADH2, in turn, donate these electrons to
other molecules in other locations within the cells. (3)
Oxygen is the final electron acceptor (oxidizing agent) in
a chain of oxidation-reduction reactions that provide
energy for ATP production.