Biochemistry and Pathophysiology Lectures
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Learning objectives
Explain the basic principles of enzyme kinetics and energy metabolism.
Explain the biochemical processes of carbohydrate, fat, and protein metabolism in
the human body.
Explain how biochemical processes are regulated to maintain homeostasis.
Integrate the key metabolic processes active in the human body during starvation,
obesity, alcohol abuse, and physical sports.
Apply the principles of metabolic regulatory systems at the molecular, cellular,
organ, and systemic levels to medical cases involving metabolic derangement.
Analyse in a team the molecular mechanism of action of an assigned drug.
Give an oral presentation and write an essay, both in scientific English.
Apply biochemical knowledge and data from current biomedical research to answer
research questions regarding important human diseases.
Topics
Connecting theme: development of new drugs
- Metabolism: Basic concepts and design.
- Signal-transduction pathways.
- The Citric Acid Cycle:
Glycolysis and gluconeogenesis.
Pentose Phosphate Pathway.
Oxidative Phosphorylation.
- Glycogen metabolism:
Fat metabolism (including fatty acid metabolism).
Protein turnover and amino acid catabolism.
Hemostasis and Thrombosis.
- The integration of metabolism:
Regulation of gene expression.
Lecture 1 – Enzymes and Enzyme Inhibitors
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Overview:
Basic concepts and enzyme kinetics.
Suicide inhibitors.
The family of CYP enzymes.
Assumed knowledge of enzymes  Chapter 5 (10th edition) of Biochemistry
Catalysis takes place at a particular site of the enzyme called the active site. They catalyze
reactions by stabilizing transition states, the highest-energy species in reaction pathways.
By selectively stabilizing a transition state, an enzyme determines which one of several
potential chemical reactions actually takes place. Carbonic anhydrase that catalyzes the
hydration of carbon dioxide is one of the fastest enzymes known. Proteolytic enzymes have
the biochemical function to catalyze the hydrolysis of a peptide bond. These enzymes are
different in their degree of substrate specificity when compared to other enzymes. For
example: Papain will cleave any peptide bond with little regard to the adjacent side chains
while Thrombin (an enzyme that participates in blood clotting) only catalyzes the hydrolysis
of Arg-Gly bonds in particular peptide sequences.
Many enzymes require cofactors for activity.
Generally, these cofactors can execute chemical reactions that cannot be performed by the
standard set of twenty amino acids. An enzyme without its cofactor is referred to as an
apoenzyme; the complete, catalytically active enzyme is called a holoenzyme.
Apoenzyme + cofactor = holoenzyme
Cofactors can be divided into two groups: metals and small organic molecules (coenzymes).
Coenzymes are usually derived from vitamins and can be either tightly or loosely bound to
the enzyme. Tightly bound coenzymes are called prosthetic groups. Loosely bound
coenzymes are more like cosubstrates because they bind to the enzyme and are released
from it.
Gibbs free energy is a useful thermodynamic function for understanding enzymes.
Enzymes speed up the rate of chemical reactions, but the properties of the reaction depend
on energy differences between reactants and products. Gibbs free energy (G) is a
thermodynamic property that is a measure of useful energy, or the energy that can do work.
To understand how enzymes work, we need to consider two thermodynamic properties of
the reaction:
1. The free-energy difference (∆G) between the products and reactants.
This will determine whether the reaction will take place spontaneously.
2. The energy required to initiate the conversion of reactants into products.
This will determine the rate of the reaction.
Enzymes can only affect the second thermodynamic property.
The free-energy change of a reaction (∆G) tells us if the reaction can take spontaneously:
- A reaction can take place spontaneously only if ∆G is negative. Such reactions are said
to be exergonic.
- A system is at equilibrium and no net change can take place if ∆G is zero.
- A reaction cannot take place spontaneously if ∆G is positive. An input of free energy
is required to drive such a reaction. These reactions are termed endergonic.
- The ∆G of a reaction depends only on the free energy of the products (the final
state) minus the free energy of the reactants (the initial state).
Free energy of the products – free energy of the reactants = ∆G
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The ∆G provides no information about the rate of a reaction. The rate of a reaction
depends on the free energy of activation (∆G‡), which is largely unrelated to the ∆G
of the reaction.
To determine the free-energy change (∆G) for an enzyme-catalyzed reaction we need to
consider the nature of both the reactants and the products as well as their concentrations.
Consider the reaction:
𝐴+𝐵 ⇌𝐶+𝐷
The ∆G of this reaction is given by:
∆𝐺 = ∆𝐺° + 𝑅𝑇 𝑙𝑛
[𝐶][𝐷]
[𝐴][𝐵]
In which ∆G is the standard free-energy change, R is the gas constant, T is the absolute
temperature, and [A], [B], [C], and [D] are the molar concentrations of the reactants. ∆G is
the free-energy change for this reaction under standard conditions – that is, when each of
the reactants A, B, C, and D is present at a concentration of 1.0 M. A simple wat to
determine ∆G is to measure the concentrations of reactants and products when the
reaction has reached equilibrium. At equilibrium, there is no net change in reactants and
products; in essence, the reaction has stopped and ∆G = 0.
The ∆G can be calculated using the following equation:
∆𝐺° = −𝑅𝑇𝑙𝑛𝐾′𝑒𝑞
[𝐶][𝐷]
In which K’eq = [𝐴][𝐵]. So, let’s use an example. Calculate ∆G and ∆G for the isomerization of
dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (GAP). At equilibrium,
the ratio of GAP to DHAP is 0.0475 at 25C (298 K) and pH 7. Hence, K’eq = 0.0475. The
standard free-energy change:
∆𝐺° = −𝑅𝑇𝑙𝑛𝐾′𝑒𝑞
= −8.315(𝑥10−3 ) 𝑥 298 𝑥 ln(0.0475)
= +7.53 𝑘𝐽 𝑚𝑜𝑙 −1 (+1.80 𝑘𝑐𝑎𝑙 𝑚𝑜𝑙 −1
Under these conditions, the reaction is endergonic. DHAP will not spontaneously convert into
GAP. Now calculate ∆G for this reaction when the initial concentration of DHAP is 2 x 10-4 M
and the initial concentration of GAP is 3 x 10-6 M.
3 𝑥 10−6 𝑀
−1
∆𝐺 = 7.53 𝑘𝐽 𝑚𝑜𝑙 + 𝑅𝑇 𝑙𝑛
2 𝑥 10−4 𝑀
−1
= 7.53 𝑘𝐽 𝑚𝑜𝑙 − 10.42 𝑘𝐽 𝑚𝑜𝑙 −1
= −2.89 𝑘𝐽 𝑚𝑜𝑙 −1 (−0.69 𝑘𝑐𝑎𝑙 𝑚𝑜𝑙 −1 )
This negative value for the ∆G indicates that the isomerization of DHAP to GAP is exergonic
and can take place spontaneously when these species are present at the preceding
concentrations. Note that ∆G for this reaction is negative, although ∆G is positive. Whether
the ∆G for a reaction is larger, smaller, or the same as ∆G depends on the concentrations of
the reactants and products. The criterion of spontaneity for a reaction is ∆G, not ∆G. This is
important because reactions that are not spontaneous based on ∆G, can be made
spontaneous by adjusting the concentrations of reactions and products.
Enzymes alter only the reaction rate and not the
reaction equilibrium. The product formed is the same
whether or not the enzyme is present, but in the
example, the amount of product formed in seconds
when the enzyme is present is much faster than in the
absence of the enzyme.
Enzymes accelerate reactions by facilitating the formation of the transition state.
Enzymes serve as catalysts by decreasing the free energy of
activation of chemical reactions. Enzymes accelerate
reactions by providing a reaction pathway in which the
transition state (the highest-energy species) has a lower
free energy and hence is more rapidly formed than in the
uncatalyzed reaction.
As seen in figure 8.3: the curve of the uncatalyzed (∆G‡) is much
higher than the curve of the catalyzed (∆G‡).
The first step in catalysis is the formation of an enzyme-substrate complex. Substrates are
bound to enzymes at active-site clefts from which water is largely excluded when the
substrate is bound. The active site of an enzyme contains the amino acid residues that
directly participate in the making and breaking of bonds. These residues are called the
catalytic groups. Although enzymes differ widely in structure, specificity, and mode of
catalysis, several generalizations concerning their active sites can be stated.
The first generalization is that the active site is a threedimensional cleft, or crevice, formed by groups that come from
different parts of the amino acid sequence. Amino acid residues
far apart in the amino acid sequence may interact more
strongly than adjacent residues in the sequence, which may be
sterically constrained from interacting with one another.
This can be seen in figure 8.6 on the right.
The second generalization is that the active site takes up a
small part of the total volume of an enzyme. Although most of
the amino acid residues in an enzyme are not in contact with
the substrate, the cooperative motions of the entire enzyme
help to correctly position the catalytic residues at the active
site. All amino acids in the protein, not just those at the active site, are required to form a
functional enzyme.
The third generalization is that active sites are unique microenvironments. Water is usually
excluded unless it is a reactant. The nonpolar microenvironment of the cleft enhances the
binding of the substrate as well as catalysis. Nevertheless, the cleft may also contain polar
residues, some of which may acquire special properties essential for substrate binding or
catalysis.
The fourth generalization is that substrates are bound to enzymes by multiple weak
attractions. The noncovalent interactions in ES complexes are much weaker than covalent
bonds. These weak reversible contacts are mediated by electrostatic interactions, hydrogen
bonds, and van der Waals forces. Van der Waals forces become significant in binding only
when numerous substrate atoms simultaneously come close to many enzyme atoms
through the hydrophobic effect.
The fifth (and last) generalization is that the specificity of binding depends on the precisely
defines arrangement of atoms in an active site. Because the enzyme and the substrate
interact by means of short-range forces that require close contact, a substrate must have a
matching shape to fit into the site. Enzymes are flexible and the shape of the active site can
be markedly modified by the binding of a substrate, a process called induced fit. Moreover,
the substrate may bind to only certain conformations of the enzyme, which is called
conformation selection. Thus, the mechanism of catalysis is dynamic, involving structural
changes with multiple intermediates of both reactants and the enzyme.
The specificity of enzyme-substrate interactions arises
mainly from hydrogen bonding, which is directional, and
from the shape of the active site, which rejects molecules
that do not have a sufficiently complementary shape.
Enzymes facilitate formation of the transition state by a
dynamic process in which the substrate binds to specific
conformations of the enzyme, accompanied by
conformational changes at active sites that result in
catalysis.
The enzyme-substrate reaction has two parameters:
1. The interaction of the enzyme with the substrate (binding)  Km
2. The conformation of the bound substrate (the catalysis)  Vmax, kcat
Inhibitors of Michaelis-Menten enzymes (single-domain enzymes)
There are two types of inhibitors that bind to enzymes: inhibitors that bind reversibly to
enzymes and inhibitors that bind irreversibly to enzymes.
Reversible bound inhibitors can be subdivided in two categories:
1. Competitive inhibitors in which the substrate and inhibitor bind to the same
substrate binding site. This gives an increased apparent Km but the Vmax remains
unchanged. Examples are ibuprofen, statins, bortezomib, azoles, and transition-state
analogs (like oseltamivir).
2. Noncompetitive inhibitors in which the inhibitor binds at the allosteric sites. This
gives a decreased apparent Vmax but the Km remains unchanged. An example is
echinocandins.
Inhibitors that bind irreversibly to enzymes have a decreased V max because of the fewer
active enzymes. An example is acetylsalicylic acid also called aspirin. There is another class
of irreversibly binding inhibitors called suicide inhibitors, like penicillin.
Allosteric inhibitors (small metabolites) bind allosteric enzymes (which are multidomain
enzymes; a single subunit with regulatory and catalytic domains or multi-subunit enzymes,
such as dimers, trimers, and tetramers) reversibly at an allosteric site (a site other than the
active site) and change the enzyme conformation from a more-active, relaxed (R) state to a
less-active, tense (T) state. Allosteric enzymes do not obey the Michaelis-Menten kinetics.
Allosteric inhibition can refer to the following types of inhibition:
- Noncompetitive inhibition in which the inhibitor binds to an allosteric site, inhibiting
the enzyme activity. The substrate can still bind to the enzyme, but the enzyme
cannot catalyze the reaction.
- Competitive inhibition in which the inhibitor binds to an allosteric site and prevents
the substrate from binding to the enzyme.
This means that all noncompetitive inhibitors are allosteric inhibitors, but not all allosteric
inhibitors are noncompetitive inhibitors.
Summary figure of the types of inhibitors:
Inhibitors
Reversible
Competitive
Irreversible
Noncompetitive
Aspirin
‘Suicide’ inhibitors
Allosteric
Competitive
Noncompetitive
Suicide inhibitors (also called mechanism-based inhibitors) are modified substrates that
modify the active site of an enzyme. At first, they bind reversibly to the enzyme at the same
method that a substrate uses. Which causes the catalytic mechanism of the enzyme to
produce a chemically reactive intermediate that inactivates the enzyme through a covalent
modification. This is why suicide inhibitors are irreversible. An example of a suicide inhibitor
is MAO, which is an inhibitor for the enzyme monoamine oxidase (important in the synthesis
of neurotransmitters). Monoamine oxidase deaminizes dopamine and serotonin resulting in
a decreased concentration of these molecules in the brain. The prosthetic group, flavine, of
the monoamine oxidase oxidases MAO which then covalently binds to this prosthetic group,
resulting in the inactivation of the enzyme.
Cytochrome P450 (CYP)
A cytochrome is a protein that transfers electrons, using heme as its prosthetic group. The
iron ion (Fe) of a cytochrome alternates between a reduced (+2) and an oxidized (+3) state
during electron transport. Cytochrome P450 (CYP) is a family of cytochromes that absorbs
light maximally at 450 nm when complexed in vitro with exogenous carbon monoxide. CYP
proteins can be divided into two groups:
1. Those that metabolize xenobiotic (drugs, pollutants, agrochemicals).
2. Those that participate in key biosynthetic pathways (biosynthesis of sterols or
vitamin D).
The reaction mechanism of CYP enzymes is as followed:
RH + O2 + NADPH + H+  ROH + H2O + NADP+
Enzyme: CYP
Prosthetic group: heme
Co-enzyme: NADPH
Substrate: RH
Co-substrate: O2