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Biochemistry Essays 1-7
Dental medicine (Медицински университет в Пловдив)
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Biochemistry Essays
1. Proteins – functions and structure. Amino acids – types and classification. Biologically
important oligopeptides and polypeptides. Molecular forms of proteins (hetero-, iso-,
and alloproteins). Levels of protein structures. Protein properties, classification types
and denaturation.
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Structural – all cellular and extracellular structures contain proteins – membranes,
cytoplasm, ribosomes, chromatin, extracellular matrices.
Catalytic – all enzymes are proteins
Transport roles; gases – hemoglobin (O2, CO2), myoglobin (O2). Mineral cations –
transferrin (Fe3+), ceruloplasmin (Cu2+). Organic anions and lipids – retinol binding
protein (retinol, Vit A), transcortin called also corticosteroid-binding protein
(cortisol), albumin (bilirubin, free fatty acids (FFA))
Regulatory functions; hormones – most of the hormones of the pituitary gland
include growth hormone (GH), thyroid-stimulating hormone (TSH), folliclestimulating hormone (FSH), luteinizing hormone (LH). Growth factors – FGF, EGF,
VEGF, PDGF. Cytokines – Ils, IFNs.
Defense – antibodies, lectins, components of complement, blood-clotting factors
Contracting (motor) function – actin, myosin, troponin, tropomyosin.
Production of energy – proteins of electron transport chain.
Isoproteins (isoenzymes) – variants of a protein. Found in each individual of the
species and have the same functions. However they have different localization
(different tissues of subcellular structures), different structure, thermostability,
electrical charge, electrophoretic mobility etc.
Aloproteins (aloenzymes) – variants of a protein found in different individuals of the
same species – have the same function. Often they are results of variant alleles of
the gene (polymorpins, SNPs)
Heteroproteins (heteroenzymes) – protein variants with the same function, but
found in different species. There is usually great homology in the structure due to
evolutional connection between species.
There are 20 alpha amino acids that are involved in protein synthesis – proteogenic.
Essential amino acids – humans are incapable of synthesizing half of the 20 common
amino acids, and these essential amino acids must be provided in the diet: Valine,
Leucine, Isoleucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan,
Histidine
Conditionally essential: Arginine
Several of the proteogenic amino acids also serve functions distinct from the
formation of peptides and proteins, e.g. tyrosine in the formation of thyroid
hormones, cateholamines or glutamate acting as a neurotransmitter.
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Several other amino acids are found in the body in free or in combined states (i.e. not
associated with peptides or proteins). These non-proteins associated amino acids
perform specialized functions – ornithine, citrulline, homocysteine.
When an amino acid is dissolved in water, it exists in solution as a dipolar ion or
zwitterion. A zwitterion can act as either an acid (proton donor) or a base (proton
acceptor).
Amino acids are classified as polar and non-polar. Polar includes; uncharged Rgroups, charged R-groups (negatively or positively charged). Non-polar includes
aliphatic R-groups and aromatic R-groups.
Non-polar with aliphatic R-groups; alanine, valine, leucine, isoleucine, methionine,
proline and glycine.
Non-polar with aromatic R-groups; phenylalanine, tryptophan and tyrosine.
Polar with uncharged R-groups; cysteine, serine, threonine, asparagine and
glutamine.
Polar with negatively charged R-groups; aspartic acid (aspartate) and glutamic acid
(glutamate)
Polar with positively charged R-groups; lysine, histidine and arginine.
Uncommon amino acids are found in proteins – they are derived from common
amino acids; 4-hydroxyproline, gamma-carboxyglutamate, 5-hydroxylysine, 6-Nmethyllysine, desmosine (specific for elastin).
Peptides and proteins are polymers of alpha amino acids bound with a peptide bond
Oligopeptides – from 2 to 20 AA – glutathione (3 AA), antidiuretic hormone
(vasopressin – 9 AA), oxytocin (9 AA), the most active gastrin (gastrin-14, 14 AA), one
form of cholecystokinin (CCK-8, 8 AA), enkehalin (5 AA).
Polypeptides – from 20 to 100 AA – Insulin (51 AA), glucagon (29 AA), gastrin-34 (34
AA), CCK-58, CCK-33.
Proteins – over 100 AA
Peptides with biological functions:
Peptides with hormonal activity: insulin (51 AA), glucagon (29 AA), vasopressin (9
AA), oxytocin (9 AA), somatostatin (14 AA), corticotropin (39 AA)
Releasing factors from hypothalamus: thyrotropin releasing factor (thyroliberin),
somatotropin releasing factor (GH releasing factor)
Natural opiates: enkephalins, endorphins
Tissue hormones: gastrin, VIP (Vasoactive intestinal peptide), SP (substance P),
cholecystokinin
Toxic compounds: phalloidin, amanitin (8 AA)
Glutathione – natural cellular antioxidant, provides 2H in the reductive reactions,
plays a role in metabolism of xenobiotics and their detoxification
Proteins:
The lengths of polypeptide chains in proteins vary considerably
Some are small: human cytochrome c has 104 AA residues linked in a single chain;
bovine chymotrypsinogen has 245 residues
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Some are very large: titin (constituent of vertebrate muscle) has nearly 27000 AA
residues
Majority – middle number of AA – no more than 2000 AA residues
Some proteins consist of a single polypeptide chain, other –of two or more chains
bound covalently (disulfide bridge), but others, called multisubunit (oligomeric)
proteins, have two or more polypeptides associated noncovalently (proteins with
quaternary structure)
Levels of organization of proteins:
For levels: primary, secondary, tertiary and quaternary.
The primary structure refers to the number, type and sequencing of AA composing
the protein. It is maintained by peptide bonds (covalent bonds) – linking amino acid
residues in a polypeptide chain. The most important element of primary structure is
the sequence of amino acid residues.
Secondary structure refers to particularly stable arrangements of amino acid residues
giving rise to repeating structural patterns. It also refers to the local confirmation of
some part of a polypeptide – it is considered as common regular folding patterns of
the polypeptide backbone. A few types of stable and are seen, those being the alphahelix and beta conformations. In these structures the polypeptide backbone is tightly
packed via H-bonds between partially polarized carbonyl oxygen and amide nitrogen
of neighboring peptide bonds.
Tertiary structure describes all aspects of the three-dimensional folding of a
polypeptide. It includes longer-range aspects of amino acid sequence – AAs that are
far apart in the polypeptide sequence and are in different types of secondary
structure may interact to fold the structure of a protein.
When a protein has two or more polypeptide subunits (bound non-covalently), their
arrangement in space is referred to as quaternary structure
Fibrous and globular proteins:
Fibrous proteins have polypeptide chains arranged in long stands or sheets
Globular proteins have polypeptide chains folded into a spherical or globular shape
Fibrous proteins: usually consist of a single type of secondary structure. They are
involved in forming of structures that provide support, shape, and external
protection for vertebrates. They share properties that give strength and or flexibility
to the structures in which they occur. All fibrous proteins are insoluble in water due
to a high conc. of hydrophobic amino acid residues both in the interior of the protein
and on its surface. E.g. alpha-keratin, collagen, elastin and silk fibroin.
Globular proteins: often contain several types of secondary structure. Most enzymes
and regulatory proteins are globular. Some proteins contain two or more separate
polypeptide chains, or subunits, which may be identical or different. The
arrangement of these protein subunits in three-dimensional complexes constitutes
quaternary structure. The association of the subunits in the quaternary structure
occurs through weak non-covalent bonds. Proteins with quaternary structure insert
their function when the complex is assembled.
Interactions in a protein:
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Hydrogen bonding: polypeptides contain numerous proton donors and acceptors
both in their backbone and in the R-groups of the amino acids. The environment
(water) in which proteins are also found contains H-bond donors and acceptors of
the water molecule
H-bonding, therefore occurs not only within and between polypeptide chains but
with the surrounding aqueous medium
Hydrophobic forces: proteins are composed of amino acids that contain either
hydrophilic or hydrophobic R-groups
It is the nature of the interaction of the different R-groups with the aqueous
environment that plays the major role in shaping protein structure.
The hydrophobicity of certain amino acid R-groups tends to drive them away from
the exterior of proteins and into the interior.
Electrostatic forces; charge-charge, charge-dipole and dipole-dipole
Charge-charge interactions that favor protein folding are those between oppositely
charged r-groups.
Substantial for protein folding are the charge-dipole interactions. This refers to the
interaction of ionized r-groups of amino acids with the dipole of the water molecule
too
The slight dipole moment that exist in the polar r-groups of amino acid also
influences their interaction with water
The majority of the amino acids found on the exterior surfaces of globular proteins
contain charged or polar r-groups
Van der waals forces: there are both attractive and repulsive van der waals forces
that control protein folding
Although van der waals forces are extremely weak, it is the huge number of such
interactions that occur in large protein molecules that make them significant to the
folding of proteins.
Strong covalent bonds in proteins:
Disulfide bridges – appears between the -SH groups of two cysteine residues of one
or different polypeptide chains of a protein. It is important for the maintenance of
tertiary structure.
Protein denaturation and folding:
All proteins after being synthesized must fold during and following synthesis to take
up its native conformation
The loss of protein structure results in loss of function
A loss of three-dimensional structure sufficient to cause the loss of function is called
denaturation
The denatured state is not necessarily the state of complete unfolding of the protein
Most proteins are denatured by heat, which affects weak interaction in a protein (Hbonds)
The very heat-stable proteins of thermophilic bacteria may function at the
temperature of hot springs (100 degrees centigrade)
Proteins can be denatured by extremes of pH, by certain organic solvents (alcohol or
acetone), by certain solutes such as urea and guanidine hydrochloride, or by
detergents.
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Organic solvents, urea and detergents act primarily by disrupting the hydrophobic
interactions that make up the stable core of globular proteins; extremes of pH alter
the net charge on the protein, causing electrostatic changes.
2. Nucleotides – composition and structure. Free nucleotides of biologic significance.
Nucleic acids – composition and structure. DNA and RNA. Levels of organization of
DNA – nucleosomes and chromosomes. Types of RNA molecules – mRNA, tRNA,
rRNA, miRNA.
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Have a nitrogenous base, pentose sugar and a phosphate group.
The nitrogenous base is either a purine (adenine and guanine) or a pyrimidine
(thymine, cytosine and uracil)
Pentose sugar is either a deoxyribose or a ribose
Functions of nucleotides:
Building blocks of nucleic acids
Energy transfer- di- and triphosphates (ATP)
Metabolic role; uridine nucleotides (carbohydrates), cytidine nucleotides
(phospholipids), guanosine nucleotides (gluconeogenesis, protein synthesis)
Components of complex coenzymes (NAD+/NADH, NADP+/NADPH, FAD/FADH2,
Coenzyme A)
Regulators (ADP/ATP)
Allosteric effectors
Second messengers (cAMP, cGMP)
Primary structure of nucleic acids
The sequence of the nucleotides is linked together by 3’, 5’ – phosphodiester bonds
in polynucleotide chains
Linear chains in eukaryotes (except cyclic mitochondrial DNA)
2 different ends – 5’ and 3’
Direction 5’ -> 3’
Primary structure of DNA: 5’ end, Guanine, Thymine, Adenine, Cytosine, 3’ end.
Primary structure of RNA: 5’ end, Guanine, Uracil, Adenine, Cytosine, 3’ end.
DNA vs RNA
DNA: double stranded molecule with a long chain of nucleotides. RNA: a single
stranded molecule and has a shorter chain of nucleotides
DNA: A-T, G-C. RNA: A-U, G-C.
DNA: found in the nucleus. RNA: found in the nucleus and cytoplasm
DNA: sugar is deoxyribose, nitrogenous bases are ATGC. RNA: sugar is ribose,
nitrogenous bases are AUGC.
DNA: transmission of hereditary material. RNA: the main job or RNA is to transfer the
genetic code needed for the creation of proteins from the nucleus to the ribosome.
DNA:
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Is a double stranded helix
It consists of two sugar-phosphate backbones on the outside, held together by
hydrogen bonds between pairs of nitrogenous bases on the inside.
Complementary, A-T, G-C. Chargaff’s rule states A=T and G=C.
Antiparallel
Most DNA double helices are right-handed, only one type of DNA, called Z-DNA, is
left-handed.
In the double-stranded DNA molecules the genetic information resides in the
sequence of nucleotides on one strand, the template strand. This is the strand of
DNA that is copied during nucleic acid synthesis. The opposite stand is considered
the coding strand because it matches the RNA transcript that encodes the protein.
Negative net charge of DNA molecules
Formation of minor and major grooves – sites for regulation of gene expression
through interaction with proteins
Intrastrand hydrophobic and pi-interactions between the bases, ‘stacking
interactions’.
Forms of DNA; Alpha, twists right, 11 bases per turn, RNA and DNA. Beta, twists
right, 10 bases per turn, DNA only. Z-DNA, twists left, 12 bases per turn, DNA only.
DNA denaturation and hyperchromic effect (increase of absorbance of DNA at 260nm
during denaturation)
DNA packing; short region of DNA double helix (2nm), ‘beads on a string’ form of
chromatin (11nm), 30nm chromatin fiber of packed nucleosomes (solenoid), section
of chromosome in extended form (300nm), condensed section of chromosome
(700nm), entire mitotic chromosome (1400nm). Net result: each DNA molecules has
been packaged into a mitotic chromosome that is 10,000 fold shorter than its
extended length.
RNA
Types; mRNA, tRNA, rRNA, snRNA, miRNA, siRNA.
mRNA: start codon – AUG. Stop codons – UAA, UGA, UAG. Cap - 5’ UTR – (start)
coding sequence (stop) – 3’ UTR – Poly A tail. (UTR- untranslated regions)
tRNA:
rRNA:
mRNA, tRNA and rRNA in protein synthesis.
3. Enzymes – general concept of enzyme catalysis. Chemical nature of the enzyme
molecule. Cofactors, coenzymes and prosthetic groups. Active site of an enzyme.
Enzyme specificity – definition. Enzyme classification and nomenclature.
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Enzymes are biological catalysts responsible for performance of almost all chemical
reactions in the body and for homeostasis of living organisms
Because of their significant role, their assessment and pharmacological regulation
are key elements of diagnosis and therapy:
Enzymes are localized in virtually all tissues and body’s fluids:
Intracellular enzymes – catalyze metabolic processes
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Enzymes of plasma membrane – catalyze processes in the cells in response to the cell
signals, and/or the transport of the compounds through the membrane
Enzymes of circulation system (plasma, lymph) – blood clotting, fibrinolysis,
activation of biologically effective compounds (angiotensin II), metabolism of LPC
(lipoprotein complexes)
Due to the enzymes – the chemical reactions in the body can occur at physiological
conditions: temperature less than 40 degrees centigrade, approx. pH 7, atmosphere
pressure 1 atm.
Like all catalysts, enzymes change reaction rate of both the forward and reverse
reaction – hence they do not change the equilibrium nor the equilibrium constant, K.
They do not result in greater yield of products
Enzymes are highly efficient – small quantity of enzymes can catalyze conversion of a
great amount of compounds
Enzymes remain unchanged during the reactions
The particles of the compounds S (substrate) in a closed (isolated) system have
different energy – distribution of the molecules depending on the energy – according
to the Maxwell-Boltzmann distribution curve.
Free energy (G0), the standard biochemical free energy change – at physiological
condition. The difference between the energy levels of the ground state and the
transition state is the activation energy, change in Gibbs free energy. A higher
activation energy corresponds to a slower reaction.
Activation energies are barriers for chemical reactions
Reaction rates can be increases by raiding the temperature via increasing the number
of molecules with sufficient energy to overcome the energy barrier
Catalysts enhance reaction rates by lowering activation energies
This is possible because the enzyme catalyzed reaction proceeds through reaction
intermediates: E+S -> ES -> EP -> E+P
Each step of the reaction has lower activation energy, than non-catalyzed reaction
One of the stop has the lowest reaction rate – rate limiting step, which determines
the rate of the whole reaction
6 classifications of enzymes:
Oxidoreductases – act on many chemical groupings to add or remove hydrogen
atoms or electrons
Transferases – transfer functional groups between donor and acceptor molecules.
Kinases are specialized transferases that regulate metabolism by transferring
phosphate from ATP to other molecules.
Hydrolases – add water across a bond, hydrolyzing it
Lyases – add water, ammonia or carbon dioxide across double bonds, or remove
these elements to produce double bonds
Isomerases – carry out many kinds of isomerization: L to D isomerizations, mutase
reactions (shifts of chemical groups) and others.
Ligases – catalyze reactions in which two chemical groups are joined (or ligated) with
the use of energy from ATP.
The enzyme’s name is composed by the names of the substrate(s), the product(s)
and the enzyme’s functional class, and ending in -ase.
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Chemical nature of enzymes:
The macromolecule component of enzymes are proteins
Only small subsets are RNA molecules – ribozymes.
Enzymes composed of only one protein are known as simple enzymes
Complex enzymes are composed of protein plus a relatively small organic molecule:
complex enzymes are also known as holoenzymes – the protein component is known
as the apoenzyme, while the non-protein part is known as the cofactors (coenzyme
or prosthetic group)
Prosthetic group describes a complex in which the small organic molecule is bound to
the apoenzyme by covalent bonds
When the binding between the apoenzyme and non-protein components is noncovalent, the small organic molecule is called a coenzyme.
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Enzyme specificity:
Reaction specificity
Specificity to substrate type:
String specificity – arginase, glucokinase
Specificity to the group – monoesterase, pepsin, trypsin
Specificity to the bond – lipase
Specificity to the steric isomers – L-AAO, D-AAO
Specificity to the geometric isomers – fumarase
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Active site catalysis and structure:
The extreme substrate specificity and high catalytic efficiency of enzymes reflect the
existence of an active center
Substrates bind to the active site at a region complementary to a portion of the
substrate
Binding of the substrate to the active site – several weak bonds; sometimes
covalently bonded.
The active site – 3D structure of the enzyme molecule, cleft-like or a pocket
It is formed during folding of the tertiary or quaternary structure of the enzyme
molecule
In the active site there are many amino acid residues coming to diverse portions of
the polypeptide chain
In the complex enzymes – the cofactors are also involved in the active sites.
Amino acid residues in active sites; Catalytic – take part in the reaction, Contact –
take part in binding the substrate to AS, Assistant (additional) – assist the catalytic
and contact groups, Conformational – associated in folding of the 3D structure of
enzymes
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Lock and key hypothesis – the conformation of the substrate is complementary to the
conformation of the active site of the enzyme
Induced fit hypothesis – both in active site and substrate induced conformational
changes are induced during the catalytic process.
4 catalysis mechanisms:
Catalysis by bond strains (tension) – induced structural rearrangements
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Catalysis by proximity and orientation – enzyme-substrate interactions orient reactive
groups and bring them into proximity with one another
Catalysis involving proton donors (acids) and acceptors (bases): the ionizable
functional groups of aminoacyl side chains and (where present) of prosthetic groups
contribute to catalysis by acting as acids or bases.
Covalent catalysis – in catalysis covalent bonds are formed.
4. Enzyme kinetics – influence of the concentration of the enzyme or its substrate on
the rate of an enzyme catalyzed reaction. Principles for determination of the enzyme
activity. Enzyme units. Influence of the temperature and pH on the rate of an enzyme
catalyzed reaction. Irreversible and reversible inhibition. Competitive and noncompetitive inhibitors.
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Time-scale dependence of the enzymatic reactions:
The reaction rate of an enzyme reaction is calculated either by decrease of the
substrate (S) (-ds/dt), or by the increase level of the products(s) (P) (dp/dt) for a
period of time
The initial rate (initial velocity), designated V0 is that rate when the [S] is much
greater than the concentration of enzyme [E]. When only the beginning of the
reaction is monitored (often the first 60 seconds or less), changes in [S] can be
limited, and [S] can be regarded as constant.
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Enzyme concentration and initial rate:
The initial velocity (V0) is dependent on the concentration of the enzymes – there is
linear proportional correlation of the V0 to the concentration of the enzymes
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Measurement units for enzyme quantity and concentration:
International unit (U) – 1.0 unit (1U) of enzyme activity is defined as the amount of
enzyme causing transformation of 1.0 micromol of substrate per 1 minute at 25 oC
under optimal conditions of measurement
The term activity refers to the total units of enzyme in a solution
The specific activity is the number of enzyme units per milligram of total protein. The
specific activity is a measure of enzyme purity: it increases during purification of an
enzyme
Catal (cat) – as the amount of enzyme causing transformation of 1.0 mol of substrate
per 1 sec at 25oC
1 cat = 6.107 U, t.e. 1U = 16.67 ncat
Concentration of the enzymes – the international units per a volume (liter) plasma or
other biological fluid (U/I)
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Substrate concentration affects reaction rate:
The concentration of substrate [S] present will greatly influence the rate of product
formation, termed the velocity (v) of a reaction
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Studying the effects of [S] on the velocity of a reaction is complicated by the
reversibility of enzyme reactions, e.g. conversion of product back to substrate
To overcome this problem, the use of initial velocity measurements are used. At the
start of a reaction, [S] is in large excess of [P], thus the initial velocity of the reaction
will be dependent on substrate concentration
Substrate concentration affects the reaction rate – Michaelis-Menten Kinetics
Vmax – the initial velocity when the concentration of the substrate is enough to fully
saturate the active sites of all enzymes
Km – this concentration of the substrate when the initial velocity of the reaction is
half the Vmax (Vmax/2). The dimension of Km is the same as substrate (mmol/l etc.)
Dual nature of the Michaelis-Menten equation: When S is low, the equation for rate
is 1st order in S. When S is high, the equation for rate is 0 order in S. The MichaelisMenten equation describes a rectangular hyperbolic dependence of v on S.
V1 = Vmax[S]/Km+[S]
When [S] = Km: V = Vmax[S]/Km+[S] = Vmax[S]/[S]+[S] = Vmax/2
When [S] >> Km: V = Vmax[S]/Km+[S] = Vmax[S]/[S] = Vmax
When [S] << Km: V = Vmax[S]/Km+[S] = Vmax[S]/Km = (Vmax/Km)[S]
Double reciprocal rearrangement of the equation: to avoid dealing with curvilinear
plots of enzyme catalyzed reactions, biochemists Lineweaver and Burk introduced an
analysis of enzyme kinetics based on the double-reciprocal rearrangement and plot
of the Michaelis-Menten equation.
Meaning of Km:
If V0 is set equal to 1/2Vmax, then the relation Vmax/2 = Vmax[S]/Km+[S] can be
simplified to Km+[S] = 2[S] or Km = [S].
Hence, at one half of the maximal velocity, the substrate concentration at this
velocity will be equal to the Km
Km is presented as the ration of the constants of the reactions of dissociation of ES to
that of forming the ES. Thus, Km is similar to the dissociation constant for the ES
complex and can be used as a relative measure of the affinity of the enzyme to the
substrate
Uses of Km:
Experimentally, Km is a useful parameter for characterizing the number and/or types
of substrates that a particular enzyme will utilize.
It is also useful for comparing similar enzymes from different tissues of different
organisms (hexokinase vs glucokinase)
Also, it is the Km of the rate-limiting enzyme in many of the biochemical metabolic
pathways that determines the amount of product and overall regulation of a given
pathway
Clinically, Km comparisons are useful for evaluating the effects that mutations have
on protein function for some inherited genetic diseases.
Kinetic parameters are used to compare enzyme activities
Summary of the interpretation of Vmax and the Km:
Km – Michaelis constant – reflects the affinity of the active sites – inverse correlation
(the higher Km is, the lower the affinity to the S exist). Km usually ranges from 10 -2 to
10-5 mol/l.
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Vmax – reflects the stability of ES complex – inverse correlation (the lower Vmax is,
the higher the stability of ES complex exists).
The values of Vmax will vary widely for different enzymes and can be used as an
indicator of an enzymes catalytic efficiency. It does not find much clinical use.
Physical-chemical factors affecting the reaction rate
Effects of temperature:
Optimum temperature depends on the length of the temperature effect
Enzymes have different sensitivity to the temperature; simple enzymes are less
sensitive, they are more stable. Enzymes with (S-S) bonds are more stable
Optimum temperature ranges from 35oC – 45oC
Effects of pH:
Enzymes have an optimum pH (or pH range) at which their activity is maximal; at
higher or lower pH, activity decreases
Optimum pH depends on; presence and pKa of ionizable functional groups in the
active sites, and presence and pKa of ionizable functional groups in the substrate
molecules.
5. Regulation of the enzyme activity. Regulation by altering the absolute amount of the
enzyme. Regulation by altering the activity of the enzyme – proenzymes, reversible
covalent modification, allosteric regulation, etc.
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Irreversible inactivation of enzymes:
Usually cause an inactivation by covalent modification of enzyme structure
The kinetic effect of irreversible inhibitors is to decrease the concentration of active
enzyme, this decreasing the maximum possible concentration of ES complex – thus
they lead to decreased reaction rates
Irreversible inhibitors are usually considered to be poisons and are generally
unsuitable for therapeutic purposes
i.e. cyanide is a classic example of an irreversible enzyme inhibitor by covalently
binding mitochondrial cytochrome oxidase
inhibitors of cofactors, specific irreversible inhibitors, non-specific inhibitors
Reversible inhibitors:
Competitive inhibitor - binds specifically at the catalytic site, where it competes with
the substrate – kinetic effect: Vmax is unchanged; Km, as defined by [S] required for
½ maximal activity, is increased (indicates a direct interaction of the inhibitor in the
active site). Binds only to free enzymes, competes with substrate for binding in a
dynamic equilibrium, inhibition is reversible by substrate, the degree of inhibition
depends on the ratio inhibitor/substrate, many drugs are antimetabolites and are
competitive inhibitors.
Non-competitive inhibitor – binds E or ES complex other than the catalytic site –
kinetic effect: Km appears unaltered; Vmax is decreased proportionately to inhibitor
concentration (inhibitor affects rate of reaction by binding to site other than
substrate active-site). Substrate binding altered, by ESI complex cannot form
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products, inhibition cannot be reversed by substrate, the degree of inhibition
depends of the quantity of inhibitor.
Uncompetitive inhibitor – binds only to ES complexes at locations other than the
catalytic site – Kinetic effect: apparent Vmax decreased; Km, as defined by [S]
required for ½ maximal activity, is decreased. Es structure, make inhibitor-binding
site available, inhibition cannot be reversed by substrate.
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Activators of enzyme reactions:
Thiol compounds – they affect via their reductive activity (glutathione, cysteine).
Activators of proteolytic activity of enzymes (proteinases)
Cations: most of the enzymes require metal ions
Anions (less frequently) – Cl-, activator of salivary amilase
Coenzymes
Allosteric activators
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Multi enzyme systems composed by free individual enzymes, dissolved inn
cytoplasm, matric of mitochondria or nucleoplasm. They function independently and
bind in multifunctional complexes by the intermediate compounds: the product (P)
of one enzyme is substrate of the ext substrate. E.g. glycolysis, gluconeogenesis.
Multi-enzyme complexes: the enzymes are associated in stable complexes and carry
out their catalytic activity only when they are part of the complex. E.g. pyruvate
dehydrogenase complex.
Multi-enzyme systems, bound to cell organelles: in addition to the enzymes, there
are also lipids, and integral membrane proteins, assisting the structure of membrane
and the function of the enzymes. E.g. electron transport chain in mitochondria.
Regulation of multi-enzyme systems (metabolic pathways):
Most often the regulatory enzymes catalyze the first reaction or reaction of the
beginning of the pathway, or at the branching point of the chain.
Inhibitors: very often, they are final products of the pathway, retro-inhibition,
negative feedback regulation loop
Activators: quite often they are substrates of the pathway, positive feedback loop
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Regulation of enzymes:
Regulation of the amount of enzymes- the amount of the enzyme depends on the
rate of turnover:
Genetic regulation – induction and suppression of the synthesis – can be regulated
The rate of degradation – can be regulated
Regulation of catalytic activity of enzymes:
Allosteric regulation – fast but short lasting effects. It is dependent on the concrete
metabolic state of the cells. Often regulated by feedback mechanisms.
Allosteric enzymes are oligometric proteins (with quaternary structure), most often
with catalytic and regulatory subunits.
In addition to the active site they possess one or more allosteric centers – these
centers bind different modulators of enzyme activity (inhibitors or activators).
Allosteric modulators have no structural similarity to the substrate
Allosteric centers have different levels of affinity to the inhibitors/activators.
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Homotropic allosteric enzymes – the substrate may have a role of allosteric activator.
The stability of ES complex is not changed (Vmax=constant), but the affinity of the
active site to the substrate is increases
Heterotrophic allosteric enzymes – the modulators are compound different from the
substrates and they are specific to the allosteric site. Vmax is not changed, but only
the affinity of AS to the substrate is changed.
In some heterotrophic allosteric enzymes, changes of Vmax appears but the affinity
to the substrate is not changed.
Allosteric enzyme – kinetics: graphic association of the velocity and concentration of
the substrate – s-shaped curve (sigmoid binding curve) is diagnostic of cooperative
binding. Specific constants – Vmax and K0.5xK0.5 – similar as Km represents the affinity
of the allosteric enzyme to the substrate.
Covalent modification – fast effect, mediated by hormones, growth factors and other
extracellular signals.
Regulatory enzymes exist in two states (bound and unbound with a modifying
group). These forms have different activity:
Modifying groups: phosphate (phosphorylation), adenylate (AMP, adenylation),
uridylate (UMP, uridylation), ADP-ribose (ADP-ribosylation), acetate (acetylation),
methyle (methylation)
Phosphorylation: of Ser/Thr residues in AS or of Tyr in the active site of enzymes.
Phosphorylation requires ATP. It is catalyzed by protein kinases (Ser/Thr protein
kinases, or Tyr protein kinases). Reversible reaction – dephosphorylation by enzymes,
protein phosphatases.
Proteolytic activation by degradation – the enzymes of gastrointestinal tracks (GIT),
enzymes involved in blood clotting, fibrinolysis, ECM degradation
Some enzymes and other proteins are regulated by proteolytic cleavage of an
enzyme precursor
For some enzymes, an inactive precursor called a zymogen is cleaved to form the
active enzyme
Many proteolytic enzymes (proteases) of the stomach and pancreas are regulated in
this way
The enzymes involved in blood clotting and ECM degredation.
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Protein-protein binding – some enzymes are activated or inhibited by binding with
other binding proteins.
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Induction/suppression of the enzyme synthesis:
There are two types of enzyme, depending on the level of expression:
Constitutive – enzymes that are permanently expressed in the cells and the rate of
enzyme synthesis does not depend on inducers or suppressors. These enzymes
usually catalyze reaction, which are life-determining
Inducible – the enzyme synthesis is changed depending on the presence of inducers
of suppressors
Genetic regulation – induction, suppression:
Slow, but long lasting effect
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It is controlled by the metabolic state of the cell
Mainly by exogenic for the cell factors (hormones, growth factors, hypoxia, etc.)
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Control of half-life of enzymes:
The half-life of a molecule (protein, enzyme) is the period of active enzyme existence
– the time of bioavailability of the enzyme
The half-life depends on:
The ratio between proteases/antiproteases
The ratio between oxidants/antioxidants in the enzyme environmental (oxidative
state of the cell)
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6. Clinical significance of enzymes: functional and non-functional plasma enzymes. Role
in the diagnosis of myocardial infarction and hepatitis. Diagnosis significance of
isoenzymes (creatine phosphokinase and lactate dehydrogenase). Genetically
determined enzymopathies (gout, Lesch-Nyhan syndrome)
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Principals of enzyme diagnostics:
Determination of enzyme activity (conc.) in body fluids (plasma, serum,
cerebrospinal fluid, urine, gastric juice) and comparison to the referent (normal)
range for health people
The enzyme conc. in serum (or other biological fluids) represents the ration between
biosynthesis, release of enzymes during the physiological degradation (apoptosis) of
cell and degradation of the enzymes.
In the sera there are 2 main groups of enzymes: functional plasma enzymes, and
non-functional plasma enzymes (secreted enzymes and cellular enzymes).
Functional plasma enzymes:
Enzymes or proenzymes, which exist either permanently or temporarily in the
plasma of health individuals. They have physiological functions in the plasma
Examples – LPL (lipoprotein lipase), LCAT (lecitine-cholesterol acyl transferase),
ceruloplasmin (ferroxidase), pro-enzymes of most factors of blood clotting and
fibrinolysis
Most of these enzymes are synthesized in the liver and are secreted by active
transport
Decreased activity (conc.) in plasma – liver diseases (decreased synthesis)
Non-functional plasma enzymes:
They do not have physiological function in the plasma
In healthy individuals – the conc. is low in comparison to the tissues
Increased values above the referent values – indicators of tissue damage
If mitochondrial isoenzymes are present – indicator for stronger tissue damage
Two types; secreted and cellular.
Secreted:
Normally exist in exocrine secretes (the secretes of exocrine glands) – they are
synthesized in an organ and are secreted at the place of action. During this process a
small amount of the enzymes appear in the blood
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Cellular:
Are localized in cytoplasm, mitochondrion, or other structures of the cells – they
appear in the blood physiologically in small amounts (mainly cytoplasmic
isoenzymes)
Increased activity and especially the presence of mitochondrial isoenzymes –
indicators of cell damage
The degree of increase – proportional to the degree of damage
Changes of the specific isoenzymes – indicator for damage of the specific organ
The rate of elimination in the blood depends on the presence and quantity of
inhibitors
Influence on the activity – gender, age, muscle activity, pregnancy, drugs.
Diagnostic role of CK (Creatine kinase)
Located in – myocardium, skeletal muscle, brain, lung, thyroid gland. Most in the
cytoplasm, less in mitochondria
Three isoenzymes; CK1-BB (brain, prostate, lung), CK2-MB (myocardium) – to 6%
from CK of health, CK3-MM (skeletal muscle) – 94-100% of KK
Referent values: Males: 10-80 U/I. Females: 10-70 U/I.
Most often in diagnosis of: myocardial infarction (MB), progressive muscle dystrophy
(MM), polymyositis (MM), dermatomyositis (MM), surgical treatment (MM), brain
insult and brain tumors (BB), meningitis and encephalitis (BB), prostate and lung
cancer (BB)
Diagnostic role of LDH (Lactate dehydrogenase)
Cytoplasmic enzyme – located in many tissues: liver, pancreas, skeletal muscle, heart,
kidney, brain, erythrocytes, leukocytes, skin, glands.
5 isoenzymes – tetrameric protein of 2 types of subunits: H and M LDH1, LDH2
(myocardium, erythrocytes); LDH4, LDH5 (liver and skeletal muscle)
Referent values 240 U/I
Most often in diagnosis of: myocardial infarction (LDH1, less LDH3), myopathy
(LDH2), myocarditis (LDH1), hemolytic anemia (LDH1 AND LDH2), acute leukosis
(LDH3), metastatic tumors (LDH3), virus hepatitis, acute hepatocellular disorders
(LDH5)
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Serum enzymes in myocardial infarction:
LDH; Beginning of increase: 6-12h, Max of enzyme activity: 24-60h, Normalization: 715d
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Enzymopathy:
Each change in activity of an enzyme (especially those catalyzing the rate-limiting
reaction) leads to impairment of dynamic equilibrium in the cells and organism
This may result in conditions with pathological symptoms – metabolic disorders
On the base of the origin of the enzyme defect by Abderhalden (1958),
enzymopathies are inborn (inherited, primary, idiopathic) or acquired (secondary)
Inborn enzyme defects are due to abnormal molecule structure of the enzymes,
which on turn are determined by the changed structure of the genes encoding them
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Enzymopathies are diseases from the larger group of molecule diseases (diseases
caused by changes in the structure and further – biological functions of the proteins
or other biological structures
Mutations in the genes encoding the proteins result in changes in the structure of
the protein molecule, and then changed of biological functions of the enzymes
(catalytic activity)
In some cases there are no changes of the catalytic activity, but only changes of
stability and other physical and chemical characteristics
When there is significant changes of catalytic activity – this results in development of
pathological symptoms.
Results from mutations, do not change the activity of the enzyme:
The enzyme variant has decreased or lacks catalytic activity,
has changed affinity of the substrate,
has decreased stability,
has changed affinity to co-enzymes,
has changed sensitivity to allosteric modulators
Metabolic consequences:
In most cases there is significant decrease of the enzyme activity of some metabolic
step, which results in ‘metabolic block’.
In other cases there is ‘transport block’ – due to gene defect of membrane transport
proteins
In some cases there is increased enzyme activity, due to enzymopathy:
If the defect is in an anabolic pathway – metabolic overproduction
If the defect is in a catabolic pathway – increased metabolism.
When the metabolic block is in biosynthetic pathway of important biological product
– deficiency disease e.g. Type 0 glycogen storage disease (GSD0) – marked decrease
in liver glycogen content due to deficiency of hepatic glycogen synthase.
If the product P of the pathway is feedback inhibitor of an enzyme in the beginning
of the pathway (e.g. E2) – there is loss of this regulation
The regulatory reaction and the following reactions occur with increased rate – as a
result – increased conc. of the metabolites of these reactions
Example: Lesch-Nyhan syndrome – the deficiency of HGPRT leads to low levels of
GMP and IMP. The inhibitory effect of GMP and IMP drops on de novo synthesis of
proteins
If the product P is not feedback inhibitor of enzymes of the beginning pathway; there
is increase of the metabolite (D), which is substrate of that enzyme in deficiency. If
the reactions preceding the metabolic block reversible – increase of all metabolites
of those reactions.
If the metabolites preceding the metabolic block are with low molecular weight –
they are able to cross the plasma membrane into body fluids (blood, urine) – their
conc. increases.
If the metabolite preceding the metabolic pathway (including the substrate of the
pathway) are high molecular weight compounds, they are deposed in the cells –
storage diseases.
Often the metabolites are deposed in lysosomes – Lysosomal storage diseases.
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7. Water-soluble vitamins – chemical structure, metabolism and biochemical
significance. Avitaminoses and hypervitaminoses.
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Common characteristics:
Vitamins are a diverse group of organic molecules required in very small quantities in
the diet for health, growth and survival
They generally cannot be synthesized by mammalian cells -> must be supplied in the
diet (exception – ninacine from the amino acid tryptophan, but synthesis is not in
sufficient quantities to meet our needs)
The vitamins are of two distinct types: water soluble and fat soluble
The absorption and transport of fat soluble vitamins is together with lipids
The most prominent function of water soluble vitamins is as cofactors for enzymatic
reactions
Some of the fat soluble vitamins act as hormones
The absence of a vitamin from the diet or an inadequate intake results in
characteristic deficiency signs and finally, to death
Excessive intake of many vitamins, mainly fat soluble and also some water soluble,
may cause deleterious effects
Vitamin B1 (thiamine):
A pyrimidine and thiazole rings which are coupled by a methylene bridge
Cofactor form – thiamin pyrophosphate, TPP
Thiamin is rapidly converted to its active from TPP in the brain and liver by specific
enzymes, thiamin diphosphotransferase
Role of TPP in enzymatic reactions:
In the processes of oxidative decarboxylation of alpha-keto acids
In the processes of transfer of 2-carbon-atom residues in pentose phosphate
pathway (transketolase) of glucose degradation
The C2 of the thiazole ring is the active atom.
Dietary reference intakes – 0.5mg/1000cal, 1.1mg/d-F; 1.2mg/d-M
Sources – unrefined grains; yeast, yeast extract, pork, grains (whole grains), bean
food, seeds, nuts.
Absorption – proximal small intestine via 2 mechanisms: if <5mg/d – active transport.
If >5mg/d – passive transport. Alcohol inhibits the active transport
Deficiency: “beriberi” (chronic peripheral neuritis, polyneuritis). Dry form –
progressive peripheral neuropathy (neuritis), muscle pain, acute muscle weakness,
contractile pain, irritability. Wet form – edemas, anorexia, weight loss, apathy, cardiomyopathy, lung bleeding, heart insufficiency. Syndromes of Wernicke-Korsakov, often
in alcoholics, acute symptoms of encephalopathy – common features: whole
disorientation
Vitamin B2 (riboflavin):
Isoalloxazine ring and ribitol
Coenzyme forms – flavin mononucleotide (FMN) and flavin adenine dinucleotide
(FAD)
The enzymes that require FMV or FAD as cofactors are termed flavoproteins
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The enzymes are involved in a wide range of redox reactions: without participation of
oxygen (aerobic dehydrogenases) succinate dehydrogenase, mitochondrial glucose3P-dehydrogenase, Acyl-CoA dehydrogenase. With oxygen – oxidases, amino acid
oxidase (AAO), monoamine oxidase (MAO), xanthine oxidase.
During the course of the enzymatic reactions involving the flavoproteins the reduced
forms of FMV and FAD are formed, FMNH2 and FADH2, respectively.
Dietary reference intakes: 1.5-2mg/day
Sources – yeast extract, liver and kidney, eggs, dairy products, heart, fish
Absorption: it is taken with the food, in the stomach as FAD and FMV is released in
the intestines, absorption via active transport, it is stored in the liver.
Deficiency: riboflavinosis, very rare. Due to insufficient digestion, lack of appetite. In
alcoholics, due to impaired transport.
Angular stomatitis, glossitis, peripheral neuritis, secondary pellagra.
Vitamin PP (B3) – niacin (nicotinic acid and nicotinamide)
Pyridine ring, two forms.
Niacin is not a true vitamin since it can be derived from the amino acid tryptophan
Cofactor forms: nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine
dinucleotide phosphate (NADP+)
Function: cofactors for numerous dehydrogenases (DH), catalyze the oxidation
(dehydrogenation) without participation of oxygen (anaerobic dehydrogenases:
lactate DH, malate DH, glutamate DH, hydroxibutirate DH). NAD+ provides ADPribose in DNA reparation by ADP-ribosylation by poly(ADP-ribose)polymerase.
Dietary reference intakes: 7mg/day in children. 20mg/day in adults
Sources: yeasts, whole rice, whole grains, liver, kidney, chicken, beef, fish
Deficiency: pellagra (diarrhea, dermatitis, dementia – neurological symptoms).
Dermatitis in areas exposed to sunlight (UV damage of DNA) – decreased reparation.
Therapy of pellagra by replacement of maize with grains and diet rich of proteins
(due to endogenous synthesis of NAD+ from tryptophan)
Deficiency of vitamins B2 and B6 – secondary pellagra, if the meet of niacin is
supplied by endogenous synthesis from Try.
Niacin has therapeutic application – in high doses (1-2g/day) – results in decreased
concentration of plasma [Chol], [LDL], [VLDL], [TAG]
Vitamin B6 (pyridoxine, pyridoxal, pyridoxamine)
Cofactor forms: pyridoxal 5’-phosphate and pyridoxamine 5’-phosphate
Approximately 80% of the body’s total vitamin B6 is present as pyridoxal phosphate
in muscle, mostly associated with glycogen phosphorylase
Function: coenzyme for many coenzymes involved in amino acid metabolism,
especially in; transamination (AsAT, AIAT), decarboxylation (glutamate decarboxylase,
ornitin, decarboxylase, DOPA decarboxylase), non-oxidative deamination of AAs: Ser,
Thr, Cys. Synthesis of heme – 5-Ale synthetase. Catabolism of tryptophan – Trp
pyrolase, kynurinenase. In the structure of glycogen phosphorylase.
Dietary reference intakes: 1.5-2.5mg/day
Sources: chicken, fish, pork, eggs, fortified cereals
Deficiency: seborrheic dermatitis, microcytic anemia, epileptic form
Convulsions, depression, confusion
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Toxicity: if 10-150mg/day- neuritis.
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Vitamin H
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