Biochemistry

Extended Syllabus in English

Prepared by: Radovan Hynek and Olga Valentová

Lecture 1 INTRODUCTION - ORGANIZATION OF LIVING

SYSTEMS Biochemistry describes living organisms on molecular level by chemical approaches. Each organism and even the smallest cells consist of thousands inorganic and organic compounds, the letter varying from small molecules to large biopolymers. All biological processes like vision, digestion, motion, immunity, disease and even thinking are based on the action and interaction of these molecules. Thus the knowledge of chemical structure as well as their biological function is necessary. The respective roles of chemistry and biology in achieving the goals of biochemistry are readily apparent. Of the same importance in understanding the processes in living system is the energy flow (bioenergetics) as some molecular events in the cell require energy while others release energy. Biochemistry is devided into two levels:

1. Conformational: discovering the chemical composition of organisms, structure and three dimensional organization of the molecules, organization of supramolecular structure, relation between structure and function of the molecules

2. Informational: describes metabolism, bioenergetics and physiological processes on molecular level

Biochemistry is divided into many sub-domains according to the field of interest: Molecular genetics studying the transfer of genetic information on molecular level (compare to Mendelian genetic) Pathobiochemistry - biochemistry of diseases Clinical biochemistry – analysis of body fluids as a part of desease diagnostics Biotechnology – studies technological applications that use biological systems or their parts. Xenobiochemistry (farmacological biochemistry) - deals with the fate of drugs and toxins in organisms. Biophysical chemistry – uses the approaches of physical chemistry to solve biological problems

Bioorganic chemistry – study of biologically active organic compounds COMPONENTS AND ORGANIZATION OF LIVING ORGANISMS Living organisms differ from inanimate objects in many aspects: they are very complex with high degree of organization, able to extract energy from nutrients, regulate their functions, actively respond to the changes of their environment, grow and reproduce. From elements to biomolecules Bulk elements essential for life: C,H,O,N,P and S make up to 92%of the dry weight of living objects. Elements in trace quantities essential for life: Ca, Na, K, Mg, Fe, I and Cl Other trace elements: As, B, Mo, Cu, Zn etc. Table I. Molecular composition of different types of living organisms Component Rel. mol.

weight Amount in organism (g/100 g) Number molecular

species in bacterial cells

human plant bacteria

water 18 60 75 70 1 proteins 104-106 18 4 15 3 000

DNA >106 <1 <1 1 1 RNA 4.104-106 1,5 1 6 1 000

saccharides 102-106 0,5 16 2 250

lipids 750-1 500 16 1 2 50

Other organic compounds

100-500 1 1 2 500

Inorganic compounds

Approx. 60

3 2 1 15-20

Water is more than solvent in living systems All components mentioned in Table I will be discussed in more details in the following chapters except water. It is worthwhile to emphasise its unique role in biological systems here: - water represents about 60 to 70 % of the fresh weight of living organisms - all reactions occurring in living organisms are performed in water solution - water is a reactant or product of many biochemical reactions - photolytic cleavage of water molecule is one of the principal reaction on the Earth Processes running in living organisms are base on non-covalent interactions

Table II Overview of non-covalent interactions in living systems

Type of interaction Example Energy (kJ/mol)

Hydrogen bridges: water (ice)

-O-H...O= 17

Peptide bond =N-H...O=C 15 electrostat. interactions ion-ion

-COO-...+H3N- 20-30

Interaction of permanent dipols

| | Cδ+=Oδ-...Cδ+=Oδ-

| |

2

Londonovy dispersní interakce

two aliphatic carbons 0,11

Stacking interactions?? two aromatic rings 6

hydrofobic interaction two methyl groups 1,2

Molecular recognition is the result of an exact fit between the surfaces of two molecules. Complementary molecules form a complex that displays certain biological activity (enzyme-substrate, hormone-receptor, antibody-antigene etc). This phenomenon is the basis of all processes in living organisms. Organization of living organisms Small molecules – not very many (hundreds) metabolites or monomers from which the biopolymers are built (amino acids, monosacharides, purine and pyrimidine bases, fatty acids). Biopolymers – much more diverse than small molecules, thounds of different biopolymers in one cell. They are built from monomers: proteins from aminoacids, nucleic acids from monosaccharide ribose or deoxyribose, nucleic base and phosphate group, polysaccharides are composed from monosaccharides. Supramolecular assemblies – clusters composed from thousands of biopolymer molecules highly organized ( cytoskeleton, ribosomes, chromatine). The exception are the biological membranes composed from phospholipids. Cytoskeleton - three dimensional fibrous matrix extended throughout inside of the eukaryotic cell, gives the shape to the cell, enables the movement and guides the internal movement of organelles. The long fibers of the cytoskeleton are polymers of subunits. The fibres are primarily composed from proteins and are of three types: microtubules composed from protein tubulin and microfilamnets composed of actine and finally

The eukaryotic cytoskeleton. actin filaments are shown in red, microtubules in green, and the nuclei are in blue.

intermediate filaments varies from cell to cellThe primary types of fibers comprising the cytoskeleton are microfilaments, microtubules, and intermediate filaments. Biological membranes

The cell membrane consists primarily of a thin layer of amphipatic phospholipids which spontaneously arrange so that the hydrophobic "tail" regions are shielded from the surrounding polar fluid, causing the more hydrophilic "head" regions to associate with the cytosolic and extracellular faces of the resulting bilayer. This forms a continuous, spherical lipid bilayer.

The arrangement of hydrophilic heads and hydrophobic tails of the lipid bilayer prevent polar solutes (e.g. amino acids, nucleic acids, carbohydrates, proteins, and ions) from diffusing across the membrane, but generally allows for the passive diffusion of hydrophobic molecules. This affords the cell the ability to control the movement of these substances via transmembrane protein complexes such as pores and gates. Membranes serve diverse functions in eukaryotic and prokaryotic cells. One important role is to regulate the movement of materials into and out of cells. The phospholipid bilayer structure (fluid mosaic model) with specific membrane proteins accounts for the selective permeability of the membrane and passive and active transport mechanisms. Transmembrane proteins serve also as receptors of various signals .. Beside these integral mebrane proteins there are many proteins associated with the mebrane surface (peripheral membrane proteins) with various functions.

Viruses – consist of DNA or RNA molecule in the protein envelope. Viruses cannot exist independently and are usually not considered as a life-form , in this intention they can be considered as supramolecular assemblies as well.

After suplamolecular assemblies the higher level of organization is the fundamental unit of life, the cell. Generally living organisms are divided to two basic groups: prokaryotes and eukaryotes. Prokaryotes Simple, unicellular organisms, mainly bacteria and blue/green algae with neither the distinct nucleus or intracellular compartmentalization. They are the most abundant organisms on the earth. Eukaryotes This class of living organisms includes animals, plants and fungi, protozoan, yeast and some algae. The comlex eukaryotic cells are much larger than prokaryotic, with diameter ranging between 10 to 100 µm. They are surrounded by plasma membrane and except the animal cell also with cell wall. Inner space of the cell is compartmentalized to organelles, membrane enclosed packages of organized molecules that perform specialized functions. Eukaryotic cells are of very different shape and size. Plant cell

Lecture 2

AMINO ACIDS AND PEPTIDES

The amino acids are the building blocks for proteins - nearly all proteins studied are made from the twenty "standard" amino acids we will look at now. All of the standard amino acids are alpha amino acids (except for proline, an imino acid). That is they have an amino group alpha to the carboxyl group (they are 2-amino acids). Amino acids share the basic structure below and occur in two optical forms:

COO- COO-

⏐ ⏐

D: H⎯ C⎯NH3+ L: NH3+ ⎯ C⎯H

⏐ ⏐

R R

Only amino acids of L forms (coded in DNA) are building blocks of proteins

L α-Amino Acids Found in Proteins

Side chains (indicated in blue in the diagram). orange area are nonpolar and hydrophobic magenta box are acidic ("carboxy" group in the side chain). blue box are basic ("basic" group in the side chain).

Amino Acid Classifications

Each of the 20 α-amino acids found in proteins can be distinguished by the R-group substitution on the α-carbon atom. There are two broad classes of amino acids based upon whether the R-group is hydrophobic or hydrophilic.

The hydrophobic amino acids tend to repel the aqueous environment and, therefore, reside predominantly in the interior of proteins. This class of amino acids does not ionize nor participate in the formation of H-bonds. The hydrophilic amino acids tend to interact with the aqueous environment, are often involved in the formation of H-bonds and are predominantly found on the exterior surfaces proteins or in the reactive centers of enzymes.

Acid-Base Properties of the Amino Acids

The α-COOH and α-NH2 groups in amino acids are capable of ionizing (as are the acidic and basic R-groups of the amino acids). As a result of their ionizability the following ionic equilibrium reactions may be written:

R-COOH <——> R-COO– + H+

R-NH3+ <——> R-NH2 + H+

The equilibrium reactions, as written, demonstrate that amino acids contain at least two weakly acidic groups. However, the carboxyl group is a far stronger acid than the amino group. At physiological pH (around 7.4) the carboxyl group will be unprotonated and the amino group will be protonated. An amino acid with no ionizable R-group would be electrically neutral at this pH. This species is termed a zwitterion.

Like typical organic acids, the acidic strength of the carboxyl, amino and ionizable R-groups in amino acids can be defined by the association constant, Ka or more commonly the negative logrithm of Ka, the pKa. The net charge (the algebraic sum of all the charged groups present) of any amino acid, peptide or protein, will depend upon the pH of the surrounding aqueous environment. As the pH of a solution of an amino acid or protein changes so too does the net charge. This phenomenon can be observed during the titration of any amino acid or protein. When the net charge of an amino acid or protein is zero the pH will be equivalent to the isoelectric point: pI.

[ ][ ][ ]K

H A

HAA=

+ - [ ] [ ][ ]H KHAAA

+-

= [ ][ ]pH pKA

HAA= +

−

log

At neutral pH (pH =7) both the acid and amine groups will be ionized to give the so-called zwitterion form. Note that there is no pH at which the amino acid structure will have no ionized groups! Note the titration behavior of amino acids, and be able to draw the structure for an amino acid at each point in the curve.

Functional Significance of Amino Acid R-Groups

In solution it is the nature of the amino acid R-groups that dictate structure-function relationships of peptides and proteins. The hydrophobic amino acids will generally be encountered in the interior of proteins shielded from direct contact with water. Conversely, the hydrophilic amino acids are generally found on the exterior of proteins as well as in the active centers of enzymatically active proteins. Indeed, it is the very nature of certain amino acid R-groups that allow enzyme reactions to occur.

The imidazole ring of histidine allows it to act as either a proton donor or acceptor at physiological pH. Hence, it is frequently found in the reactive center of enzymes. Equally important is the ability of histidines in hemoglobin to buffer the H+ ions from carbonic acid ionization in red blood cells. It is this property of hemoglobin that allows it to exchange O2 and CO2 at the tissues or lungs, respectively.

The primary alcohol of serine and threonine as well as the thiol (–SH) of cysteine allow these amino acids to act as nucleophiles during enzymatic catalysis. Additionally, the thiol of cysteine is able to form a disulfide bond with other cysteines:

Cysteine-SH + HS-Cysteine <——> Cysteine-S-S-Cysteine

This simple disulfide is identified as cystine. The formation of disulfide bonds between cysteines present within proteins is important to the formation of active structural domains in a large number of proteins. Disulfide bonding between cysteines in different polypeptide chains of oligomeric proteins plays a crucial role in ordering the structure of complex proteins, e.g. the insulin receptor.

• Nonpolar side chains: these will tend to be found on interior of protein, except that glycine and alanine are so small that they can fit into interior or on surface. Compare these amino acids: note how these side chains build in size from gly (glycine), ala (alanine), val (valine), to leu (leucine), then have two which have about same size but different shapes: ile (isoleucine) and met (methionine - met has a nearly identical shape to the linear analogue of leucine, norleucine). Met of course also has possibility of liganding metal ions through sulfur. Next have phe (phenylalanine) and trp (tryptophan). These are aromatic, which enables stacking interactions with other aromatic groups as well as being very hydrophobic. Trp also has an amine group which needs to form a hydrogen bond. Thus trp is often found with the -NH at the surface but with the remainder in a hydrophobic cleft. If trp is interior it will generally hydrogen bond with another functional group. Finally pro (proline) is also hydrophobic, but its main characteristic of interest is its tendency to put a near right angle in the direction of a peptide chain. It thus generally disrupts particular structural elements of proteins. As such it is often near the surface, since it forces structural elements to turn at the surface (defining the surface).*

• Uncharged Polar side chains: These side chains will generally occur on the surfaces of proteins because of their polarity and hydrogen-bonding characteristics. If they occur on the interior they must generally H-bond with other interior functional groups. The definition of "uncharged" is based on a pH of 7. There are four side-chains, ser (serine), thr (threonine), asn (asparagine), and gln (glutamine), which are neutral under all conditions of pH. (Note that asn and gln are simply the amide forms of asp and glu. It is thus often difficult to determine whether a given residue was a asp or asn etc. in chemical analysis of peptides, since the treatment breaking peptide bonds also will generally break the amide bonds of asn and gln.) Tyr (tyrosine) and cys (cysteine) are uncharged at pH 7, but both ionize at higher pH's (respective pKa's = 9.5-10.9 & 8.3-8.6). Finally, his (histidine - imidazolium grp), has a pKa of 6.4-7.0 and is thus partially charged (positive) at pH 7, and will be charged at low pH's.

• Charged Polar side chains: These four side-chains will have very strong tendencies to be on the surface - it costs a great deal of energy to bury an ionic charge in a non-polar interior! It turns out that the sum of the acidic groups in a protein, asp (aspartate) + glu (glutamate), is usually equal to the sum of the sum of the basic groups, lys (lysine - amino grp) + arg (arginine - guanidinium grp). This is expected since we want a net neutral particle at its operating pH (usually around pH 7)

Peptides and Amino Acid Chemistry

• Peptide bond formation: Peptide bond is simply an amide bond between the alpha carboxyl and amino groups of amino acids. If we write the reacting groups in their unionized (acid and amine) forms, then we can see the reaction takes place with the loss of the elements of water, via an attack of the lone-pair electrons of the amine on the carbonyl carbon of the carboxyl group: •

• The peptide bond is formed with the elimination of water, giving a planar bond between the carboxyl carbon and the amino nitrogen. This is due to the partial double bond character on the amide/peptide bond as seen in the shorter bond length (0.133 nm vs. 0.146 nm). This bond is nearly always trans in proteins due to steric interactions of the amide hydrogen and oxygen, except for proline.

Examples of peptides

Glutathione (abbreviated GSH) is a tripeptide composed of glutamate, cysteine and glycine that has numerous important functions within cells. Glutathione serves as a reductant; is conjugated to drugs to make them more water soluble; is involved in amino acid transport across cell membranes (the γ-glutamyl cycle); is a substrate for the peptidoleukotrienes; serves as a cofactor for some enzymatic reactions and as an aid in the rearrangement of protein disulfide bonds.

Synthesis of Glutathione (GSH) Structure of GSSG

The role of GSH as a reductant is extremely important particularly in the highly oxidizing environment of the erythrocyte. The sulfhydryl of GSH can be used to reduce peroxides formed during oxygen transport. The resulting oxidized form of GSH consists of two molecules disulfide bonded together (abbreviated GSSG). The enzyme glutathione reductase utilizes NADPH as a cofactor to reduce GSSG back to two moles of GSH. Hence, the pentose phosphate pathway is an extremely important pathway of erythrocytes for the continuing production of the NADPH needed by glutathione reductase. In fact as much as 10% of glucose consumption, by erythrocytes, may be mediated by the pentose phosphate pathway.

Other biologically aktive peptides: insulin, oxytocin, vasopresin, endorfins etc.

Lecture 3

PROTEIN STRUCTURE AND FUNCTION Proteins are commonly large (MW > 6,000), globular molecules serving many functions

Levels of protein structure:

• Primary structure : the linear order or sequence of peptide bonded amino acid residues, beginning at the N-terminus. (Characteristic bond type: covalent.)

• Secondary structure: the steric relations of residues nearby in the primary structure which give rise to local regularities of conformation. These structures are maintained by hydrogen bonds between peptide bond carbonyl oxygens and amide hydrogens. The major secondary structural elements are the alpha helix and the beta strand. (Characteristic bond type: hydrogen.)

• Tertiary structure (3°): the steric relations of residues distant in the primary sequence; the overall folding pattern of a single covalently linked molecule. (Characteristic bond type: hydrophobic; others: hydrogen, ion-pair, van der Waals, disulfide.)

o Domains: independent folding regions within a protein. The group/pattern of secondary structures forming a Domain's tertiary structure is called a Fold. (Characteristic bond type: hydrophobic; others: hydrogen, ion-pair, van der Waals.)

• Quarternary structure: the association of two or more independent proteins via non-covalent forces to give a multimeric protein. The individual peptide units of this protein are referred to as subunits, and they may be identical or different from one another. (Characteristic bond type: hydrophobic; others: hydrogen, ion-pair, van der Waals.)

Levels of protein structure:

Protein Folding

Primary structure specifies tertiary (& therefore quaternary) structure. This is known from in vitro denaturation/renaturation studies of small proteins.

• Denaturation means to unfold to non-functional state, often achieve a "random coil" in solution.

o Denatruration is cooperative, that is takes place all at once as seen in denaturation curves

• Renaturation means to return to the properly folded, natural, and functional state.)

The classic study involved Ribonuclease: Reduce (break) -S-S- bonds, denature with urea to random coil. Now can renature by gently removing denaturant (urea) and oxidize -S-S- bonds. X-ray diffraction image is also the same! Note - no gremlins, no magic, done in "test tube."

Other small proteins, such as Myoglobin and proinsulin, fold up spontaneously in the same manner as Ribonuclease. However, insulin fails to fold correctly, since a peptide essential to folding has been cleaved off.

Accessory Folding Proteins. The ribonuclease renaturation-type experiment has not been repeated with large proteins, which seem to require the participation of "folding catalysts," to aid their folding: the Chaperones.

Chaperones

• Renaturation-type experiment has not been repeated with large proteins. • Many proteins are aided in folding process by "folding catalysts." These so-called

chaperones appear to stabilize unfolded conformations, allowing time to find correct folding pattern.

• Some chaperones require ATP energy to function. A number of different type: o The so-called Heat-shock proteins (Hsp70) are a family of chaperons

The Chaperonins (Hsp60 or GroEL and Hsp10 or GroES) are barrel-like proteins providing an internal folding environment. GroEL is large enough to accomodate a protein with >600 residues.

Thermodynamics notes to protein folding

Let's look at folding in another way: You might guess a protein would fold to lowest free energy conformation. Problem: is there time? ("Levinthal's Paradox", formulated by Cyrus Levinthal in 1968) Stryer calculation (very conservative): Assume 100 aa residue protein with 3 possible conformations/residue; then get 3100or 5 x 1047 possible conformations. If search at a rate of one structure/10-13sec then get (5 x 1047)(10-13)= 5 x 1034 sec or 1.6 x 1027 years to search (and thus to fold protein).

Obviously from these calculations not searching all possible conformations (or we have the process wrong!), so cannot say protein achieves the lowest global free energy, but rather a local free energy minima and by compaction, they may unfold and try other combinations until stable associations result.

Protein functions Proteins have numerous functions – e.g.: They serve as enzymatic catalysts, are used as transport molecules (hemoglobin transports oxygen) and storage molecules (iron is stored in the liver as a complex with the protein ferritin); they are used in movement (proteins are the major component of muscles); they are needed for mechanical support (skin and bone contain collagen-a fibrous protein); they mediate cell responses (rhodopsin is a protein in the eye which is used for vision); antibody proteins are needed for immune protection; control of growth and cell differentiation uses proteins (hormones) etc. Demonstration of protein functions on examples:

Myoglobin

Myoglobin is a 153 residue globular protein in the globin family. Eight alpha helices form its single domain (myoglobin fold) tertiary structure; about 80% alpha helix (high for globular proteins). Interior almost exclusively hydrophobic residues, with water excluded from interior. Surface has mix of hydrophobic and hydrophilic residues, with ionizable groups on surface.

Myoglobin functions to store and facilitate the diffusion of oxygen in muscle. Oxygen binds to a heme {Fe (II)-protoporphyrin IX} prosthetic group. Four of iron's six ligands are to heme nitrogens, with a fifth to a histidine nitrogen. The final ligand bond goes to oxygen. Breathing motions (see below) are necessary to allow the exchange of oxygen, since the heme is in a closed pocket.

Protein Dynamics

"Breathing" motions:

• Atomic fluctuations (10-15- 10-11 sec; 0.001 - 0.1 nanometer) Myoglobin example [overhead v&v 8.9, 8.10]

• Collective motions of covalently linked atoms, from aa R-groups to domains (10-12 - 10-3 sec; 0.001 - >0.5 nanometer)

• Triggered conformational changes: in response to ligand binding, covalent modification etc.

How do we know about the mobility of protein structures?

• X-ray diffraction studies of proteins with and without ligand bound • NMR (phe, his ring protons/carbons show up on edges of signal envelope) • H-exchange • Antibody binding: make antibodies to normally interior aa residues, over time protein

ppt forms as interior groups momentarily exposed.

Oxygen Binding

Myoglobin

Let's look at binding in terms of saturation, Y, where if Y = 1 every site of every Myoglobin is occupied by an oxygen molecule (thus if Y = 0.5, then 50% of the myoglobin are binding oxygen and 50% are "empty"). Mb/Hb binding curve :

Reviewing the curve in terms of saturation, Y, if Y = 1 then every site of every Myoglobin is occupied by an oxygen molecule (thus if Y = 0.5, then 50% of the myoglobin are binding oxygen and 50% are "empty").

Can describe binding as dissociation equilibrium,then:

MbO2 Mb + O2 ; &

for saturation. Substituting, , the equation of a hyperbola. If expressed as

pressures, then where P50 = pO2 @ 50% saturation. Note that the binding curve for Mb is indeed hyperbolic in shape.

Hemoglobin

Hemoglobin is an alpha-alpha-beta-beta oligomeric protein: its quaternary structure consists of a tetramer of myoglobin like subunits. The two types of chain are slightly shorter than myoglobin chains (alpha= 141 aa residues, beta= 146 aa residues). There are extensive contacts between an alpha and a beta subunit to give a dimer. The dimers have additional contacts to give the tetramer. Oxygen binding results in a change of conformation in Hb. The change of conformation affects the binding of oxygen {oxygen binding is reduced in the "blue" form due to steric hindrance between the oxygen and the heme}.

What about Hb oxygen binding? Obviously more complex. The sigmoid shape (s-shape) of the curve indicates cooperativity. That is, if one site binds, another is more likely to as well (it cooperates with the first site).

Lecture 4

ENZYMES: COMMON CHARACTERISTIC AND CLASSIFICATION

The enzymes are catalysts for biochemical reactions in living organisms. They direct and regulate the thousands of reactions providing for energy transformation, synthesis and metabolic degradation. As catalysts generally, the enzymes lower the activation energy of the reaction.

Compared to chemical catalyst they have some unique features: enzyme work under mild, physiological conditions with high efficiency and specificity and their activity can be regulated. Basic terms: substrate – reactant of the enzyme catalyzed reaction, product of the catalyzed reaction active site – the region that contains catalytic groups, binds the substrate, and then carries out the reaction catalytic site (catalytic groups) - groups of atoms (i.e atoms from the side chains of amino acids or cofactors or metal ions) in the enzyme molecule which are directly involved in the chemical change of the substrate. Tab Reaction rates of the decomposition of hydrogen peroxide in presence of different catalysts

Catalyst Reaction rate (mol.l-1.s-1)

Ea (kJ.mol-1)

non 10-8 71,1 HBr 10-4 50,2 Fe(OH)2-triethylen tetraamine

103 29,3

Catalase 107 8,4

Basically the enzymes are proteins , but can contain also a nonproteinaceous part - cofactors which are necessary for their function. Cofactors are either covalently bound to peptide chain - prosthetic group ( FAD, lipoamide, biotin) or noncovalently associated with protein – coenzyme (NAD+, CoA, ATP).

Enzyme nomenclature – classification of enzymes A systematic scheme for classification of enzymes was established in 1972 by the International Union of Biochemistry. Each enzyme is designated by EC number with four numbers indicating class (x), subclass (y), subsubclass (z) and ordinal number

EC x.y.z.i Enzymes are divided into six classes according to the type of catalyzed reaction:

Class Reaction catalyzed Typical reaction Enzyme example(s) with trivial name

EC 1 Oxidoreductases

Catalyze oxidation/reduction reactions; transfer of H and O atoms or electrons from one substance to another

AH + B → A + BH A- + B → A + B-

Dehydrogenase, oxidase

EC 2 Transferases

Transfer of a functional group from one substance to another. The group may be methyl-, acyl-, amino- or phosphate group

AB + C → A + BC Transaminase, kinase

EC 3 Hydrolases

Hydrolysis of substrate AB + H2O → AOH + BH

Lipase, amylase, peptidase

EC 4 Lyases

Non-hydrolytic addition or removal of groups from substrates. C-C, C-N, C-O or C-S bonds may be cleaved

RCOCOOH → RCOH + CO2 or [x-A-B-Y] → [A=B + X-Y]

Decarboxylase

EC 5 Isomerases

Intramolecule rearrangement, i.e. isomerization changes within a single molecule

AB → BA Isomerase, mutase

EC 6 Ligases

Join together two molecules by synthesis of new C-O, C-S, C-N or C-C bonds with simultaneous breakdown of ATP

X + Y+ ATP → XY + ADP + Pi

Synthetase

Subclasses represent the type of substrate and subsubcalss the special features of the enzyme (i.e. acceptor).

Cofactors

NAD+, FAD, CoA,

Lecture 5 ENZYME KINETICS

Study the rate of the conversion of substrate to products (S → P):

Michaelis-Menten model for one substrate reaction:

(k-2)E + S ES E + P

k-1

k+1 k+2(k-2)

E + S ES E + PE + S ES E + Pk-1

k+1 k+2

Initial reaction rate depends on the substrate and initial enzyme concentration

v k E Sk k

kS

oo = . [ ] . [ ]

+ ]-

2

1 2

1+ [

v k E SK S

oo

M = . [ ] . [ ]

]2

+ [

KM = Michaelis konstant – equal to substráte concentration at which the velocity of the reaction is half maximal, has a dimension of a concentration,

Michaelis-Menten equation

v V SK S

oM

= . [ ]]

lim

[+

Vlim = k2 . [Eo]

Maud Menten

Reversible substrate binding

Irreversible product formation

VE

k ko

catlim

[ ]= =2

kcat- turnover number (molecular activity ) of the enzyme - maximum number of molecules of the substráte that could be converted to produkt by one molekule of enzymr in one second

Substrate binding

"Lock and key" model

Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve. The "lock and key" model has proven inaccurate, and the induced fit model is the most currently accepted enzyme-substrate-coenzyme figure.

Induced fit model

In 1958, Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the presence of substrate induces conformational changes in the protein molecule especially in the active site region, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme. The region of active side not only recognizes the substrate molecule but also orients it in such a way that facilitates the catalytic reaction.

Stabilization of the transition state

Mechanism of catalysis

( example: acid catalysis by serine proteases)

Factors affecting enzyme acitivity

Enzyme inhibition

Inhibitors are specific agents that interfere with binding of a substrate to the active site or with conversion of the enzyme-substrate complex into products:

Immobilized enzymes

An enzyme fixed by physical or chemical means to a solid support–e.g., a bead or gel to confine a reaction of interest to a particular site. Immobilized enzyme preparation can be used repeatedly and continuously. They are used in biotechnologies.

Immobilization of enzymes

Binding to the matrix

Entrapment

Adsorption Covalent bond In the gel matrix encapsulation

Effect of immobilization on the enzyme:

1. Inactivation by reactants or products of immobiliztion reaction 2. Conditions of the immobilization reaction 3. binding forces or bonds fix the enzyme molekule in an inactiv or not fylly aktive

conformation 4. covalent bond formed with functional residuem of the aktive site 5. Orientation of the enzyme molekule limits the substrate Access to aktive site 6. influence of the funcional groups of the matrix (e.g. charged, hydrophobic).

Influence of the charge of matrix on the pH optimum of the immmobilized enzyme a. positively chrged matrix b/ native enzyme c. negatively charged matrix

Aplication of enzymes in technology

Food and non-food industry

Clinical biochemistry (diagnostics and determination of analytes)

Pharmaceuticals

Research (genetic engineering etc.)

Enzymes used:

Hydrolases (80%) / glycosidases and proteases, lipases

Isomerases (12% - glucose isomerase)

Oxidoreductases and others (5%)

Sources of enzymes for technology

Microbial enzymes, bacterial and fungi, extremophiles, recombinant enzymes

Animal and plant – less than 5% , limitations

Lecture 6

NUCLEIC ACID STRUCTURE

Nucleic acids - deoxyribonucleic acid (DNA), ribonucleic acid (RNA) are biopolymers built form monomer units nucleotides

Nucleotide (nucleoside mono, di, tri – phosphate)

Sugar – ribose (RNA), 2’- deoxyribose (DNA) Nitrogenous base :

– purine - adenine (A), guanine (G) – pyrimidine – cytosine (C), thymine (T), uracil (U) (replaces thymine in RNA) –

nucleoside

adenine guanine cytosine

thymine uracil

Character of nucleobases : Weak bases, planar, keto-enol tautomerism spectral characteristics - UV Nucleotide functions

- Building blocks of nucleic acids - intracellular energy transfer - donor of phosphate group - activation of intermediates for biosynthesis (UDP glucose, CDP choline) - structural components of cofactors,

vitamins (NAD(P)+. FAD, PAPS, CoA, cobalamine)

- Regulation (second messengers, neuromodulators - cAMP, cGMP)

- Therapeutics (antivirotics )

DNA structure Primary structure – nucleotides linked with 3’,5’-phosphodiester bond in the direction from5’ to 3’ end. Invariant backbone is formed by alternating phosphate and deoxyribose units. Heterocyclic bases linked to the covalent backbone by N-glycosidic bonds on the 1’ C atom of the monosaccharide. The primary structure is recorded by using the letter symbols - the following structure then as … TCAG…

Cyclic AMP 3’-azido-2’,3’ dideoxythymidin (AZT )

Secondary structure - double helix, antiparalel strands, base pairing according to complementarity of bases , fixed with hydrogen bonds

In a DNA molecule, the two strands are not parallel, but intertwined with each other. Each strand looks like a helix. The two strands form a "double helix" structure, which was first discovered by James D. Watson and Francis Crick in 1953. In this structure, also known as the B form, the helix makes a turn every 3.4 nm, and the distance between two neighboring base pairs is 0.34 nm. Hence, there are about 10 pairs per turn. The intertwined strands make two grooves of different widths, referred to as the major groove and the minor groove, which may facilitate binding with specific proteins.

Tertiary structure - supercoiled DNA

- circular chromosome in prokaryotes, - linear chromosome in eukaryots – nucleosome, histones

RNA structure Single stranded with ribose and uracil instead of thymine in the primary structure Partial secondary structure - double helices, bulges, loops, hairpin turns formed within the single strand rRNA - ribosomal (~80% of RNAs in the organisms), forms ribosomes together with proteins mRNA - messenger (~15% of RNAs), linear molecule, transcribed from DNA template, translated to proteins tRNA – transfer RNA (~5%), the smallest molecules of approx 75 to 95 nucleotides, contain modified purine and pyrimidine bases, displays the three-dimensional structure with double helices and loops. Carries an anticodon (three nucleotide bases) and a specific amino acid which is tranferred to the growing polypeptide chain in proteosynthesis . The identity of the amino acid is determined by the sequence of nucleotides in the anticodon.

Lecture 7

STORAGE AND UTILIZATION OF GENETIC INFORMATION

Genetic information flow: 1) From DNA to DNA during its transmission from generation to generation - replication 2) From DNA to Protein during its phenotypic expression in an organism. Transcription: DNA to RNA. Occassionally, genetic information flows from RNA to DNA (reverse transcription). Translation: RNA to protein (irreversible). Replication of DNA When cell is dividing, complete genetic information has to be copied an given to daughter cell. Each chromosome hast to be duplicated, process is called replication. This process is semiconservative : two original strands are separated and each acts as a template for the synthesis of a new strand on the principle of the complementarity of bases A=T and G≡C. Phases of the replication: Initiation, elongation, termination and processing Replication fork, replication bubble

Topoisomerase - removes supercoils ahead of the replication fork Helicase – separates two nucleic acid strands

DNA primase - synthesizes a short RNA segment (called a primer) complementary to a ssDNA template. DNA polymerases cannot initiate the synthesis of a DNA strand without an initial primer with free 3’OH group. DNA polymerase - key enzyme: dNTP + (DNA)n → (DNA)n+1 + PPi, can synthetize polynucleotide chin only in the direction 5‘ → 3‘ Leading strand - continuously synthetized strand Lagging strand - discontinuously synthetized short oligonucleotides Okazaki fragments DNA ligase - forming bonds between Okazakiho fragments to finish the synthesais of lagging strand DNA sequencing - determination of primary structure of DNA Polymerase chain reaction - a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Gene expression Genetic information encoded in the sequence of nucleotides in DNA has to be transcribed to the sequence of aminoacids in polypeptide chain. It undergoes in two steps, tracription fo the DNA sequence to the single stranded RNA (messenger, mRNA) and translation of the nucleotide sequence to the correct sequence of aminoacids to produce protein for which a given gene is responsible. Transcription (DNA → RNA) RNA polymerase - key enzyme, synthetizes all types of RNA, catalyzes all steps of the process: recognizes the initial site for synthesis, unwinds the double helix of DNA, catalyzes the addition of nucleotides, recognizes the termination site. Has no proof reading ability. Postrancriptional processing Prokaryotic mRNA- without processing Eukaryotic mRNA Splicing – excision of introns (intervening seguences)

Poly A tail on the 3’ end protection against rapid destruction by poly A binding proteins Capping of the pre-mRNA involves the addition of 7-methylguanosine (m7G) to the 5' end. The cap protects the 5' end of the primary RNA transcript from attack by ribonucleases that have specificity to the 3'-5' phosphodiester bonds. Translation (RNA → protein) Order of aminoacids in polypetide chain is given by order of nucleotides in DNA, resp. mRNA Genetic code 4 nucleotide bases has to code for 20 amino acids - 43 = 64 combinations – triplets

Proteosynthesis Occurs on ribosomes (small and big subunit ) Iniation factors tRNA activation reaction → aminoacyl tRNA Mechanism of polypeptide chain synthesis Termination Postranslation modification of proteins

Regulation of gene expression Constitutive genes, inducible genes, lac Operon Brief overview of gene technologies Recombinant technology

Lecture 8

BASIC CONCEPT OF METABOLISM AND ENERGY CONVERSION

Metabolic Pathways

Catabolism: degradation of molecules to provide energy

Anabolism: reactions using energy to synthesize new molecules for growth etc.

Metabolic pathway is a series of enzyme-catalyzed reactions, initiated by a flux-generating step and ending with either the loss of products to the environment, to a stored product (a metabolic 'sink') or in a reaction that precedes another flux-generating step (that is, the beginning of the next pathway)." Where a flux generating step is a non-equilibrium reaction that generates the flux going through the pathway and to whose rate all other reactions of the pathway conform. Note that by this definition some pathways may be inter-organ while others may take place in single compartment. We will explore this definition/concept as we look at metabolism.

Characteristics of pathways:

• Irreversible • First committed step (flux generating step) • Regulated • Localized in eukaryotes • Catabolic and anabolic pathways are generally distinguished by coenzymes and/or

compartmentalization.

The flux through a metabolic pathway is invariably controlled or regulated, most commonly by Feedback Inhibition, but also through Feed-forward activation. Regulation is one of the things that makes biochemistry "biological" and it will be a focus in our study.

The stages of catabolism

For convenience we can breakdown catabolism into four hierarchical levels:

• Stage I: Hydrolysis of polymers to monomeric units (fat fatty acids and glycerol, protein amino acids, etc.)

• Stage II: breakdown of products of Stage I to pyruvate, acetyl CoA, and/or intermediates of the Kreb's Cycle

• Stage III: Breakdown of Acetyl CoA by the Kreb's Cycle into carbon dioxide and water with the production of reducing equivalents (NADH etc.)

• Stage IV: Oxidation of the reducing equivalents by oxygen with the production of ATP via the Electron Transport System.

Reactions in Metabolism

Organic Reaction Mechanisms: We can categorize all common biological reactions into four groups:

• Group-transfer reactions (transfer of an electrophile [acyl {RCOX}, phosphoryl {OPO3X2-}, and glycosyl groups] between nucleophiles [alcohols, amines, thiols, etc.])

• Redox Reactions • Eliminations {eliminate H2O, NH3, ROH or RNH2}, Isomerizations, Rearrangements • C-C bond formation or breakage (condensation and cleavage reactions).

High Energy Compounds

Look at ATP:

Each of the phosphoric acid anhydride bonds is unstable. That is hydrolyzing either will release a lot of energy.

So why ATP? First, we want a compound with intermediate hydrolysis energy so it can pick up energy from some reactions and deliver to others. Second we want a kinetically stable molecule which is thermodynamically unstable. Thus acetic acid anhydride would not work: it is thermodynamically unstable to hydrolysis, but it is also kinetically unstable, with the carbonyl carbons wide open to water attack. Phosphoric acid anhydride is equally unstable, but is is sterically protected from water attack - in order to react quickly we need a catalyst - perfect.

ATP is sometimes referred to as a "Hi Energy" compound. High energy in this case does not refer to total energy in compound, rather just to energy of hydrolysis. Thus ATP is unstable to hydrolysis, or has a large negative G for hydrolysis. For biochemistry High Energy is defined in terms of ATP: if a compound's free energy for hydrolysis is equal to or greater than ATP's then it is "High Energy," if its free energy of hydrolysis is less than ATP's then it is not a "hi energy" compound. Note that ATP has two hi energy anhydride bonds.

Thermodynamics in Metabolism

Remember, the cool thing about thermo is that it is pathway independent - we can tell how much energy is available in an M&M by burning it in pure oxygen in a stainless steel container and tell you how far you can run on that M&M!

The tragedy of thermo, the other side of the coin, is that it tells nothing about the details - thermo gives us no idea about how the energy is used, or what steps are involved in its loss.

Remember also that for chemists and biologists the thermodynamic term generally of most interest is the Free Energy for a reaction, that is the energy available to do work.

• The free energy is defined as: G = Gproducts- Greactants = H - T S. • When the free energy is negative we say the reaction is spontaneous, which simply

means the reactants are favored in the reaction equation as written. • Note when a reaction is at equilibrium then the G is zero.

Since free energy depends on conditions, chemists tabulate free energies under Standard Conditions, ( G°): 298 K, 1 atm., with all concentrations at 1 M.

For biological systems we define a slightly different standard free energy with [H+]= 10-7 M (pH=7), G° '.

For non-standard conditions we can find the free energy of a reaction using:

G = G° ' + RT lnQ.

For the special case of equilibrium, the free energy is zero, so

G° ' = -RT lnK',

Reaction must be exergonic ∆G < 0

Endergonic reaction can not run ∆G > 0 and must be realised by different way

Solution:

Lecture 9

ELECTROTRANSPORT SYSTÉM, CITRIC ACID CYCLE

Electrotransport system

NADH and FADH2 carry protons (H+) and electrons (e-) to the electron transport chain located in the membrane. The energy from the transfer of electrons along the chain transports protons across the membrane and creates an electrochemical gradient. As the accumulating protons follow the electrochemical gradient back across the membrane through an ATP synthase complex, the movement of the protons provides energy for synthesizing ATP from ADP and phosphate. At the end of the electron transport system, two protons, two electrons, and half of an oxygen molecule combine to form water. Since oxygen is the final electron acceptor, the process is called aerobic respiration.

ATP Production during Aerobic Respiration by Oxidative Phosphorylation involving an Electron Transport System

Citric acid cycle

The citric acid cycle , also known as the tricarboxylic acid cycle (TCA cycle) and the Krebs cycle is a series of enzyme-catalysed chemical reactions of central importance in all living cells that use oxygen as part of cellular respiration.

TCA cycle

In eukaryotes, the citric acid cycle occurs in the matrix of the mitochondrion. In aerobic organisms, the citric acid cycle is part of a metabolic pathway involved in conversion of carbohydrates, fats and proteins into carbon dioxide and water to generace a form of usable energy. Other relevant reactions in the pathway include those in glycolysis and pyruvate

oxidation before the citric acid cycle, and oxidative phosphorylation after it. In addition, it provides precursors for many compounds including some amino acids and is therefore functional even in cells performing fermentation.

Lecture 10

METABOLISM OF CARBOHYDRATES

Wider metabolic context of glycolysis

The Energy Derived from Glucose Oxidation

Aerobic glycolysis of glucose to pyruvate, requires two equivalents of ATP to activate the process, with the subsequent production of four equivalents of ATP and two equivalents of NADH. Thus, conversion of one mole of glucose to two moles of pyruvate is accompanied by the net production of two moles each of ATP and NADH.

Glucose + 2 ADP + 2 NAD+ + 2 Pi ——> 2 Pyruvate + 2 ATP + 2 NADH + 2 H+

The NADH generated during glycolysis is used to fuel mitochondrial ATP synthesis via oxidative phosphorylation, producing either two or three equivalents of ATP.

Glycolysis

Regulation of Glycolysis

The reactions catalyzed by hexokinase, PFK-1 and PK all proceed with a relatively large free energy decrease. These non-equilibrium reactions of glycolysis would be ideal candidates for

regulation of the flux through glycolysis. Indeed, in vitro studies have shown all three enzymes to be allosterically controlled.

Regulation of hexokinase, however, is not the major control point in glycolysis. This is due to the fact that large amounts of G6P are derived from the breakdown of glycogen (the predominant mechanism of carbohydrate entry into glycolysis in skeletal muscle) and, therefore, the hexokinase reaction is not necessary. Regulation of PK is important for reversing glycolysis when ATP is high in order to activate gluconeogenesis. As such this enzyme catalyzed reaction is not a major control point in glycolysis. The rate limiting step in glycolysis is the reaction catalyzed by PFK-1.

Alternative pathways

Gluconeogenesis

Gluconeogenesis is the biosynthesis of new glucose, (i.e. not glucose from glycogen). The production of glucose from other metabolites is necessary for use as a fuel source. The primary carbon skeletons used for gluconeogenesis are derived from pyruvate, lactate, glycerol, and the amino acids alanine and glutamine. The liver is the major site of gluconeogenesis.

The relevant reactions of gluconeogenesis are depicted. The enzymes of the 3 bypass steps are indicated in green along with phosphoglycerate kinase. This latter enzyme is included since when functioning in the gluconeogenic direction the reaction consumes energy. Gluconeogenesis from 2 moles of pyruvate to 2 moles of 1,3-bisphosphoglycerate consumes 6 moles of ATP.

Lecture 11

PHOTOSYNTHESIS Photosynthesis is an important biochemical process in which plants, algae, protistans, and some bacteria convert the energy of sunlight to chemical energy and store it in the bonds of sugar, glucose.

Ultimately, nearly all living things depend on energy produced from photosynthesis for their nourishment, making it vital to life on Earth. It is also responsible for producing the oxygen that makes up a large portion of the Earth's atmosphere. Organisms that produce energy through photosynthesis are called photoautotrophs. Plants are the most visible representatives of photoautotrophs, but it should be emphasized that bacteria and algae as well contribute to the conversion of free energy into usable energy.

Light-dependent reactions The reaction of all light-dependent reactions in oxygenic photosynthesis is:

12H2O + 12NADP+ + 18ADP + 18Pi → 6O2 + 12NADPH + 18ATP

The light-dependent reactions, or light reactions, are the first stage of photosynthesis. In this process light energy is converted into chemical energy, in the form of the energy-carriers ATP and NADPH. In plants, the light-dependent reactions occur in the thylakoid membranes of the chloroplasts and use light energy to synthesize ATP and NADPH.

The photons are captured in the antenna complexes of photosystem I and II by chlorophyll and accessory pigments (see diagram at right). When a chlorophyll a molecule at a photosystem's reaction center absorbs energy, an electron is excited and transferred to an electron-acceptor molecule through a process called Photoinduced charge separation. These electrons are shuttled through an electron transport chain that initially functions to generate a chemiosmotic potential across the membrane, the so called Z-scheme shown in the diagram. An ATP synthase enzyme uses the chemiosmotic potential to make ATP during photophosphorylation while NADPH is a product of the terminal redox reaction in the Z-scheme.

Water photolysis

The NADPH is the main reducing agent in chloroplasts, providing a source of energetic electrons to other reactions. Its production leaves chlorophyll with a deficit of electrons (oxidized), which must be obtained from some other reducing agent. The excited electrons lost from chlorophyll in photosystem I are replaced from the electron transport chain by plastocyanin. However, since photosystem II includes the first steps of the Z-scheme, an external source of electrons is required to reduce its oxidized chlorophyll a molecules. This role is played by water during a reaction known as photolysis and results in water being split

to give electrons, oxygen and hydrogen ions. Photosystem II is the only known biological enzyme that carries out this oxidation of water. Initially, the hydrogen ions from photolysis contribute to the chemiosmotic potential but eventually they combine with the hydrogen carrier molecule NADP+ to form NADPH. Oxygen is a waste product of light-independent reactions, but the majority of organisms on Earth use oxygen for cellular respiration, including photosynthetic organisms.

Calvin cycle The Calvin cycle or Calvin-Benson-Bassham cycle is a series of biochemical reactions that take place in the stroma of chloroplasts in photosynthetic organisms. It was discovered by Melvin Calvin, James Bassham and Andrew Benson at the University of California, Berkeley.[1] It is one of the light-independent (dark) reactions, used for carbon fixation.

Steps of Calvin cycle

1. The enzyme RuBisCO catalyses the carboxylation of Ribulose-1,5-bisphosphate a 5-carbon compound, by carbon dioxide (a total of 6 carbons) in a two-step reaction. The initial product of the reaction is a six-carbon intermediate so unstable that it immediately splits in half, forming two molecules of glycerate -3-phosphate, a 3-carbon compound. (also: 3-phosphoglycerate, 3-phosphoglyceric acid, 3PGA)

2. The enzyme phosphoglycerate kinase catalyses the phosphorylation of 3PGA by ATP (which was produced in the light-dependent stage). 1,3 bisphosphoglycerate and ADP are the products. (However, note that two PGAs are produced for every CO2 that enters the cycle, so this step utilizes 2 ATP per CO2 fixed).

3. The enzyme G3P dehydrogenase catalyses the reduction of 1,3BPGA by NADPH (which is another product of the light-dependent stage). Glyceraldehyde 3-phosphate (also G3P, GP, TP, PGAL) is produced, and the NADPH itself was oxidized and becomes NADP+. Again, two NADPH are utilized per CO2 fixed.

The enzymes in the Calvin cycle are functionally equivalent to many enzymes used in other metabolic pathways such as gluconeogenesis and the pentose phosphate pathway, but they are to be found in the chloroplast stroma instead of the cell cytoplasm, separating the reactions. They are activated in the light (which is why the name "dark reaction" is misleading), and also by products of the light-dependent reaction. These regulatory functions prevent the Calvin cycle from being respired to carbon dioxide. Energy (in the form of ATP) would be wasted in carrying out these reactions that have no net productivity.

The sum of reactions in the Calvin cycle is the following:

3 CO2 + 6 NADPH + 5 H2O + 9 ATP → Glyceraldehyde 3-Phosphate(G3P) + 2 H+ + 6 NADP+ + 9 ADP + 8 Pi

It should be noted that hexose (six-carbon) sugars are not a product of the Calvin cycle. Although many texts list a product of photosynthesis as C6H12O6, this is mainly a convenience to counter the equation of respiration, where six-carbon sugars are oxidized in mitochondria. The carbohydrate products of the Calvin Cycle are three-carbon sugar phosphate molecules, or "triose phosphates," to be specific, glyceraldehyde-3-phosphate (G3P).

Comparison photosynthesis and respiration

Lecture 12

METABOLISM OF LIPIDS Lipids are a broad group of naturally occurring compounds either non-polar or amphipathic defined by low solubility in water. Triacyl glycerols (fats) Glycerol esterified with three fatty acids, serve as metabolic energy store of important fuel molecules fatty acids: Common saturated fatty acids in organisms Symbol Common

name formula Melting

point (°C)

12:0 lauric CH3(CH2)10COOH 44.2 14:0 myristic CH3(CH2)12COOH 52 16:0 palmitic CH3(CH2)14COOH 63.1 18:0 stearic CH3(CH2)16COOH 69.6 20:0 arachidonic CH3(CH2)18COOH 75.4 Common unsaturated fatty acids in organisms Symbol Common

name fomula Melting

point (°C) 16:1∆9 Palmitoleic CH3(CH2)5CH=CH-(CH2)7COOH -0.5 18:1 ∆9 oleic CH3(CH2)7CH=CH-(CH2)7COOH 13.4 18:2 ∆9,12 linoleic CH3(CH2)4(CH=CHCH2)2(CH2)6COOH -9 18:3 ∆9,12,15 linolenic CH3CH2(CH=CHCH2)3(CH2)6COOH -17 20:4 ∆5,8,11,14

arachidonic CH3(CH2)4(CH=CHCH2)4(CH2)2COOH -49

Waxes – esters of long-chain primary alcohols and long-chain fatty acid, this class of molecules confers water-repellant character to animal skin, to the leaves of certain plants, and to bird feathers. Polar lipids Phospholipids (glycerophospholipids) and sphingolipids constituents of biological membranes due to their amphiphilic character. Non polar region of the phospholipid molecule is formed by two acyl chains of fatty acids, polar part of the molecule by the phosphate (phosphatidic acid) esterified with various alcohols (choline, ethanolamine, serine, inositol) Sphingolipids are based on aminoalcohol sphingosine (instead of glycerol in glycerophospholipds) modified with one acyl chain and polar part is formed by hydrogen ( ceramide) phosphocholine (sphingomyeline) or saccharide residue (glucosylcerebroside or ganglioside).

Polar lipids in contact with water spontaneously form the lipid vesicles, micelles or bilayers, basic process of biological membrane formation. Isoprenoids – derived from isoprene units (2-methy-1,3-butadiene) Biologically active compounds and could be divided to two groups sterols and terpenes Steroids are characterized by fused ring system. Cholesterol is the best known steroid found in plasma lipoproteins and serving as membrane lipid or a precursor of steroid hormones, (testosterone, estradiol, cortisol) or vitamin D. Terpenes are a class of lipids formed from combinations of two or more molecules isoprene, linear and cyclic compounds – carotene, retinol, squalene, lycopene, gibberelic acid etc. Eicosanoids – derivatives of arachidonic acid of three subclasses: prostaglandins, thromboxanes, leukotrienes and lipoxins– they posses hormonlike signaling function (regulation of blood pressure and blood clotting, generarion of inflammation, fever and pain) Lipid metabolism Metabolism of triacylglycerol Diatary or synthetized in liver and stored in adipocytes or myocytes. Digestion of dietary triacylglycrols starts in small intestine, emulsified with bile salts, cleaved by pancreatic lipase to monoacylglycerol and free fatty acids, transported to intestinal mucosa, whwre they are resynthetized to triacylglycerols and transported in the form of lipoprotein particles (chylomicrons) by blood stream to the stores. Katabolism of triacylglycerols Cleaved by lipase to glycerol and free fatty acids Glycerol is further metabolised to the intermediates of glucose metabolism

Fatty acids are activated in cytoplasm by the reaction with CoA with concomitant cleavage of ATP (fatty acid –CoA ligase) Activated fatty acid is transferred to carnitine and acylcarnitin is transported by palmitoyl transferase I and II located in the inner mitochondrial membrane to the mitochondrial matrix where acyl carnitine reacylates the mitochondrial CoA. Acyl CoA then undergoes the degradation through the β-oxidation, a spiral pathway. In each turn the acyl chain undergoes a four step sequence of dehydrogenation, hydration, second dehydrogenation and thiolytic cleavage of C-C bond forming the acetyl CoA and acyl CoA of the fatty acid shorter by two carbon atoms Reduced cofactors NADH + H+ and FADH2 are reoxidized in electron transport chain coupled with oxidative phosphorylation (formation of ATP) in the inner mitochondrial membrane Acetyl CoA could be further metabolized in citric acid cycle. The overall energy yield (number of ATP molecules) of complete oxidation of the

fatty acid with n carbon atoms is then: 212

52

12 −⎟⎠⎞

⎜⎝⎛ −+ nn

β-oxidation of unsaturated fatty acids β-oxidation of fatty acids with odd number of carbon atoms Metabolism of keton bodies Biosynthesis of fatty acids Fatty acid synthesis from acetyl CoA occurs in cytoplasm. Acetyl CoA is carboxylated to malonyl CoA (-OOCCH2COSCoA) by acetyl CoA carboxylase. Fatty acid synthesis is catalyzed by fatty acid synthase complex (in animals) and begins with acetyl CoA and malonyl CoA which are bound to acyl carrier protein (ACP), part of the enzyme complex. These two molecules undergous the condensation reactin with concomitant decarboxylation followed by reduction, dehydrogenation and another reduction. These reactions can be considered as the reverse of β-oxidation. The reduction agent is NADPH + H+. Product of the first cycle is the four carbon butyryl CoA which enters the first reaction and condensate with another malonyl CoA. During each run the chain elongates by two carbons. In animal cells the process stops at 16 carbon palmitic acid. Elongation of palmitate as well as introduction of double bonds must be carried out by other enzyme systems. Biosynthesis of triacylglycerol – precursor is glycerol 3-phosphate which is acylated by acyl CoA to monoacylglycerol 3-phosphate and subsequently to diacylglycerol 3-phosphate, this is dephosphorylated to diacylglycerol and acylated to triacylglycerol. Glycerophospholipid biosynthesis – pathway for the synthesis vary with type of the organism. Starts with daicylglycerol or diacylglycerol 3-phosphate. CDP serves as carrier of polar head (CDP-choline, CDP-ethanolamine. Isoprenoids (steroids) – mevalonate pathway

Lecture 13

METABOLISM OF NITROUS COMPOUNDS

Nitrogen in the nature

• N2 abundant, fixation by bacteria = reduction to NH3 (NH4

+) • nitrification- soil (NH4

+) oxidized to nitrite and nitrate • plants and many bacteria convert nitrate and nitrite to (NH4

+) and amino acids, etc. • animals get amino acids from plants • denitrification- conversion of nitrates to N2

Metabolism of nitrogen

Urea cycle

The urea cycle takes place in the liver. The steps in the urea cycle occur in two places in the cells of the liver: the mitochondria and the cytosol within the cytoplasm.

The first three steps in the urea cycle (N-acetylglutamine Synthase, Carbamyl Phosphate Synthetase and Ornithine Transcarbamylase) occur in the mitochondria of the cell. The mitochondria contain the metabolic pathways involved in the metabolism of the carbohydrates, lipids and amino acids. The special pathways involving heme and urea synthesis are also located in the mitochondria. It generates most of the cell's ATP that provides the energy for the metabolic pathways to occur.

The last three steps of the urea cycle (Argininosuccinate Synthetase, Argininosuccinate Synthase Lyase, and Arginase) occur in the cytosol. Cytosol is the aqueous solution that makes up the cytoplasm. It contains thousands of enzymes involved in intermediate metabolism and ribosomes making proteins.

Central role of glutamate:

Four of the amino acids: glutamate, aspartate, alanine and glutamine are present in mammalian cells at much higher concentrations than the other 16. All four have major metabolic functions in addition to their roles in proteins, but glutamate occupies the prime position.

Glutamate and Glutamine

Glutamine Synthetase Ubiquitous- all organisms

1. glutamate + ATP --> γ-glutamyl phosphate + ADP 2. γ-glutamyl phosphate + NH4

+ --> glutamine + Pi

Glutamate Synthetase Plants and bacteria; animals use transamination to α-KG during AA catabolism

α-KG + glutamine + NADPH + H+ --> 2 glutamate + NADP+

sum; α-KG + NH4+ + NADPH + ATP --> l-glutamate + NADP+ + ADP + Pi

Glutamate Dehydrogenase mitochondria, 1 mM Km for NH4

+ makes reverse likely

α-KG + NH4+ + NADPH --> L-glutamate + NADP+ + H2O

Glutamate and aspartate function as excitatory neurotransmitters in the central nervous system, and glutamate is partly responsible for the flavour of food. (It is the mono sodium glutamate listed on processed food labels.) However, glutamate also occupies a special position in amino acid breakdown, and most of the nitrogen from dietary protein is ultimately excreted from the body via the glutamate pool.

Glutamate is special because it is chemically related to 2-oxoglutarate ( = a-ketoglutarate) which is a key intermediate in the Krebs cycle. Glutamate can be reversibly converted into 2-oxoglutarate by transaminases or by glutamate dehydrogenase. In addition, glutamate can be reversibly converted into glutamine, an important nitrogen donor, and the most common free amino acid in human blood plasma.

Transamination reactions:

Most common amino acids can be converted into the corresponding keto acid by transamination. This reaction swops the amino group from one amino acid to a different keto acid, thereby generating a new pairing of amino acid and keto acid. There is no overall loss or gain of nitrogen from the system - it is simply a question of "robbing Peter to pay Paul".

Transamination reactions are readily reversible, and the equilibrium constant is close to 1. One of the two substrate pairs is usually glutamate and its corresponding keto acid �-oxoglutarate. All transaminases require pyridoxal phosphate or pyridoxamine phosphate (both derived from vitamin b6) as an essential cofactor.

The reaction mechanism is shown on the next page. The substrates bind to the enzyme active centre one at a time, and the function of the pyridoxal phosphate is to act as a temporary store of amino groups until the next substrate comes along. In the process the pyridoxal phosphate is converted into pyridoxamine phosphate, and then back again. Enzymologists call this a "ping pong" mechanism, and it leads to a characteristic pattern of parallel lines in a double reciprocal plot of 1/V versus 1/S1 at various S2 concentrations.

The condensation between the alpha amino group and the aromatic aldehyde to form a "Schiff base" makes the alpha carbon atom chemically reactive, so the isomerisation of the Schiff base takes place very easily. In practice the pyridoxal form of the coenzyme condenses with the epsilon amino group of a lysine residue in the enzyme protein when no amino acid is bound, and the free aldehyde form of the coenzyme has only a transitory existence. Many of the enyzmes that metabolise amino acids require pyridoxal phosphate as a cofactor. Unexpectedly, this compound also serves in a completely different manner in the active centre of glycogen phosphorylase.

Glycogenic and ketogenic amino acids

The carbon skeletons from the majority of amino acids are degraded to Krebs cycle intermediates after removal of the amino group by transamination. This means that they can give rise to blood glucose via the gluconeogenic pathway (Dr Bonnett’s lectures). They are termed 'glycogenic' amino acids, because it was observed many years ago that they made diabetic glycosuria worse. In contrast to this 'ketogenic' amino acids exacerbated diabetic ketoacidosis, and these amino acids are degraded to compounds such as acetoacetate and acetyl-CoA. 'Mixed' amino acids are degraded to both Krebs cycle acids and to acetyl-CoA. The situation is summarised in the following table:

Phenylketonuria:

Phenylalanine is normally metabolised by conversion to tyrosine. The enzyme responsible for this conversion is phenylalanine hydroxylase, a mixed function oxygenase with a tetrahydrobiopterin cofactor:

Half of the oxygen molecule re-appears in the tyrosine -OH group and the other half is reduced to water. The "dihydrobiopterin" in the above reaction is an isomer of the folic acid compounds involved in one-carbon metabolism. It is recycled back to tetrahydrobiopterin using NADH:

"dihydrobiopterin" + NADH = tetrahydrobiopterin + NAD

Approximately one person in 45 American whites is a carrier for a defective phenylalanine hydroxylase gene, or (less frequently) the dihydrobiopterin cofactor. These mutations are particularly common in people of Celtic origin, but are less frequent in Eastern Europe. Unless treated they are seriously mentally defective and excrete large quantities of phenylpyruvate in the urine. This compound gives the disease its name, and is formed by the transamination of phenylalanine, a reaction that is normally insignificant. These patients are often tyrosine deficient, and have abnormally light skin pigmentation, because they have insufficient tyrosine to synthesise melanin in normal amounts.

When this condition was first recognised in the 1930's, a significant proportion of all long term patients in mental institutions proved to be undiagnosed phenylketonuriacs. Nowadays the condition can be readily diagnosed by a heel-prick blood test performed on all new-born babies.

Treatment consists of a very low phenylalanine diet, supplemented with extra tyrosine that the patients cannot synthesise from phenylalanine. This diet must be instituted at birth, but can be discontinued after a few years when brain maturation is completed. For obvious reasons it must be restarted during pregnancy. The artificial sweetner aspartame is a phenylalanine derivative, and this fact is declared on soft drinks cans to assist those following the special diet.

Lecture 14

REGULATION OF METABOLIC PATHWAYS Regulation of methabolic pathways will be shown directly on examples from former lectures.

Discussion will be focused on different levels of regulation

- allosteric effect - regulation by phosphorylation

Example of regulation after being scared by angry bear