Biological Chemistry Lehningers 6Th Ed. V2

Front 1

Protein structure
- primary: covalent backbone description of a polymer (macromolecule); inctlude sequence of monomeric subunits and any intrachain covalent bonds.

- secondary: local spatial arangement of the mainchain adjacent atoms in a segment of polypeptide chain (accounts also for polynucleotide structure).

- tertiary: Three dimensionel conformation of a polymer in its native folded state.

- quarternary: Three dimensionel structure of a multisubunit protein; particularly the manner in which the subunits fits together.

Back 1

- Primary: Description of all covalent bonds (mainly peptide and disulfide) linking amino acid residues in a polypeptide chain is its primary structure. Most important element is the sequence of amino acid residues.

-Secondary: refers to particularly stable arrangements of amino acid residues giving rise to reoccurring structural patterns.

Tertiary: Describes all aspects of the three-dimensional folding of a polypeptide.
- Includes longer-range aspects of amino acid sequence.
- The location of bends (including beta turns) in the polypeptide chain and the direction and angle of these bends are determined by the number and location of specific bend-producing residues: Pro, Thr, Ser and Gly.

Quarternary: The arrangement of the polypeptide subunits in a protein with to or more polypeptide subunits; these may be identical or different.

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Specific Activity

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- The number of enzyme units per milligram of total protein.

- A measurement of enzyme purity; it increases during purification of an enzyme, and becomes maximal and constan when the enzyme is pure.

- SPECIFIC ACTIVITY INCREASES EVEN AS TOTAL ACTIVITY FALLS.

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Protein homology

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- Similarities in sequence and function indicate that the proteins in question are members of a family of proteins that share common ancestor; the members of protein families are called HOMOLOGOUS PROTEINS or HOMOLOGS.

HOMOLOGS: Gene or protein that possess a clear sequence and functional relationship to each other.

HOMOLOGOUS PROTEIN: Proteins having similar sequence, occurring in ALL cells; Occurs during meiosis and mitosis in eucaryotes.

Front 4

Titration curves - Isoelctric point

Back 4

Titration curve: a plot of pH versus the equivalents of the corresponding bace-pair added during titration of an acid.

Isoelectric pH/point: The pH at which a solute has no net electric charge, and thus does not move in an electrical field.

pKa= a measure of the tendency of a group to give up a proton.

pI = the isoelectric point; the amino acid is present largely as the zwitterion. The net electric charge is 0.

pI = ½*(pK1 + pK2)

Front 5

Buffers - Buffering regions

Back 5

- Buffers are aqueous systems that tend to resist changes in pH when small amounds of acid (H+) or base (OH-) are added.

- Buffering region: extending from 1 pH unit on either side of the pKa.

Front 6

Colligative properties.

Back 6

Colligative = tied together.
- Properties of a solution that depend on the number of solute particles per unit volume; fx freezingpoint depression.

- Solutions alter certain physical properties of water: vapor pressure, boiling point, melting point (freezing point) and osmotic pressure.

- The concentration of water is lower in solutions than in pure water.

Front 7

Henderson-Hasselbach equation

Back 7

- This equation fits the titration curve of all WEAK ACIDS.

With this you can:
1) calculate pKa, given pH and the molar ratio of proton donor and acceptor.

2) calculate the pH, given pKa and the molar ratio of proton donor and acceptor.

3) calculate the molar ratio of proton donor and acceptor, given the pH and pKa.

Front 8

Acid Dissociation Constant.

Back 8

- Equilibrium constants for ionization reactions, are called ionization constants, or acid dissociation constants; Ka.

STRONG ACID = HIGH Ka
WEAK ACID = LOW Ka

Front 9

Ionic product

Back 9

Kw = ion product of water.
- The product of the concentrations of H+ and OH- in pure water.

Front 10

Charge of amino acid side chains

Back 10

- In general the charge of Gly, Pro, Leu, Met, Tyr, Ser, Cys, Ala, Val, Ile, Phe, Trp, Thr, Asn, Gln, and His, are the same in three different pH environments:
pH 2 = +1
pH 7 = 0
pH 10 = -2

In general Asp, Lys, Arg and Glu has the same charge in three different pH environments:
pH 2 = 0
pH 7 = -1
pH 10 = -3

Front 11

Spectral properties of Amino Acids - Chirality

Back 11

- The alfa carbon is bonded to four different groups, a carboxyl-, amino-, R-group and a hydrogen atom; it is a CHIRAL CENTER.

- L-Amino acids are those with the α-amino group on the left side of the chiral atom, and the D-Amino acids have the α-amino group on the right side of the chiral atom.

- The amino acid residues in protein molecules are EXCLUSIVELY L-STEREOISOMERS.

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Zwitterion

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- When an amino acid, lacking an ionizable R-group, is dissolver in water at neutral pH, it exists in solution as the DIPOLAR ION, or zwitterion.

- A zwitterion can act as either an acid or base.

- Substances having dual nature are amphoteric and are often called ampholytes.

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Peptide Bonds

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- Are formed by a DEHYDRATION reaction (in the amino backbone) between the C-terminal of the first peptide, and the N-terminal of the second peptide; called a SWUBSTITUTED AMIDE LINKAGE.

- ALL PEPTIDE BONDS ARE IN THE PLANAR TRANS-CONFIGURATION.

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Prostetic Groups - Conjugated Proteins

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- Some proteins contain PERMANENTLY ASSOCIATED CHEMICAL COMPONENTS in addition to amino acids, these are called CONJUGATED PROTEINS.

- The NON-AMINO ACID PART of a conjugated protein is called its PROSTHETIC GROUP.

- Conjugated proteins are classified on the basis of the chemical nature of their prosthetic group; fx lipoproteins - lipids.
glycoproteins - sugar groups.

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Amino Acid Sequencing

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- Offer insights into its three dimensionel structure and its function, cellular location and evolution; furthermore at offers insights when comparing two different species (as B. subtilis and E. coli) by inserting gaps in their protein sequence for a better alignment.

- Individual species are assigned to families based on the degree of similarity in amino acid sequence; share at least some structural and functional characteristics.

Front 16

Peptide Fragment ordering

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- Some proteases only cleaves the peptide bond adjacent to particular amino acid residues, and thus fragment a polypeptide chain in a predictable and reproducible way; A few chemical reagents also cleave the peptide bond adjacent to specific residues.

Front 17

Classical Sequencing

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1. A large protein would be cleaved into fragments twice, using a different protease or cleavage reagent each time so that fragment endpoints would be different.

2. Both sets of fragments would be purified and sequenced.

3. The order in which the fragments appeared in the original protein could then be determined by examining the overlaps in sequence between the two sets of fragments.

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The Edman degradation method

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The sequencing of some amino acids from the amino terminus (using Edman Chemistry) is often sufficient to confirm the identity of a known protein that has just been purified, or to identity an unknown protein purified on the basis of an unusual activity.

- In the Edman degradation system the amino-terminal residue is labeled, cleaved and identified in each successive cycle.

1. The polypeptide is treated with a reagent that labels the amino-terminal residue, then that residue is removed and identified.
2. In the next cycle, the identity of the new amino-terminal residue is similarly determined, and the procedure is repeated until the entire sequence is determined.

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Mass Spectromotry in sequencing

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1. Molecules to be analyzed, are reffered to as analytes, are first ionized in a vacuum.

2. When the newly charged molecules are introduced into an electric and/or magnetic field, their paths through the field are a function of their mass-to-charge ratio, m/z.

3. This measured property of the ionized species can be used to deduce the mass (m) of the analyte with very high precision.

- The m/z measurements are made on molecules in the gas phase, and the heating or other treatment needed to transfer a macromolecule to the gas phase usually caused its rapid decomposition.

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Peptide synthesis

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Front 21

Covalent structure - covalent bond

Back 21

Covalent bonds: are formed by sharing one or more pairs of electrons.

Front 22

Intermolecular interactions

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- Weak non-covalent interactions will hold the protein in its functional shape – these are weak and will take many to hold the shape.

- The peptide bond (covalent) allows for rotation around it and therefore the protein can fold and orient the R groups in favorable positions

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Fibrous proteins.

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- the polypeptide chains are arranged in long strands or sheets.

- Are insoluble in water, a property conferred by a high concentration of hydrophobic amino acid residues both in the interior ande exterior of the protein.

- Usually consists largely of a single type of secondary structure, and their tertiary structure is relatively simple.

- Function: provide support, shape, and external protection to vertebrates.

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Tandem Mass spectromotry - The difference in mass from peak to peak identifies the amino acid that was lost in each case, thus revealing the sequence of the peptide.

Back 24

1. A solution containing the protein under investigation is first treated with a protease or chemical reagent to hyrdolyze it to a mixture of shorter peptides.

2. The mixture is then injected into a device that is essentially two mass spectrometers in tandem;

1. In the first MS, the peptide mixture is sorted so that only one of the several types of peptides produced by cleavage emerges at the other end.

2. The second MS, measures the m/Z ratios of all the charged fragments. This process generates one or more sets of peaks. A given set of peaks consists of all the charged fragments that were generated by breaking the same type of bond (but at different points in the peptide). One set of peaks includes only the fragments in which the charge was retained on the amino-terminal side of the broken bonds; another includes only the fragments in which the charged was retained on the carboxyl-terminal side of the broken bonds.

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Beta-turn

Back 25

- Connect the ends of two adjacent segments of an antiparallel Beta sheet.

- The peptide groups of the central two residues DO NOT participate in any inter-residue hydroen bond.

- Gly + Pro residues often occur in beta turns.

- Are often found near the surface of a protein, where the peptide groups of the central two amino acid residues in the turn can hydrogen-bond with water.

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Beta-sheet

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- Beta conformation: the backbone of the polypeptide chain is extended into a zigzag rather than a helical structure.

- Beta sheet: The arrangement of several segments side-by-side, all of which are in the beta-conformation.

- Hydrogen bonds form between adjacent segments of polypeptide chain within the sheet.

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Protein analysis

Back 27

- CRUDE EXTRACT: First step in any protein purification procedure is to break open releasing the proteins into a solution.

- FRACTIONATION: the extract is subjected to treatments that separate the proteins into different FRACTIONS based on one property the fractions share. Hereafter the fractions are subjects to a specific analysis method, the different methods are:

Salting out - selective precipitation.

Dialysis - separating by taking advantage of protein size.

Chromatography - Charge, size, binding affinity and other properties (see figure).

Gel-filtration (PAGE) - size.

Electrophoresis - number of different proteins degree of purity of a preparation.

Front 28

SDS-PAGE (Sodium Dodecanoyl Sulfate Polyacryl Amid Gel Electrophereses)

Back 28

- A process for the sparation of proteins which is based on the SIZE of the protein.

- A protein will bind 1.4 times its weight of SDS nearly one molecule of SDS for each amino acid residue.

- The bound SDS contributes a large net negative charge, rendering the intrinsic charge of the protein insignificant and conferring on each protein a similar charge-to-mass ratio.

- SDS binding partially unfolds proteins, such that most SDS-bound proteins assume a similar rodlike shape

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PAGE (Polyacryl Amid Gel Electrophereses)

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- A process for the separation of proteins which is based on the migration of charged proteins in an electric field.

- Proteins can be visualized as well as separated, permitting one to quickly estimate the number of different proteins in a mixture or the degree of purity of a particular protein preparation.

- Can be used to determine crucial properties of a protein such as its isoelectric point and approximate molecular weight.

- Is carried out in gels made up of cross-linked polymer polyacrylamide.

Front 30

Salting out

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- The name for the effect, in which the solubility of proteins is lowered in the presence of some salts.

- The addition of certain salts in the right amount can selectively precipitate (udfælde) some proteins, while others remain in solution; Ammonium sulfate is particularly effective and is often used to salt out proteins.

- The precipitated proteins is removed by low-speed centrifugation.

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Dialysis

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- A procedure that separates proteins from small solutes by taking advantage of the proteins' larger size.

- Dialysis retains large proteins within the membranous bag or tube while allowing the concentration of other solutes in the protein preparation to change until they come into equilibrium with the solution outside the membrane.

Front 32

Chromatograpy

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COLUMN chromatography: A method for frationating proteins which takes advantage of differences in protein charge, size, binding affinity and other properties.
- A porous solid material (the stationary phase) is held in a column, and a buffered solution (the mobile phase) migrates through it.

ION-EXCHANGE chromatography: Exploit differences in the sign and magnitude of the net electric charge of proteins at a given pH.
- The column matrix is a synthetic polymer (resin) containing bound charged groups; Those with bound anionic groups are called cation exchangers, and those with bound cationic groups are called anion exchangers.

AFFINITY chromatography: Based on binding affinity.
- The beads in the columns have a covalently attached ligand; When a protein mixture is added to the column, any protein with affinity for this ligand binds to the beads, and its migration through the matrix is retarded.

Front 33

Gel-filtration

Back 33

SIZE-EXCLUSION chromatography (gel filtration):
- Separates proteins according to size; The solid phase consists of cross-linked polymer beads with engineered pores or cavities of a particularly.

- Large proteins cannot enter the cavities and so take a shorter (and more rapid) path through the column, around the beads.

- Small proteins enter the cavities and are slowed by their more labyrinthine path through the column.

Front 34

Super secondary structure (motif)

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- Consists of two or more elements of secondary structure, and the connection between them.

- it is a recognizable folding pattern.

- A motif is NOT a hierarchical structure element falling between secondary and tertiary structure.

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Secondary Structure

Back 35

- The term refers to any chosen segment of a polypeptide chain and describes the local spatial arrangement of its main-chain atoms without regard to the positioning of its side-chains og its relationship to other segments.

- The most prominent types of secondary structure are; alfa-helix, beta-conformation, and beta-turns (and therfore also beta-sheets).

- A regular secondary structure occurs when each dihedral angle (phi (ɸ) or psi (ψ)) remains the same or nearly the same throughout the segment.

- No regular pattern = random coil.

Front 36

Alpha-helix

Back 36

- The simplest arrangement the polypeptide chain can assume, that maximizes the use of internal hydrogen bonding.

- The polypeptide backbone is tightly wound around an imagenary axis drawn longitudinally through tha middle of the helix, and the R-groups of the amino acid residues protrude outward form the helical backbone.

- The right-handed alpha-helix is the most common form.

3.6 residues per turn, 1 amino acid residue is 1,5 Å.
The rise along the helical axis is 5,4 Å.

Front 37

Primary structure

Back 37

Primary structure = amino acid sequence of a protein.

- The primary structure of a protein determines how it folds up into its unique three-dimensionel structure, and this in turn determines the function of the protein.

- Different amino acid sequences = different functions.

Front 38

Dihedral Angles

Back 38

- Phi and Psi angles
The important dihedral angles in a peptide are defined by three bond vectors (w not considered) connecting four consecutive main-chain (peptide backbone atoms).

ɸ (phi) involves th C-N-Ca-C bonds (rotation occurring about N-Ca)

ψ (psi) involves the N-Ca-C-N bonds (rotation occurring around Ca-C)

The N-Ca and Ca-C bonds can rotate to define the dihedral angles, ψ and ɸ.

Front 39

Ramachandran plot

Back 39

- A graph were ψ is plotted versus ɸ; By doing this the allowed values for ɸ and ψ become evident, and the allowed structures in the peptide backbone becomes evident.

- Also used to test the quality of three-dimensional protein structures.

Front 40

Molten Globule

Back 40

- The molten globule state is an intermediate conformational state between the native and the fully unfolded states of a globular protein.

- The characteristics of the molten globule state are:
1. the presence of a native-like content of secondary structure;

2. the absence of a specific tertiary structure produced by the tight packing of amino acid side chains;

3. compactness in the overall shape of the protein molecule, with a radius 10 to 30% larger than that of the native state;

4. the presence of a loosely packed hydrophobic core that increases the hydrophobic surface area accessible to solvent.

- Levinthal’s paradox: The length of time in which a protein chain finds its folded state is many orders of magnitude shorter than it would be if it freely searched all possible configurations.

Front 41

Structural motifs

Back 41

- A recognizable folding pattern involving two or more elements of secondary structure and the connection(s) between them; The lowest form of structure, hereafter there is domain, and lastly it's a fold.

- For proteins: a three-dimensional structural unit formed by a particular sequence of amino acids, found in proteins and which is often linked with a particular function.

- For nucleic acids: a particular, usually short, nucleotide sequence that forms a recognition site usually to which other proteins bind.

- Certain specific orderings of secondary structure that may have a functional role and include β-α-β helix-turn-helix, leucine zippers, calcium binding EF hands, zinc fingers and longer orderings that take on a structural domain such as the β-barrel and the immunoglobulin fold.

Front 42

The Prion Hypothesis

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- Prions are hypothesized to infect and propagate by refolding abnormally into a structure which is able to convert normal molecules of the protein into the abnormally structured form.

- All known prions induce the formation of an amyloid fold, in which the protein polymerises into an aggregate consisting of tightly packed beta sheets;
- This altered structure is extremely stable and accumulates in infected tissue, causing tissue damage and cell death.
- This stability means that prions are resistant to denaturation by chemical and physical agents, making disposal and containment of these particles difficult.

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Oxidation state of Fe and O2 binding

Back 43

....

Front 44

Noncooperative and Coopoerative binding of O2

Back 44

....

Front 45

Sigmoid binding curve

Back 45

A diagnostic of cooperative binding

Front 46

Hill plot

Back 46

Has a slope of n - reflects NOT the numbers of binding sites but the degree of interactions between them.

nH - the Hill coefficient; is a measure of the degree of cooperativity

nH = 1 --> ligand binding is NOT cooperative.
nH > 1 --> positive cooperativity in ligand binding
nH = n --> completely cooperative
nH < 1 --> negative cooperativity; binding of one molecule of ligand IMPEDES the bindings of other.

Front 47

Myoglobin - principles of structure

Back 47

Myoglobin is a monomeric (single chained) protein that has 153 amino acids residues.

- It consists of eight α-helicies connected through the turns with a Oxygen binding site.

- It has a globular structure; van der Waals interactions make a significant contribution in stabilizing hydrophobic interactions because the nonpolar side chains in the core are so close together.

- Myoglobin contains a heme (prosthetic) group which is responsible for its main function (carrying of oxygen molecules to muscle tissues); the flat heme groups rests in a crevice, or pocket, where the accesibility of the group to solvent is highly restricted.

- Myoglobin can exist in the oxygen free form, deoxymyoglobin, or in a form in which the oxygen molecule is bound, called oxymyoglobin.

- Myoglobin is a protein found in muscles that binds oxygen with its heme group like hemoglobin.

Front 48

Heme group

Back 48

- Heme group consists of protoporphyrin organic component and an iron atom located in its center.

Front 49

Induced fit

Back 49

- A mechanism where the enzyme itself usually undergoes a change in conformation when the substrate binds, induced by multiple weak interactions with the substrate; it permits tighter binding.

- The motions can effect a small part of the enzyme near the active site or can involve changes in the positioning of entire domains.

Front 50

Levinthal's paradox

Back 50

- The length of time in which a protein chain finds its folded state is many orders of magnitude shorter than it would be if it freely searched all possible configurations; therefore there must be some kinds of "shortcuts" the protein makes.

Front 51

Binding-, active- and catalytic site

Back 51

Binding site: a site on the protein were a ligand binds; this side is only complementary to the ligand that should bind there, either in size, shape, charge, and hydrophobic/hydrophilic character.

The ligand-binding site is called the CATALYTIC SITE or ACTIVE SITE.

Front 52

Globular protein.

Back 52

- Have no systematic structures; the three-dimensionel structure of a typical globular protein can be considered an assemblage of polypeptide segments in the alpha-helical and beta-conformation, linked by connecting segments.

- There may be single chains, two or more chains which interact in the usual ways or there may be portions of the chains with helical structures, pleated structures, or completely random structures.

- Globular proteins are relatively spherical in shape as the name implies.

- Common globular proteins include egg albumin, hemoglobin, myoglobin, insulin, serum globulins in blood, and many enzymes.

Front 53

X-ray and NMR - tools for structural analysis

Back 53

X-ray: Provide little information about molecular motion within the protein.
- the proteins must be crystallized (LIMITATION).

NMR: Can be carried out on macromolecules in solution.
- Can illuminate the dynamic side of protein structure, including conformational changes, protein folding and interactions with other molecules.

Front 54

Domains

Back 54

- Regions of a polypeptide chain that can fold stably and independently.

- A domain from a large protein will retain its native three-dimensionel structure even when separated from the remainder of the polypeptide chain.

- Small proteins usually is a domain.

Front 55

Folding

Back 55

- The peptide bond allows for rotation around it and therefore the protein can fold and orient the R-groups in favorable positions.

Folding is generally hierarchical; initially, regions of secondary structure may form, followed by folding into motifs and hereafter domains.

- The folding into native conformations can either happen spontaneously, but more often with the assistance of chaperones; if the protein is not properly folded it can leed to the formation of inactive aggregates.

Front 56

Denaturation

Back 56

- Defined as a loss of three-dimensionel structure sufficient to cause loss of function.

- Most proteins is denatured by heat, but also extreme pH or denaturing reagents.

Front 57

Chaperones

Back 57

- Proteins that interact with partially folded or improperly folded polypeptides, facilitating correct folding pathways or providing microenvironments in which folding can occur.

- There are two major families of chaperones; Hsp70 and Chaperonins.

Front 58

Hsp70

Back 58

- Molecular weight near 70.000; More abundant in cells stressed by elevated temperatures (heat shock proteins).

- Bind to regions of unfolded polypeptides that are rich in hydrophobic residues; Thus they "protect" both proteins subject to denaturation by heat and new peptide molecules being synthesized (and not yet folded).

- Also block the folding of certain proteins that must remain unfolded until they have been translocated across a membrane.

- Binds to and release polypeptides in a cycle that uses energy from ATP hydrolysis and involves several other proteins.

Front 59

Chaperonins

Back 59

- Use energy from ATP hydrolysis to maintain proteins in the necessary folded configuration for proper function, thus having ‘foldase’ activity

- Any of a group of 60 kD cytosolic chaperone proteins—e.g., heat shock protein 60, hsp60, GroEL—found in prokaryotes, the equivalent of the eukaryotic hsp60, mitochondria and plastids.

- Other postulated roles for chaperonins include protein transport, oligomer assembly, DNA replication, mRNA turnover, and protection of the cell from various stresses; some chaperonins have auto-foldase activities

Front 60

The BPG effect

Back 60

- The interaction of 2,3-bisphosphoglycerate (BPG) with hemoglobin molecules further refines the function of hemoglobin, and provides an example of heterotropic allosteric modulation (a regulatory molecule that is not also the enzyme's substrate).

- BPG binds at a site distant from the oxygen-binding site and regulates the O2-binding affinity of hemoglobin in relation to the pO2 (oxygen partial pressure) in the lungs.

- BPG is important in the physiological adaption to the lower pO2 at high altitudes

- 2,3-bisphosphoglycerate is known to greatly reduce the affinity of hemoglobin for oxygen - there is an inverse relationship between the binding of O2 and the binding of BPG; another binding process for hemoglobin:

HbBPG + O2 ↔ HbO2 + BPG

Front 61

The principles of the sequential and concerted model

Back 61

- Two models for cooperative binding of ligands to proteins with multiple binding sites, used to investigate allosteric enzymes. The two models are not mutually exclusive;The concerted model may be viewed as the "all-or-one" limiting case of the sequential.

- Concerted model: assumes the subunits of a cooperativity binding protein are functionally identical, that each subunit can exist in (at least) two conformations, and that all subunits undergo transition from one conformation to the other simultanously.
- The ligand can bind to either conformation but binds to each with different affinity.

Sequential model: The ligand binding can induce a change of conformation in an individual subunit. A conformational change in one subunit makes a similar change in an adjacent subunit, as well as the binding of a second ligand molecule, more likely.
- There are more potential intermediate states in this model than in the concerted model.

Front 62

Hemoglobin in Adults and Fetus'

Back 62

Fetal hemoglobin:
- has a somewhat higher affinity for CO than adult hemoglobin; because a fetus must extract oxygen from its mother's blood, to do this, it must have higher affinity for O2 than the maternal hemoglobin.
- The fetus synthesizes y subunits rather than Beta-subunits forming alpha2y2 hemoglobin; This tetramer has a much lower affinity for BPG than normal adult hemoglobin and a correspondingly higher affinity for O2.

Adult hemoglobin:
- Contains two types of globin, two alpha-chains and two beta-chains.

Front 63

Hemoglobin - The principle of the structure.

Back 63

- A tetramene protein containing four heme prosthtic groups, one associated with each polypeptide chain; roughly spherical.

- The quarternary structure features strong interactions between unlike subunits.

- Hydrophobic interactions predominate at all the interfaces, but there are also many hydrogen bonds and a few ion pairs (or salt bridges).

- Hemoglobin exists in TWO INTERCHANGEABLE structureal states, T and R; The T-sate is most stable when oxygen is not bound. Oxygen binding promotes transition to the R-state.

- Has a hybrid S-shaped, or sigmoid binding curve for oxygen.

Front 64

Ligand and substrate

Back 64

- The functions of many proteins involve the reversible binding of other molecules. A MOLECULE BOUND REVERSIBLY BY A PROTEIN IS CALLED A LIGAND.

- A ligand may be any kind of molecule including another protein.

- THE MOLECULES ACTED UPON BY ENZYMES ARE CALLED REACTION SUBSTRATES, NOT LIGANDS.

Front 65

Specificity constant - Kcat/Km

Back 65

- The best way to compare the catalytic efficiencies of different enzymes or the turnover of different substrates by the same enzyme is to compare the ratio Kcat/Km for the two reations.

- SPECIFICITY CONSTANT is the rate constant for the conversion of E+S to E+P.

- There is an upper limit to Kcat/Km imposed by the rate at which E and S can diffuse together in an aqueous solution.

- This diffusion-controlled limit is 10^8 to 10^9 M-1S-1
- Enzymes near this range are said to have achieved catalytic perfection.

Front 66

Turnover Number Kcat

Back 66

- The general rate constant; Describes the limiting rate of any enzyme-catalyzed reaction at saturation.

- If the reaction has several steps and one is clearly rate limiting, Kcat is equivalent to the rate constant for that limiting step.

- When product is clearly rate-limiting: Kcat = K3

- The constant Kcat is a first-order rate constant.

- It is equivalent to the number of substrate molecules converted to product in a given enzyme is saturated with substrate.

Front 67

Lineweaver-Burk plot.

Back 67

- A form of the Michaelis-Mentel equation that is more useful in plotting experimental data.

- The equation yields a straight line, which has a slope of Km/Vmax, an intercept of 1/Vmax on the 1/V0 axis, and an intercept of -1/Km on the 1/[S] axis.

- Allows a more accurate determination of Vmax, which can only be approximated from a simple plot of V0 versus [S].

Front 68

Km

Back 68

- The Michaelis Menten constant.

Front 69

Prosthetic Group, Holo- and Apoenzym.

Back 69

Prosthetic: A coenzyme or metal ion that is very tightly, or even covalently, bound to the enzyme protein.

Holoenzyme: A complete, catalytically active enzyme together with its bound coenzyme and/or metal ions.

Apoenzyme/Apoprotein: The protein part of such an enzyme

Front 70

The Michaelis-Menten Equation.

Back 70

- The rate equation for one-substrate enzyme-catalyzed reaction.

- It is a stament of the QUANTITIVE relationship between the initial velocity, V0, the maximum velocity Vmax, and the initial substrate concentration [S], all related through the Michaelis constant, Km.

- ALL enzymes that exhibit a hyperbolic dependence of V0 on [S] are said to follow MICHAELIS-MENTEN KINETICS.

Front 71

Lock and key, induced fit - TRANSISTION STATE.

Back 71

Induced fit: The enzyme itself usually undergoes a change in conformation when the substrate binds, induced by multiple weak interactions with the substrate.

- The enzyme mus NOT be like "lock and key" (complementary to substrate) with the substrate; an uncomplementary enzyme can "use" the "leaver effect" to "break" the substrate (illustratet as a metal rod); if this is the case, it's complementary to the transition state.

Front 72

Rate Constant

Back 72

- The rate of any reaction is determined by the concentration of the reactant (s) and by a RATE CONSTANT, k.

Front 73

General and specific Acid-Base catalysis

Back 73

General: Catalysis involving proton transfer(s) to or from a molecule other than water.

Specific: Acid or base catalysis involving the constituents of water H+ or OH-.

Front 74

Equilibrium and catalysis

Back 74

- Reaction equilibria are linked to the standard free-energy change for the reaction, ΔG’°.

- Reaction rates are linked to the activation energy, ΔG‡.

- The function of a catalyst is to increase the rate of a reaction. Catalysts do NOT affect the reaction equilibria.

Front 75

Transistion- and activation state.

Back 75

- CATALYSTS ENHANCE REACTION RATES BY LOWERING ACTIVATION ENERGIES.

- Ground state: the starting point for either the forward or reverse reaction.

-ΔG’°: the standard free-energy change at pH7.

- Transition state: NOT a chemical species with any significant stability; It is a fleeting molecular moment in which events such as bond breakage or bond formation heve proceeded to the precise point at which decay to either substrate or product is equally likely.

- Activation energy, ΔG‡: the difference between the energy levels of ground state and transition state. A HIGHER ACTIVATION ENERGY = SLOWER REACTION.

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Equilibrium constant.

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- An equilibrium such as S ⇌ P is described by an EQUILIBRIUM CONSTANT, Keq or simply K.

- Under standard conditions it's used to compare biochemical processes K'eq or K'.

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Enzyme-substrate complex - ES

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- The distinguishing feature of an enzyme-catalyzed reaction is that it takes place within the confines of a pocket on the enzyme called the ACTIVE SITE.

- Substrate: the molecule that is bound in the active site and acted upon by the enzyme.

- The surface of the active site is lined with amino acid residues with substituent groups that bind the substrate and catalyze its chemical transformation.

- Often, the active site encloses a substrate, sequestering it completely from solution.

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Initial velocity and Maximum velocity.

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- Initial rate = Initial velocity = V0: Depends on the concentration of substrate [S]; At relatively low concentrations of substrate, V0 increases almost linearly with and increase in [S]. At higher [S] V0 increases by smaller and smaller amounts in response to increase in [S].

- When increase in V0 are vanishingly small as [S] increases the plateau-like V0 region is close to the MAXIMUM VELOCITY, Vmax.

- Vmax is observed when virtually all the enzyme is present as the ES complex and [E] is vanishingly small under these conditions; the enzyme is "saturated".

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Pre-steady, Steady + steady-state

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Pre-steady state: the initial period where there is a large excess of substrate, comparing to the enzyme added in the mixture; during this state the concentration of ES builds up, but is usually too short to be easily observed.

Steady-state: In this state [ES] (and the concentrations of any other intermediates) remains approximately; the measured V0 generally reflects the steady state, eventhough V0 is limited to the early part of the reaction.

Steady state kinetics: the analysis of the initial rates.

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Bisubstrate reactions

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- An enzymatic reaction where two different substrate molecules bind to the enzyme and paticipate in the reaction.

- Bisubstrate reactions usually involve transfer of an atom or a functional group from one substrate to the other. These reactions proceed by one of several different pathways;

- Ternary complex: Both substrates are bound to the enzyme concurrently at some point in the course of the reaction, forming a non-covalent ternary complex; the substrates bind in a random sequence or in a specific order .

- Ping-Pong mechanisms: the first substrate is converted to product and dissociates before the second substrate binds, so no ternary complex is formed.

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Hexokinase - a bisubstrate enzyme

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- Can discriminate between glucose and water, eventhough water freely enters the enzyme active site, and is similar in chemical reactivity; It discriminate because of the conformation change in the enzyme when the correct substrate binds.

- When glucose is not present, the enzyme is in an inactive conformation, with the active-site amino acid side chains out of position for reaction.
- When glucose (not water) and Mg*ATP bind, the binding energy derived from this interaction induces a conformational change in hexokinase to the catalically active form.

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Effect of pH on enzyme

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Enzymes have an optimum pH at which their activity is maximal; at higher or lower pH activity decreases.

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Competitive inhibitor - reversible inhibitor

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- Competes with the substrate for the active site of an enzyme.

- Many competitive inhibitors are structurally similar to the substrate and combine with the enzyme to form an EI complex, but without leading to catalysis.

- There are three types of reversible inhibition; Competitive, uncompetitive and mixed inhibition.

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Uncompetitive inhibitor

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- One-substrate enzymes, but are in practice observed only with enzymes having two or more substrates.

- Binds at a site distinct from the substrate active site and, unlike a competitive inhibitor, binds ONLY to the ES complex.

- At high conc. substrate, V0 --> Vmax/alpha' (it lowers the measured Vmax).

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Mixed inhibitor

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- Binds at a site distinct from the substrate active site, but it binds to either E or ES.

- A mixed inhibitor usually affects both Km and Vmax.

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Protein kinases and Phophatises

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Protein kinase: The kinase the catalyses the attachment of phosphoryl groups to specific amino acid residues of a protein.

Protein phosphatases: The phosphate that catalyses the removal of phosphoryl groups from amino acid residues of a protein.

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Chymotrypsin

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- Protease, catalyzes the hydrolytic cleavage of peptide bonds.

- Specific for peptide bonds adjacent to aromatic amino acid residues (TRP, PHE, TYR)

- DOES NOT catalyze a direct attack of water on the peptide bond; instead, a TRANSIENT COVALENT ACYL-ENZYME INTERMEDIATE is formed .

- Two phases:
1. In the acylation phase, the peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme.

2. in the deacylation phase, the ester linkage is hydrolyzed and the non acylated enzyme regenerated.

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Catalytic triad

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- Ser195 is linked to His57 and Asp102 in a hydrogen-bonding network referred to as the CATALYTIC TRIAD.

- When a peptide binds to chymotrypsin, a subtle change in conformation compresses the hydrogen bond between HIS57 and ASP102, resulting in a stronger interaction, called a LOW-BARRIER HYDROGEN BOND between His57 and ASP102.
- This enhanced interaction increases the pKa of HIS57 from ca 7 (for free his) to >12, allowing the His residue to act as an enhanced general base that can remove the proton for SER195 hydroxyl group

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Allosteric enzymes

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- Are those having "other shapes" or conformations induced by the binding of modulators.

- The conformationel change converts a relatively inactive conformation (T-state) to a more active conformation (R-state).

- A regulatory enzyme with catalytic activity modulated by the noncovalent binding of a specific metabolite at a site other than the active site.

- Function through reversible, noncovalent binding of regulatory compounds called allosteric modulators or allosteric effectors, which are generally small metabolites or cofactors.

- Sigmoid kinetic behavior reflects cooperative interactions between multiple protein subunits.
V0 versus [S] = sigmoid saturation.

- Changes in structure of one are translated into structural changes in adjacent subunits.

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Lambert-Beer's law

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- the amount of light emerging from a sample is diminished by three physical phenomena:

- The amount of absorbing material in its pathlength (concentration)
- The distance the light must travel through the sample (optical pathlength OPL)
- The probability that the photon of that particular wavelength will be absorbed by the material (absorptivity or extinction coefficient)

This relationship may be expressed as:
A = εdc
A = absorbance ε = molar extinction coefficient
d = pathlength in cm c = molar concentration

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Oligomers and Protomers

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Some proteins consist of a single polypeptide chain, but others have two or more polypeptides associated noncovalently.

OLIGOMERIC: a phrase used for a protein where at least two of the individual polypeptide chains are identical.

PROTOMERS: A phrase used for the identical units in a MONOMERIC protein.

Hemoglobin have four polypeptide subunits: Two identical beta-chains and two identical alpha-chains.

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Binding of O2 and CO to heme to free hemoglobin

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- Cooperative binding of O2 to hemoglobin, is a form of allosteric binding, meaning the binding of one ligand affects the affinities of any remaining unfilled binding sites.

- Free heme molecules (heme not bound to protein) leave Fe2+ with two "open" coordination bonds; Simultaneous reaction of O2 molecule with two free heme molecules (or two free Fe2+) van result in irreversible conversion of Fe2+ to Fe3.

- In heme-containing proteins this is prevented by sequestering each heme deep within the protein structure-access to the coordination bends is restricted.

- By surrounding and sequestering heme, oxygen-binding proteins regulate the access of CO to the heme iron.

- CO coordinate to heme iron with greater affinity than O2.

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Heme and iron

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- Iron is often incorporated into a protein-bound prosthetic group called HEME.

- Heme consists of a complex organic ring structure, protoporphyrin, to which is bound a single iron atom in its ferrous (Fe2+) state.

- The iron atom has six coordination bonds, four to nitrogen atom that are part of the flat porphyrin ring system and two perpendicular to the porphyrin.

- The coordinated nitrogen atoms (which have an electron-donating character) help prevent conversion of the heme iron to the ferric (Fe2+) state.

Fe2+ binds oxygen reversibly.
Fe3+ does not bind oxygen.