Biochemistry – Dr



Biochemistry – Dr. Rosevear

Physical and chemical properties of water

- relatively high heat of vaporization

- higher melting and boiling points than liquids on I-3

- Hydrogen bonding network explains the internal cohesive properties of it

- Dipole Moment – partial charge separation

- Rough tetrahedron

- H-bonds in water are weak with a bond energy of ~20KJ/mole

- H-bonds are involved in specificity, receptor ligand interactions and enzyme-substrate

- Lifetime of an H- bond is near 10^9sec

- Micelle- have polar regions that face water exhibiting hydrophobicity and philicity

- Waters high dielectric constant contributes to its ability to form H-bonds

- Highly polar molecules readily dissolve in water

- Hydrophobic interactions govern protein folding

- Hydrophobic – non-polar; hydrophilic – polar

Protein Structure and function

- Proteins function through their conformation

- All AA’s are optically active (except Gly) they have four different substituents bonded to the alpha carbon

- Aliphatic amino acids would be expected to be in the interior of the protein (non-polar); contribute to van der waals interactions

o Glycine, Alanine, Valine, Leucne, Isoleucine, Proline

- Polar, uncharged; have S or O in the side chain, water soluble and participate in catalytic processes

o Serine, Threonine, Cysteine, Methionine, Asparagine, Glutamine

- Aromatic; all are very hydrophobic (except Tyrosine); hydroxyl group of tyr gives it some polar characteristics

o Phenylalanine, Tyrosine, Tryptophan

- Proline is the only cyclic amino acid (imino); hydrophilic and is known as a breaker of alpha helical structure

- Positively charged,

o Histidine – pH is in physiological range, Arginine, lysine-performs dual duties w/ hydrocarbons and polar head

- Negatively charged

o Aspartate, Glutamate

- Overall charge is determined by the sum of the charges on each amino acid

o PH at which ½ the acid protons are lost is the pK – carboxylic acid group is the first group to lose protons

- Henderson-Hasselbach (I-11)

- Isoelectric “zwitterionic form – net charge of 0, contains 1 positive and 1 negative

o Proteins are least soluble at zwitterionic state

- Know pK (I-12)

Higher order structure in proteins

- Primary Structure – linear order in which linked

o Joined by peptide bonds

▪ Peptide bonds are almost always planar and trans – meaning the alpha carbons are opposite each other

o If you knew the phi and psi angles of every peptide bond you could define the protein 3 dimensionally

o Stabilized by resonance hybridization

o Catalytic residues like aspartate, histidine and serine are known to be close in the final structure

- Secondary Structure – any regular repeating hydrogen bonding structures, alpha helices and beta sheets

o Alpha helix

▪ Stabilized by H-bonding between carbonyl oxygen and main chain amide hydrogens (amide hydrogen I is bound to carbonyl oxygen I+4)

▪ 3.6 residues /turn

▪ all discovered helices are right handed

▪ two other types of helices are the 3 sub 10 helix which is wound more tightly w/ 10 atoms in the closed ring – and the pi helix which is wound looser w/ 4.4 res/turn and 16 atoms

o Beta Structure

▪ Distance between adjacent amino acids is 3.5A as opposed to 1.5A

▪ Stabilized by H-bonding between carbonyl oxygens and amide hydrogens but these bonds are between polypeptide chains rather than within a single polypeptide chain

▪ Can either be parallel or antiparallel, peptide chains running in the same direction and peptide chains running in opposite directions (both N—C)

▪ Chain folds back on itself via beta turns

• Proline is highly favored for beta turn regions

o Ramachandran Plot – limits the number of psi phi angles, also illustrate the redundancy of nature

- Tertiary Structure- the total of all the noncovalent structure contained in the folded protein – the way we organize secondary structure

o Forces responsible for stabilizing

▪ H –bonding, hydrophobic interactions, ionic interactions, disulfide bonds

▪ Hydrophobic interactions in water are the most important in stabilizing protein folding

• Driven entropically

▪ Electrostatic bonds (salt bridge)

• Strongest noncovalent linkage

▪ Van der waals interaction is the distance of maximum favorable interaction between two atoms

o Stability of Proteins

▪ Delta G is positive the rxn favors unfolding – delta G is negative the reaction favors folding

▪ Major contributor to protein instability is chain entropy, which favors the unfolded state

▪ Major contributors to protein stability are hydrophobic interactions and H- bonding

▪ Folded conformation determined by the primary sequence

• Native conformation is most stable

• Large proteins fold into discrete domains

▪ Central Dogma – Conformation of a native polypeptide is entirely determined by amino acid sequence (primary sequence)

▪ Prion diseases are examples of protein misfolding diseases e.g. mad cow

- Quaternary Structure – manner in which the subunits of a multisubunit protein are arranged with respect to one another

o Subunit – subunit interaction is usually via hydrophobic and hydrogen bonding interactions

- Post-Translational Modifications

o Carbohydrates, Lipids, Cofactors, Metal Ions

Oxygen Binding Molecules

- Myoglobin – iron containing protein whose function is to bind to and carry oxygen in the muscle

o Active form of iron is its Fe 2+ state (ferrous) oxidation to Fe 3+ (ferric) will destroy Oxygen binding

▪ Fe 2+ binds up to six ligands at a time

o Oxidized – loses; Reduced – gains

o Heme group gives ability to bind oxygen

▪ Inorganic portion is iron and organic portion is the porphyrin ring

▪ Porphyin ring consists of 4 pyrrole groups

▪ 4 methyl, two vinyl, and two propionate groups are attached to the pyrrole group

▪ the middle of this Protoporphyrin IX tetrapyrole ring supplies four ligands for iron

o eight major right handed helical segments A---H

o Oxygen Binding

▪ Myoglobin is bond in an apolar crevice of the molecule

▪ Histidine F8 or 93 is directly and covalently bonded to the 5th coordination position of iron – and is called the proximal histidine

▪ Near the oxygen binding (6th position) site is Histidine E7 or 64 called the distal histidine

• Role of distal is to decrease heme’s affinity for CO

▪ The heme is in the non-polar pocket to protect the iron from oxidation

- Hemoglobin – the oxygen transport carrier in the vertebrate erythrocyte – consists of four polypeptides that are homologous to each other

o Bisphosphoglycerate is an important allosteric effector of hemoglobin

▪ BPG acts to lower the oxygen affinity of Hb

▪ Since HbF uses different subunits it binds BPG much weaker and therefore has a higher oxygen affinity

o Important Amino Acid residues in hemoglobin

▪ Histidine F8 – 92 --- proximal heme linked; Histidine E7 – 63 --- Distal; Glycine B6 – 25 --- allows the close approach of the B and E helices; Tyrosine HC2 – 145 --- cross links the H and F helices; Asp FG1 – 94 ---forms salt link with His 146

o Allosterism

▪ Hemoglobin binds H as well as CO2

▪ In presence of BPG has lower affinity for oxygen

• In presence of BPG Hb binding curve adopts sigmoidal shape

▪ Oxygenation of Hb causes a conformational change in which an alpha beta dimmer rotates

▪ Shifts from T (taut) form low oxygen affinity to R (relaxed) high oxygen affinity

▪ T form is stabilized by ionic bonds between subunits, these are absent in the R form

o Cooperativity

▪ Binding the 2nd oxygen is easier than binding the first oxygen and so on due to signaling caused by binding and the resultant conformational change

▪ Oxygen binding decreases BPG affinity and subsequent binding further decreases BPG affinity

▪ Measured using the Hill plot

• Slope of 1 – cooperativity

▪ Converse is also true, as one oxygen is released this facilitates releasing of the other oxygens

▪ Saturation curve

• If pH down = Oxy affinity Down = left shift

• If pH Up = Oxy Affinity Up = right shift

• At a high pH get deprotonation of imidazole ring

o Subunit Interactions

▪ If the subunits were separated they would display the oxy binding characteristics of myoglobin

▪ In deoxy Hb carboxy terminal residues are tightly constrained by formation of ionic bonds (salt links)

▪ T form oxygenation results in conformational changes

• Breaking of Tyr47(a-chain) -Asp99(b-chain) hydrogen bond and the formation of Asp94(a-chain) to Asn102(b-chain)

o These movements destroy the salt bridges

▪ T form stabilizers

• Increase Hydrogen ions (low pH), carbon dioxide binding, BPG

• BPG binds to deoxy form via salt links in the center of the tetrameric residue

▪ Oxygen binds to iron moving the iron into the heme plane this breaks the hydrogen bond between Val 98 and Tyr 146 --- helix H then falls away from F, resulting in the ionic bond being broken between the carbonyl of His 146 and Lys on alpha subunit, which signals the alpha that the beta has an oxygen

o Movements of the iron atom

▪ Oxygenation of the taut form moves the iron into the heme plane converting the heme from domed to planar, pulling the His F8 with it and causing several changes

- Bohr Effect

o Increase in proton concentration (lower pH) decreases oxygen affinity for Hb – hemoglobins ability to bind H+

▪ If pH low = {H} up = Oxy Affinity low

▪ Three amino acids are involved in this effect

• His146 on beta, and the two alpha amino acids on the alpha chain Asp94 and Val 98

o Protonation of His146 allows it to form a salt bridge stabilizing the T form

o Hemoglobin Protonation (favors taut form) occurs in the Tissues and deprotonation (favors relaxed form) occurs in the lungs

▪ CO2 up --- O2 down = low Oxy affinity of Hb

- Sickle Cell

o Results from mutation in Beta subunit Glutamine 6 is replaced by a Valine residue, which places a hydrophobic amino acid in the place of a hydrophilic

o Deoxygenated HbS has an abnormally low solubility, which forms a fibrous precipitate within the cell which physically deforms the cell

Collagen

- Most abundant protein in mammals, with a very high tensile strength

- Major stress bearing element of connective tissues; bone , teeth, tendon, cartilage, ligament

- Part of the fibrous matrix of skin and blood vessels

- Five types of collagen (II-2)

- Levels of Collagen Structure

o Gly every third residue ~33%, approx 20% proline or hydroxyproline ( which is formed via the hydroxylation of proline, approx 10% Ala, an 1-5% hydroxylysine

o Minor Helix – not alpha but a type II, 3 residues/turn

o Triple Helix – three type two helices to form a superhelix

o Tropocollagen – post translational modification – single collagen fiber before it forms fibrils

o Fibril Formation – self-assembly of collagen into fibrils

- Interchain bonding is present in collagen

- Hydroxyproline confers molecular stability to collagen through intermolecular hydrogen bonds

- Ascorbate is also required to maintain the iron, found in the enzyme prolyl hydroxylase, in its ferrous state, this enzyme is critical for collagen helix formation

o Lack of ascorbate at this point causes scurvy – skin and blood vessels become weak

- Steric hinderance involved in collagen helix formation only allows for glycine to be in the center

- Contains covalently linked carbohydrates which are attached to the hydroxyl lysine

- Collagen Cross Linking

o Lysyl Oxidase catalyzes the conversion of Lysine to Allysine, which forms crosslinks predominantly in the N and C termini

- Collagen Biosynthesis

o MRNA made, message translated by polysomes, (as protein is being made and before helix formation Hydroxylation and glycosylation occurs), following modification chains associate and disulfide bonds are formed in the N and C termini, triple helix formation (procollagen), moved to Golgi and released to extracellular space, N and C termini are then cleaved (collagen), which is incorporated into a fibril and cross linked by Lysyloxidase

▪ N terminal pro-sequence contains intra-strand disulfide bonds

▪ C terminal pro-sequence contains interchain disulfide bonds

o Hydroxyproline and Hydroxylysine

▪ Are formed through the oxidation of their respective amino acids

• Requires Fe containing enzyme, O2, ascorbate

- Native collagen is relatively insoluble

o Explained by the fact that it is inter and intra molecularly crosslinked

Dr. Cartwright – Nucleic Acid structure, function, and correlation

- Central Dogma

o genes are located on DNA, which are unbranched polymers called chromosomes

o DNA is transcribed into RNA which is translated into Proteins

- Building Blocks

o Nucleotide subunits (III-5)

▪ Base connected through a nitrogen atom to a five carbon sugar to which a phosphate is connected via one of the sugar hydroxyl groups

▪ Polymers are created via phosphodiester linkages

▪ Purines (the big ones) and pyrimidines (the small ones)

• Purines – Adenine and Guanine; Pyrimidines – Cytosine, Thymine, Uracil (only in RNA)

▪ Ribose sugar in RNA w/ an OH on the 2nd carbon; deoxyribose sugar in DNA w/ no oxygen at the 2nd carbon

▪ Phosphate groups are what give nucleotides their acidic nature

▪ Sugar is linked to the base via a glycosidic bond

- DNA Structure

o Double-helical, anti-parallel, hydrogen bonding

▪ A has two H bonds with T; G has three H bonds with C

▪ Watson Crick base pairing

▪ Chargaffs Rule # of A’s is always equal to # of T’s

▪ Right handed orientation

▪ ~10 base pairs/turn, at ~34 angstroms

o Can denature through

▪ Addition of alkali’s that interfere w/ H bonding

▪ Addition of heat

▪ Reversible – reannealing or renaturation

o Nucleic Acid Synthesis

▪ Polymerase reads the sequence and inserts nucleotides bearing complementary bases into a newly synthesized chain

▪ Precursors are 4 dNTP’s, Mg

▪ 5’ to 3’

▪ polymerase reads in the 3’ to 5’ direction

▪ RNA is transcribed using a Mg dependent RNA polymerase

▪ Polymerase binds to a template strand of DNA to make RNA

o Reverse transcriptase, found in a retrovirus, is able to synthesize DNA from RNA

▪ AZT is a dideoxynucleotide that seeks out reverse transcriptase and becomes a substrate for it and subsequently halts transcription because it lacks the appropriate-OH group

o Deciphering RNA

▪ Read using triplets – codons

▪ Amino acids being specified by more than one codon – degeneracy – known as synonymous codons

▪ Methionine AUG start codon; UAA,UAG, UGA stop codons

▪ Proteins are synthesized from N to C

- DNA replication and repair

o Semi-conservative

▪ One parent and one new in the new strand

o DNA replication starts at origin sites and moves away bidirectionally

o Proteins of DNA replication have helicase activity – unwinding, topoisomerase activity – cutting and rejoining the phosphodiester backbone, SSB(single strand binding) protein that binds to and keeps DNA from reannealing

o Continuous DNA synthesis is templated from the leading strand; w/ the lagging strand undergoing discontinuous replication

o A “nick” is formed between okazaki fragments because there won’t be a phosphodiester bond between the 3’ end of the ok frag and the 5’ of the newly synthesized DNA

▪ This nick is closed via DNA ligase

o Uses an RNA dependent DNA polymerase, which lays down a RNA template to begin synthesis

▪ This polymerase has exonuclease activity in that it removes the RNA template as it chugs along, chews ahead and adds behind

o DNA replication in prokaryotes

▪ Done by complex of proteins called a Replisome

▪ Several different kinds of DNA polymerase

• DNA polymerase III responsible for leading and lagging strand synthesis – highly processive

• DNA polymerase I responsible for RNA primer degradation/DNA resynthesis – carries 5’ to 3’ exonuclease activity

▪ Process

• Origin is melted by helicase and topoisomerase; Primase lays down a short RN primer, which is extended by DNA poly III on the leading and lagging strand both in the 5’ to 3’ direction; 5’ to 3’ exonuclease activity of DNA polymerase I erases the RNA primer and DNA is put in their place, on the lagging strand the nicks are filled in using DNA ligase

o DNA replication in eukaryotes

▪ Multiple origins of replication

▪ ~9 known polymerases, alpha, delta, and epsilon are involved in DNA rep in nuclei

• alpha is always in complex with DNA primase which lays down the RNA primer

• after a short amount of synthesizing polymerase delta switches with alpha and continues processive synthesis

• PCNA (proliferating cell nuclear antigen) – provides sliding clamp activity of replication

• Polymerase beta seems to be for repair of damage, gamma does mitochondrial DNA

• Eukaryotic DNA polymerases do not have 5’ to 3’ exonuclease activity

▪ Telomerase activity

• Slight overhang of the leading strand caused by shortening of the lagging strand because of its inability to process the last RNA primer

• Telomerase – extends the 3’ end of the leading strand w/ GGGTTG

▪ Mutation

• Frameshift mutation – insertion of a base and changing the reading frame

• Missense – single amino acid change

• Nonsense – switch a coding sequence to a stop sequence

▪ Fidelity

• 3’ to 5’ exonuclease activity – proofreading activity

• mismatch repair enzyme which actively seeks out mismatched pairs

o DNA damaging

▪ UV radiation interacting with thymine bases

▪ Spontaneous events – Depurination and Deamination

• Uracil DNA-glycosylase cleaves uracil residues

o Al such mutations of bases is a substrate for AP endonuclease, which clips the DNA strand and the resulting nick is extended via DNA poly I

• Called Base Excision Repair

▪ Environmental damage – radiation or chemical

• Nucleotide Excision Repair

• UV irradiation creates thymine dimmers

o Can either photo-reverse the reaction using photolyase

o Or use Nucleotide Excision Repair, which recognizes the legion and cleaves it as well as about 12 nucleotides in both directions, and then is repaired by DNA poly I and DNA ligase

▪ Xeroderma Pigmentosum

• Patients fail, to varying degrees, to repair thymine dimmers

• Patients don’t produce UV endonuclease

• There are ~7 complementation groups involved with the repair of UV damage

• Life threatening aspect of XP is the generation of carcinomas and melanomas

▪ Ames test

• Uses a bacteria mutated in the gene to synthesize histidine, which makes them unable to produce histidine

o On plates w/o histidine only a few spontaneous revertants will grow

o If a mutagen is added the histidine mutation will be more efficiently reversed

- Transcription, Translation, and Gene regulation

o RNA synthesis

▪ Extra oxygen in the sugar ring makes RNA unstable

▪ The start and stop information for making a message is not the same as the start and stop information for making a protein

▪ Synthesized by RNA polymerase

• Only one is required for bacterial RNA synthesis

o Exists in two states – holoenzyme and core enzyme(which has lost the sigma subunit

▪ W/o sigma polymerase cannot recognize the correct start sequence

▪ Acts as a transcription initiation factor

o Start signal known as a promoter

o Promoter sequences TATAAT and TTGACA are located around –10 and –35 respectively on the strand of DNA

▪ Once 10 or so bases have bound sigma dissociates – no primer is needed for this process

▪ Elongation of the RNA chain is relatively monotonous, synthesis is continuous in that the same polymerase that starts it will finish it

▪ Termination occurs at specific sites

• factor independent – require only a specific DNA sequence – have characteristic DNA sequence – long run of A’s

• Rho dependent –require an additional protein termination factor to interact with the template – Rho binds to the RNA and the RNA begins to wrap around it – Rho is a RNA-DNA helicase-unwinds hybrids

▪ Transcription unit containing several transcription units is called an operon

▪ Coupled transcription and translation in bacterial cells

▪ Bacterial mRNA is not post-transcriptionally modified

• It is ready for immediate translation

▪ Eukaryotic RNA is transcribed in the nucleus but translated in the Cytoplasm

• Initial transcript is interrupted by non-coding intervening sequences – introns

• Coding region – exons

• Splicing is the removal of introns and subsequent binding of exons

▪ Eukaryotic RNA polymerases

• RNA poly II is responsible for mRNA

o Made in nucleoplasm

• RNA poly I is responsible for rRNA

o Cleaved into two subunits – large 28s an small 18s

o Made in nucleolus

• RNA poly III is responsible for tRNA

o Brings individual amino acids to the ribosomes and aligning them correctly

o Made in nucleoplasm

o Most abundant ~90% of all RNA

• Initiation of RNA synthesis in Eukaryotes

o Certain proteins bind to the promoter before the RNA polymerase and assist in the recognition of the promoter with the help of these additional TFII factors and ATP(for energy) synthesis begins

o First to bind is the TATA binding protein

▪ TFIID binds TATA; TFIIF binds PolII which is bound by TFIIDAB (dab complex) resulting in DABFpolII, which is bound by E and the resulting complex is lastly bond by PolIIH to begin transcription

• RNA polymerase II promoter

o TATA – lies about 30 bases upstream – stimulates the efficiency of the promoter recognition

▪ Several enhancers are located w/in ~200 base pairs of the promoter to aid in influencing promoter activity

• Processing of the 5’ end

o The 5’ ends of all RNA’s are capped using the enzyme Guanyl transferase catalyzing the addition of GTP through a 5’-5’ linkage, which is then methylated

▪ Aids in splicing, transport to cytoplasm, translation, stability

o Both rRNA and tRNA are not capped

• Processing of the 3’ end

o After the polymerase has synthesized all required RNA it encounters TTATTT on the DNA sequence and ~25 bases downstream of this the RNA is cleaved and the 3’ end is polyadenylated by the poly A polymerase

▪ For regulation of translational efficiency

▪ Stability

• Removal of intervening sequences

o Essentially all introns begin with GU and end with AG

o Spliceosomes cleave the introns and ligate the exons

▪ 5’ end cleaved first by forming a 5’-2’ bond between the G residue of the intron and an internal A residue – forming a lariat structure

▪ after lariat formation the 3’ end of the intron is cleaved and the exons are joined

o histones are not spliced

o precursor is synthesized, capped, and polyadenylated before splicing

o occurs in the nucleus

• Thalassemia is a set of syndromes resulting from graded deficiencies in the production of globin alpha or beta chains

o Result from sub-normal or zero production of normal globin proteins

o Correct splicing does not take place and the absence of the normal splice acceptor activates a cryptic splice site within the second intron

▪ Translation

• All tRNA’s have the same overall three dimensional structure

o Intrastrand base pairing gives it a cloverleaf appearance

o All have the sequence CCA at their 3’ ends; the loop opposite the CCA is the anti-codon which always contains the bases that pair with the codons of the message

o Contain numerous modified bases – inosine

o Aminoacyl tRNA synthetases recognize single AA’s and the appropriate tRNA

▪ W/ ATP it joins the –COOH of the AA to the 3’ –OH of the 3’ terminal A residue of the tRNA

▪ ATP is cleaved to AMP in the process and the pyrophosphate drives the reaction

o Two methionine tRNA’s one for initiation and the other for internal incorporation

• The Ribosome

o Large subunit collective-60 and small collective 40; Two subunits large 28s(in eukar) and small 18s (in eukar) (rRNA sizes)

o Large subunit contains 5s and 5.8s (in eukar) and 5s in prokar

• Initiation

o Involves the assembly of the ribosomal subunits w/ a mRNA and an initiation tRNA w/GTP

o Small subunit binds mRNA

o Large subunit has two binding sites P and A, the initiation tRNA is the only on that can bind to the P site directly without binding the A site first

• Elongation and Termination

o After initiation a new tRNA comes up w/ an elongation factor and binds to the A site, where peptidyl transferase mediates the bonding of the amino acids (no GTP)

o Translocation occurs via an elongation factor and another GTP

o Polysomes occur when several ribosomes are reading the same message

o Protein synthesis terminates when stop codons are encountered and termination factors bind to the A site because there are no tRNA’s for stop codons

o It is then released when there is no group to bind to the P site facilitated by GTP

• Wobble pairing

o Shine-Dalgarno sequence facilitates the recognition of the appropriate start codon

o In codons the first two bases are restricted to Watson – Crick rules but the third is more loosely regulated

▪ Table 1 on V-44

▪ Wobble bases are the first ones in the anticodons and the third in codon w/ respect to 5’-3’ reading

o Table 2 components required for the four major stages of protein synthesis (V-48)

o Gene Regulation

▪ Transcriptional control (most important) – control of initiation rate, elongation or termination of transcription by RNA polymerase

▪ Translational control (post transcriptional event) – mRNA stability, protein stability

▪ Major point of control for bacteria is at the transcriptional level

• Protein synthesis can be regulated rapidly if transcription is turned off

• Bacterial genes are regulated through attenuation

o E.g. Feedback inhibition

▪ Lac Operon

• When glucose levels are low but the lactose levels are high the enzymes produced by this operon are increased 1000 fold

• One of the enzymes is beta galactosidase which splits glucose and galactose

• The actual inducer of the operon is not lactose but allolactose an isomer of lactose

• The operon contains 3 structural genes LacZ,Y, and A and these encode for galactosidase, lactose permease, and transacetylase

• Upstream is the promoter (where RNA polymerase is bound initially) and the Operator, and an inducer{i.e. lactose} (regulatory gene)

• Operator sequence shows dyad symmetry

• Two mutant classes

o Constitutive producer – produces the enzymes under all conditions

▪ The repressor gene which is a trans-acting protein that represses genes Z,Y, and A in normal genes is mutated

▪ Cis Acting protein that physically links to genes known as the operator, is mutated and the repressor is unable to bind to it and cut it off

o Permanently repressed – not inducible under any circumstances

▪ Catabolite repression – high levels of glucose

▪ If cAMP is strongly impaired no Lac Operon

▪ Catabolite Activator Protein (CAP) is absent can’t bind to camp no Lac Operon

▪ Glu up – cAMP low – CRP bind low – Lac low

o Eukaryotic Gene Regulation

▪ No more than 1% of the DNA in the human genome actually comprises the protein coding component

▪ Typical mammalian cell mRNA populations consist of 3 abundance classes

• Rare, intermediate, and highly abundant

▪ Those regulatory molecules whose role is quickly fulfilled and whose presence would be counterproductive tend to have the sequence AU3A in a number of copies

▪ Peptide and steroid hormones have been known to enhance the stability of certain messages

▪ Processing of mRNA

• The use of alternate splice donor and or splice acceptor sites to give rise to alternate mRNA molecules is well documented as a regulated process

▪ Alternative splicing and polyadenylation defects in beta thalassemia

• Most common form in Mediterranean region changes a G to an A resulting in an AG sequence further upstream than normal

• The polyadenylation effect occurs when the AATAA sequence is changed to AACAA and cleavage and poly A additions are impaired

▪ When the polymerase pauses in transcribing the trascription is stopped and these are known as paused polymerases

▪ Cis-regulatory and trans-acting factors in initiation

• Most eukaryotic polymerase II genes possess a TATA box ~30bp upstream, this is bound by TATA binding protein, and w/o this binding RNA polymerase is unable to identify the promoter

• The TATA box is the basal promoter for most eukaryotic genes – without it transcription loses its accuracy and efficiency

o The TATA box needs help from other DNA sequences w/in 100-200bp upstream – known as promoter proximal or upstream promoter elements – cis acting

o There are also several enhancers located both up and down stream

▪ These enhancer and promoter proximal regions bind to transcription factors (proteins) and when mutation occurs binding of these factors can be seriously affected

• Many transcription factors bind to there recognition sequences as dimers because the sequences have internal dyad symmetry

• Some factors can act as both enhancers and inhibitors

• The precise level of transcription in a given case is likely to be a combinatorial function of activating vs. repressing effects

o Regulatory elements interact with the RNA polymerase and each other by establishing protein protein contacts

▪ Most involve two active sites one to bind DNA and another to play a role in activation of transcription via protein protein contacts

▪ Adaptor proteins bind to other proteins to facilitate signal transmission from one DNA bound protein to another via protein protein interactions

o A looping phenomenon is the only way to explain the ability of distant of distant enhancers to have an effect

o The helix-turn-helix motif is thought to be how proteins recognize certain sequences

▪ Homeodomain makes use of this motif

o The zinc finger is used to interact with the major groove along a considerable distance of the DNA

o Two more of these are the bZip and the helix-loop-helix

o In every case of beta thalassemia known there are sequence mutations in the promoter proximal region – known as non-deletional HPFH (hereditary persistence of fetal hemoglobin)

Dr. Lieberman

Glycolysis – is the major pathway for the conversion of carbohydrates into energy

- Sugar Structures

o Glucose – six carbon sugar, if the orientation of the hydrogen and hydroxyl group around any of the carbons is altered then an isomer of glucose will form (two of which are mannose and galactose)

o Orientation around C-5 determines the L or D character in comparison to glyceraldehydes

- The pathway of Glycolysis

o Fully illustratedon (VII-10)

o Step 1

▪ Phosphorylation of glucose by hexokinase (muscle) or glucokinase (liver) with a group translocation facilitated by ATP hydrolysis to form Glucose-6-phosphate

• Hexokinase has a higher affininty for glucose than glucokinase; they are isozymes

o Hexokinase undergoes induced fit catalysis

• This reaction traps the glucose within the cell (phosphorylated compounds are membrane impermeable) and is the first irreversible reaction

o Regulation of this fr=irst irreversible step

▪ When PFK-1 is inhibited, F^P builds up, which means that G6P builds up (since they are at equilibrium)

▪ High levels of G6P inhibit hexokinase inn the muscle

▪ At low glucose levels glucokinase is virtually inactive

o Step 2

▪ Glucose-6-phosphate is isomerized to fructose-6-phosphate by the enzyme phosphohexoisomerase (glucose-6-phosphate isomerase)

▪ Aldose to ketose reaction

o Step 3

▪ Fructose-6-phosphate is now phosphorylated at the one position to generate fructose 1,6 bisphosphate, requiring ATP catalyzed by phosphofructokinase 1 and ios the major regulated step of glycolysis

▪ Irreversible and commits the sugar to being metabolized

• Regulation of this major regulatory step

o Committed through allosteric-modification through non-substrate molecules and –Covalent modification – modifying an enzymes activity by adding a group, via a covalent bond, to an amino acid residue of the protein (PFK-1 is an allosteric)

o Two major inhibitors of PFK-1 are ATP (signifying high energy) and citrate(indicative of an adequate supply of carbon skeletons)

▪ In the presence of an inhibitor 50% max value is reached at a higher substrate concentration

o Two major activators of PFK-1 are AMP (signifying low energy levels) and Fructose 2,6 bisphosphate

▪ In the presence of an activator 50% max value is reached at a lower concentration

o Regulation of fructose 2,6 bisphosphate synthesis

▪ F2,6BP is made from F6P and ATP by the enzyme PFK-2 and F26BP is broke down by F26bisphophatase

• PFK-2activity is activated by F6P, such that when F6P levels are high PFK-2 is active, and the phosphatase is inactive, so as F6P builds up PFK-2 is activated, F26BP is produced and PFK-1 is activatedand glycolysis proceeds

• When PFK-2 is phosphorylated its activity is reduced and phosphatase activity is enhanced slowing down the glycolytic pathway

o Kinase that phosphorylates PFK-2 is cAMP dependent protein kinase

▪ When glucose levels are low glucagons is secreted and activates adenylate cyclase which raises cAMP levels, which activates cAMP dependent protein kinase, which phosphorylates PFK-2, which inactivates PFK-2 kinase activity and activates PFK-2 phosphatase activity, which reduces the levels of F26BP, whichreduces activation of PFK-1, which lower glycolytic rate

▪ Muscle form of PFK-2 cannot be phosphorylated and therefore is not subject to regulation

▪ The cAMP dependent kinase consists of two distinct subunits, R(regulatory) and C(catalytic); R binds cAMP and the C phosphorylates a wide variety of proteins

o Step 4

▪ The splitting of F1,6BP into three carbon units by the enzyme aldolase forming dihydroxyacetone phosphate and glyceraldehydes-3-phosphate

▪ The enzyme requires an active amino group – lysine – which will form a covalent bond with the substrate

o Step 5

▪ Next rxn isomerizes DHAP to form G3P forming two molecules of G3P from one glucose molecule catalyzed by triose-phosphate isomerase

o Step 6

▪ Next is the oxidation of G3P to form 1,3 bisphosphoglycerate using the enzyme glyceraldehydes-3-phosphate dehydrogenase

▪ Redox rxn using the cofactor NAD+

▪ An active cysteine residue is required for the enzyme to have activity

▪ NAD & NADH used in catabolism; NADP & NADPH used in anabolism

o Step 7

▪ Next is the dephosphorylation of 1,3 BPG forming ATP and 3-phosphoglycerate catalyzed by phosphoglycerate kinase

▪ Group transfer rxn

o Step 8

▪ Next step converts 3-PG to 2-PG catalyzed by phosphoglyceromutase

o Step 9

▪ Next is the dehydration of 2-PG to form phosphoenolpyruvate catalyzed be enolase

o Step 10

▪ Final step PEP is converte4d to Pyruvate by pyruvate kinase

▪ Generates an ATP

▪ Irreversible

• Regulation of this third major step

o Catalyzed by pyruvate kinase, which exists in L(liver), M(muscle), and A(other)

o The L-form is activated by PEP and F16BP, whereas ATP and alanine inhibit (alanine can be converted to pyruvate in one step); it is also phosphorylated by cAMP dependent protein kinase

o The M-form is not subject to inhibition by phosphorylation

o When blood glucose levels are low hormones are released that activate the cAMP dependent protein kinase cascade system; kinase will phosphorylate liver PFK-2 and liver PK thereby inactivating them and slowing glycolysis

o Activation of the cAMP dependent kinase will signal the liver to begin gluconeogenesis

o Insulin signals the muscle that blood glucose levels are high

o The fate of pyruvate

▪ Can be used for the biosynthesis of various AA’s or if energy is required it can be oxidized to CO2 and HOH via the TCA cycle

▪ If O2 not available pyruvate will generate lactate

• Lactate formation occurs primarily in actively exercising muscle in order to regenerate NAD+ for glycolysis

o Cori Cycle

o RBC’s are a major source of lactate—in order to maintain glycolysis lactate is produced in orderto regenerate NAD+

o Lactate formation is a metabolic dead-end

o Enzyme Kinetics

▪ Competitive Inhibition

• Inhibitor binds to the same active site as the substrate

• V-max (y-intercept) is not altered but the Km (x-intercept) is altered by the presence of the inhibitor

▪ Non-competitive Inhibition

• Inhibitor binds to an alternate site on the enzyme changing its conformation

• Km-remains the same but the V-max is decreased

▪ Uncompetitive inhibition

• Combination of both types of inhibition in that there is a partial inhibition of substrate binding, as well as distinct effect on velocity

▪ Michaelis – Menten Equation

• VII-26

▪ V-max – how fast an enzyme works ; Km – substrate concentration at which the rxn is proceeding at half its maximal velocity

▪ The Hill Coefficient can be used for the kinetics of allosteric enzymes

• For enzymes which do not display cooperativity they have a hill coefficient of 1 (hemoglobin)

o Fructose and Galactose

▪ Fructose metabolism

• Phosphorylation of fructose by fructokinase to form fructose-1-phosphate

• The F1Pis the substrate of F1Paldolase to split F1P to DHAP and glyceraldehydes using the enzyme triose kinase burning an ATP

• Same ATP generation as glycolysis

▪ IV fructose load

• Rapid depletion of phosphate and energy supplies

o Fructokinase very active,fructose transport into liver is very fast; ATP is hydrolyzed to ADP

• Elevated levels of uric acid

o End product of purine metabolism

▪ Drop in phosphate, increase in AMP trigger purine degredation

▪ Gout – is the overproduction of uric acid

▪ Lactic Acidosis

•

▪ Hereditary Fructose Metabloism

• Defect in F1P aldolase

o Aldolase B in liver and kidney,A and C in other tissues

o F1P accumulates due to speed of fructokinase

• Hypoglycemia results from increase in F1P

o Glycogen degredation inhibited – F1P blocks glycogen phosphorylase

o Gluconeogenesis impaired

▪ Galactose Metabolism

• Phosphorylation of galactose by galactokinase yielding galactose-1-phosphate and ADP

• A transfer rxn occurs in which the UDP of UDP-glucose is transferred to make UDP-galactose and glucose-1-phosphate

• The G1P is quickly converted to Glucose 6 phosphate

o Digestion and absorption of Carbohydrates

▪ Amylase (Glu a1,4);amylopectin (amylase w/ Glu a1,6); sucrose (glu a1,2 fru); Lactose (gal b1,4 glu)

▪ Digestion of Starch

• Salivary a-amylase

o Hydrolyzes internal a1,4 bonds btw glu in amylase and amylopectin

o Endoglycosidase – cleave w/in a chain

o Exoglycosidase – requires an end of a carb to start

o Creates dextrins – oligosacchrides

• Pancreatic a amylase

o Converts carbs to 2,3,4,and 5 member glu chains

• Small Intestine

o Sucrase-isomaltase complex

▪ Hydrolyze maltose(2glu a1,4); sucrose (glu,fru), isomaltose (2glu a1,6);

• Sucrase can only hydrolyze sucrose

• Both subunits can do a1,4 glucosyl bonds

• Only isomaltase can do a1,6 glucosyl bonds

o Glucoamylase complex

▪ Two subunits both of which are exoglycosidases

▪ Hydrolyze a1,4 between glu residues in oligosacchrides

o B-glycosidase complex – Lactase

▪ Gal B1,4 glu bonds in lactose

▪ Deficiencies in lactase lead to lactose intolerance

o Trehalase

▪ Does trehalose (glu a1,1 glu) bond

• Sugars are absorbed through the intestinal epithelial cells through facilitative and Na dependent transporters

o Na dependent couples this action with the intake of sugar

o Once inside the intestinal epi cells the sugars are transported out the other side of the cell into the capillary beds via facilitative transporters

• Dietary fiber consists of polysacchrides that cannot be digested by man

o Cellulose – B1,4 bonds

o Beans - gal a1,6 bonds

o Glycolysis and Diabetes

▪ Type I is caused by lack of insulin

• This alters the transport of glucose into many tissues, but not the liver

• No insulin glucagons is not balanced and the cAMP dependent protein kinase is activated;in liover,activation of this results in phosphorylation of PFK-2 and PK, inhibiting both and shutting off glycolyisis and cutting on gluconeogenesis

• Unfortunately the other tid=ssues need insulin to bring glucose into the cell so even though there is plenty around the cells can’t tell because no insulin

o Gluconeogenesis – livers production of glucose to export

▪ This process requires that the three irreversible rxn’s of glycolysis be bypassed; it takes four rxn’s to bypass the three

• Two rxn’s are required to convert Pyruvate to PEP with pyruvate carboxylase (Pyruvate+CO2+ATP---oxaloacetate+Pi) and PEP Carboxylase (oxaloacetate+GTP---PEP+GDP+CO2) 1st rxn requires biotin

• Once PEP is formed it goes through the rest of the rxn’s until gets to F16BP where you need F16 bisphosphatase to convert it to F6P

• It then moves on to be acted on by G6phosphatase to form glucose

▪ Regulation

• Reciprocal regulation of PFK-1 and the F16BP, such that low energy (AMP) activates PFK1 and inhibits phosphatase

• Compounds that indicate high energy (ATP and Citrate) inhibit PFK1 and activate phosphatase and activate F16BP

• F26BP is the major activator of PFK1 inhibits F16bisphosphatase

• Pyruvate carboxylase is regulated reciprocally with pyruvate dehydrogenase

o How Insulin and Glucagon alter metabolism

▪ They exert their effects extracellularly

• Interact through signal transduction pathways, which a process of converting an extracellular signal to intracellular metabolic changes

o E.g. cAMP, phosphorylation

o Scatchard derivation analyzes ligand protein bindind equation on VII-40

▪ Curvilinear plot indicates that it is becoming more difficult for insulin to bind after one insulin molecule has bound – negative cooperativity

o Insulin receptor consists of 2 alpha and 2 beta subunits

▪ Alpha contains the insulin binding site

▪ Beta contains an insulin stimulated kinase activity

▪ Receptor precursor is 1370 AAs long and the first 27 contain a sequence that tells the protein synthesis machinery that this is a protein

▪ After the 27 is a cleavage site identified by R-K-R-R

▪ Beta subunit contains the transmembrane protein

▪ Alpha is entirely extracellular

• Insulin binding initiates kinase activity in beta, which phosphorylates tyrosine, which then autophosphorylates at least five tyrosine residues on the beta

• Leads to the activation of cAMP phosphodiesterase which cleaves cAMP into AMP lowering its levels and the levels of cAMP dep pro kin which brings about a cange in metabolic pathways

▪ Major substrate for the insulin receptor is the insulin receptor substrate-1/pp185(IRS-1)

o Glucagons and epinephrine are mediated through the use of a G-protein

▪ G-protein is heterotrimeric w/ alpha,beta,gamma

▪ Upon interaction alpha dissociates and binds GTP, which replaces GDP, thus activating the protein

▪ It then interacts with its target adenylate cyclase and activates it, produces more cAMP, and activation cAMP dep pro kin

▪ Upon binding the GTPase activity of the beta subunit begins and ensures that the protein will shut-off

▪ Cholera toxin – is an enzyme which transfers an ADP-ribose to G and inactivates the GTPase activity

▪ Pertussin toxin – ADP ribosylated subunit is unable to exchange GDP for GTP and the subunit is never activated

• Non insulin dependent diabetes mellitus(NIDDM)

o One type is when the patient does not allow the receptor to be processed properly, such that it does not reach the cell surface

o Another is the tyrosine kinase activity is reduced by about 90%

▪ Mutated insulin receptor is usually present at birth

o Aconthosis nigrecans – the patient develops antibodies to either insulin or their own receptor

o Through over –eating

- TCA Cycle

o Major function is to act as the final common pathway for the oxidation of carbohydrates, lipids, and proteins

o Major role in gluconeogenesis, transamination, deamination, and lipogenesis

o Oxidizes acetyl CoA to CO2 and H2O

▪ Acetyl coa is synthesized exclusively in the mitochondria – it does not have a transporter in the inner mitochondrial membrane

o When ATP and NADH levels are high the cycle slows down and when they are reduced the cycle speeds up

o Occurs totally within the mitochondrial matrix

o Produces reducing equivalents for oxidative phospho NADH and FADH2

o Mitochondria

▪ The protein porin makes the outer membrane very permeable but the inner membrane is impermeable to almost all proteins

▪ Contains several transport proteins

o TCA cycle intermediates can be used to synthesize amino acids

o Step 1

▪ Citrate synthase catalyzing the condensation of an oxaloacetate and the acetyl to give citrate

▪ First committed step – due to loss of high energy thioester bond

▪ Aldol condensation – coenzyme A is released from the product while it is still bound to the enzyme

o Step 2

▪ Designed to rearrange citrate to its isomer, Isocitrate

▪ Two step process both catalyzed by aconitase

• -OH on #3 carbon is removed and put in the #4 position making I more oxidizable

▪ aconitase treats citrate as an asymmetrical molecule by requiring it to bind three distinct sites

• catalyzes the attainment of euqilibrium between citrate, isocitrate, and cis-aconitase 90:6:4

• under cellular conditions all the rxns of the TCA cycle have a negative delta G except aconitase

• it moves on because because the isocitrate dehydrogenase rxn is extremely favorable continually reducing mitochondrial concentrations and creating a need for this aconitase rxn

o Step 3

▪ Isocitrate dehydrogenase oxidizes isocitrate to alpha-ketoglutarate

▪ First oxidation coupled to the production of CO2 and NADH

▪ Step captures 2 of the 8 available electrons

▪ Isocitrate dehydrogenase catalyzes the irreversible oxidation and decarboxylation of isocitrate yielding the first of three NADH molecules and first release of CO2

• Rate-limiting step and is a two step process with an oxidation and then a beta decarboxylation(CO2 releaser)

o Step 4

▪ Alpha –ketoglutarate dehydrogenase catalyzes an oxidative decarboxylation rxn which converts alpha ketoglutarate to succinyl-coa

▪ Involves the reduction of a second NAD to NADH and the production of CO2

▪ With the production of the CO2 the net loss of carbons donated by acetyl is complete

• These are not the carbons of the acetyl but those of the oxaloacetate

▪ Captures 2 of the 8 electrons

▪ Generates a high energy thioester bond

▪ Enzyme complex is very similar to the pyruvate dehydrogenase complex

o Step 5

▪ Succinyl coa synthetase converts succinyl coa to succinate generating GTP

▪ High energy bond is hydrolyzed

▪ Rxn is reversible

▪ Example of substrate level phosphorylation

▪ Only rxn yielding a high energy phosphate bond

▪ Succinyl-CoA is used in the synthesis of heme

o Step 6

▪ Succinate dehydrogenase(flavoprotein) converts succinate into fumarate

▪ Produces FADH2 and captures 2 of the 8 electrons

▪ Succinate dehydrogenase is the sole enzyme of the cycle that is embedded in the inner membrane of the mitochondria, where it feeds electrons into the electron transport system

▪ Strong oxidizing agent needed to insert the double bond into the saturated hydrocarbon in the form of FAD(always the electron acceptor in oxidations that remove two hydrogen atoms from a substrate

o Step 7

▪ Fumarase converts fumarate into malate by addition of H2O across the double bond

o Step 8

▪ Malate is converted to oxaloacetate by malate dehydrogenase

▪ Involves a substantial increase in free energy – unfavorable rxn

▪ Concentration of oxaloacetate is a limiting factor in the rate at which the TCA cycl can catalyze its rxn’s

▪ Will proceed because the intramitochondrial levels of oxaloacetate are extremely low because of the citrate synthase rxn

o Carbons derived from acetate can be ambiguously determined up to the point at which succinate is formed

o Free energy associated with a rxn : delta G = delta G-0’ + RT

o 10 high energy bonds are generated for every turn of the cycle

o NADH=2.5 high energy phos FADH2=1.5 high energy phos

o Acetyl CoA

▪ Obtained either by oxidative decarboxylation of pyruvate(glycolysis) or by oxidative cleavage of fatty acids

▪ TCA cycle is initiated by the addition of two carbons(acety-coa) and as the cycle proceeds two carbons are lost as CO2

▪ Pyruvate is brought into the matrix via a Symport with a proton where it undergoes decarboxylation to form acetyl-coa catalyzed by pyruvate dehydrogenase

▪ Conversion of pyruvate to acetyl coa occurs in six steps

• Step 1

o Pyruvate decarboxylase contains a thiamine pyrophosphate which assists in breaking a carbon-carbon bond

• Step 2

o Hydroxyethyl group is transferred to one of the two sulfur atoms on a lipoamide side arm of dihydrolipoyl transacetylase

o Redox rxn

o The disulfide lipoamide derivative is reduced, to form one free sulfhydryl group

o Lipoic acid is covalently linked to a lysine side chain of dihydrolipoyl transacetylase

• Step 3

o Acetyl group is transferred to a second lipoamide side arm covalently linked to dihydrolipoyl transacetylase

• Step 4

o Transfer of the acetyl group to coenzyme A is catalyzed by dihydrolipoyl transacetylase

o Formation of a new thioester bond

• Step 5

o Dihydroxylipoyl dehydrogenase catalyzes the oxidation of the reduced lipoic acid back to the oxidized form generating FADH2

• Step 6

o Is the oxidation of the reduced bound flavin by NAD+

o NADH is produced and FADH2 is converted to FAD

• Pyruvate + NAD + CoA --- acetyl-CoA + NADH + CO2 + H

o Free energy change ~ -8 kcal/mole

o Regulation of the TCA cycle

▪ Pyruvate dehydrogenase

• Conversion of pyruvate to acetyl-coa is a key irreversible step because animals are not able to convert acetyl-coa into glucose

• Activity of the pyruvate dehydrogenase complex is stringently controlled at three levels – inhinition by products, feedback regulation by nucleotides, and regulation by reversible phosphorylation

o Inhibition by end products

▪ Coenzyme A and acetyl coa compete for the same site on dihydrolipoyl transacetylase high acetyl coa levels inhibit PDH activity and high CoA levels reverse the inhibition – regulated reciprocally from pyruvate carboxylase

▪ NAD and NADH also compete for the same site on the dihydrolipoyl dehydrogenase of PDH

▪ High NADH levels reverse this inhibition

o Feedback regulation by nucleotides

▪ Pyruvate decarboxlase of PDH is allosterically inhibited by ATP and activated by AMP

▪ Energy levels are high the enzyme is inactive and when energy is required the enzyme is activated

o Regulation of PDH by reversible phosphorylation

o Kinase inactivates PDH by phosphorylating the pyruvatedehydrogenase component

o High levels of end-products acetyl-CoA, NADH and ATP all activate the kinase

o High energy levels inhibit activity whereas low levels will activate

• Regulation of Isocitrate Dehydrogenase

o Stimulated by ADP, Ca ions; inhibited by NADH and ATP

o When energy levels are high isocitrate dehydrogenase is inhibited isocitrate builds up and citrate begins to be formed increasing its cytosolic concentration which allows for the eventual production of fat and cholesterol through acetyl-coa

• Regulation of alpha ketoglutarate dehydrogenase

o Branch point metabolite because it can be transaminated to form glutamate

▪ Glutamate is needed in protein synthesis directly

o NADH inhibits, ADP, and Ca ions activate

• Regulation of oxaloacetate

o Rate limiting and is regulated through the use of anaplerotic reactions

▪ Anaplerotic Reactions

• The reactions and pathways that replenish intermediates in the TCA cycle

o Pyruvate carboxylase contains biotin and is activated by acetyl coa and is used to generate OAA for gluconeogenesis

o Production of malate from carboxylation of pyruvate catalyzed by malic enzyme

o Transaminations will convert alpha keto acids to their corresponding amino acids

▪ Pyruvate – alanine

▪ Alpha ketoglutarate – glutamate

▪ OAA – aspartate

o High energy levels in the form of ATP or NADH tend to inhibit the four important regulated steps (PDH, citrate synthase, isocitrate DH, alpha KG DH) and when energy levels are low they are activated

Oxidative Phosphorylation

- Redox thermodynamics

o Electrons are accepted from NADH and delivered to oxygen through a series of carefully controlled energy transduction steps which culminate in the formation of 1.5FADH2 to 2.5NADH ATPs per pair of electrons

o Nerst Equation -- Delta G0 = (-n)(F)(deltaE0) (E=redox potential)

o A redox pair in which the electron donor is a stronger donor than hydrogen will have a negative redox potential and vice versa

o NADH is the first electron donr – the energy released during the oxidation of NADH is theoretically enough to drive the formation of 4 ATPs

- Vectorial Metabolism

o As electrons are passed from carrier to carrier they move from one side of the inner mitochondrial membrane to the other so that the passage of electrons is oriented in space as well as time

o Components of the Electron Transport Chain

▪ Complex I and Coenzyme Q

• Also called NADH-CoQ Reductase passes electrons from NADH to coenzyme Q, it is the largest of the complexes and contains one FMN; also contains 6-7 iron-sulfur clusters that participate in the process of passing along the pair of electrons donated by NADH

• Accepts two electrons from NADH and can transfer 2 electrons to CoQ

• For every 2electrons there are 4 proton extruded

▪ Complex III (AKA CoQ-cytochrome c reductase)

• Passes electrons from CoQ to cytochrome c

• Contains two b-cytochromes(562 & 566) and one cytochrome c1, and an iron sulfur cluster

o Cytochromes heme groups that alternate between Fe+2 and Fe+3

• Cytochrome c1 can only carry one electron at a time so a reduced CoQH accepts one of the two electrons to form CoQH2

• Fully reduced CoQ then donates one of its electrons to cytob562 and then the resultant CoQH then donates its electron to cytochrome c1, which transfers that electron to cytochrome c for transfer to complex IV; the electron on 562 is transferred to 566 which gives it back to CoQ

▪ Complex IV

• Contains 2 copper containing cytochromes a and a3, which are collectively called cytochrome oxidase and alternate between their Cu1 and Cu2 states

• Electron received from cytochrome c is delivered across the membrane to oxygen, it takes four to fully reduce oxygen

▪ Complex II (AKA succinate-CoQ reductase)

• Serves as an alternate point of entry for electrons into the electron transfer chain, most importantly those derived from FAD of succinate dehydrogenase

▪ Proton Motive Force

• Each time an electron is carried across the inner membrane a proton is also transported in the same direction and extruded from the matrix and placed in the space between the membranes creating an electrochemical gradient

• FMNH2 does this in complex I and CoQH2 does this in complex III

• Creates both a charge and pH difference across the membrane

▪ ATP synthase

• Consists of a multi subunit proteolipid called Fo, which traverses the inner membrane and functions as a channel to allow protons to move in the matrix from outside and a water soluble peripheral membrane protein complex called F1(5 different subunits), which functions as a catalytic site for the synthesis of ATP

o Oligomycin plugs up the core of the Fo subunit and acts to inhibit ATP production

o Movement of two protons through the synthase can drive the formation of one ATP from ADP

▪ ADP binds to an alpha beta dimer on the F1 portin of the enzyme, reacts with inorganic phosphate and atp is formed – the dimer then rotates and another binds ADP (there are 3 dimers in the complex) unless there is an energy transfer, provided by proton flow, to the dimer ATP will be stuck

▪ ATP synthesis is not possible unless ADP is present

o Phosphate:Oxygen ratios from mitochondrial electron carriers

▪ Complex I ejects 4 protons; II ejects 2; and IV ejects 4

▪ ATP synthase requires 4 protons enter to synthesize one ATP

• For every NADH 10 protons are ejected

• Rotenone inhibits respiration if NADH is used because it inhibits the transfer of electrons from complex I to CoQ

• If Antimycin A inhibits complex III and Cyanide inhibits complex IV this would block both NADH and FADH2 respiration

▪ P:O ratios for cytosolic NADH and Shuttle reactions

• Glycerol-phosphate shuttle

o Cytosolic glycerol-3-phosphate dehydrogenase reduces DHAP to form G3P and the electrons used to reduce DHAP are derived from cytosolic NADH which s produced in glycolysis – the G3P formed can be oxidized by mitochondrial G3P dehydrogenase—this enzyme will catalyze he conversion of the newly synthesized G3P back into DHAP, and the enzyme bound FAD is reduced to FADH2 and the membrane bound FADH2 can transfer its electrons directly to CoQ one electron at a time

o This results in 1.5 ATPs from FADH2 from the NADH

• Malate-aspartate shuttle

o Cytosolic NADH transfers its electrons to cytoplasmic oxaloacetate, to generate malate – malate is transported across the mito membrane in exchange for alpha-ketoglutarate – malate in the matrix is then oxidized to OAA, which is then transaminated to form aspartic acid and alpha ketoglutarate; aspartate then leaves the mitochondria reacts with alpha ketoglutarate in a transamination to form glutamate – glutamate formed can be used to exchange for another aspartate in the mitochondria and the cycle can start anew

o Which results in 2.5 ATPs from NADH

▪ Coupling of Oxidation to Phosphorylation

• Transfer of electrons through the electron transport chain will continue as long as there is sufficient energy to extrude protons from the matrix of the mitochondria

• Addition of oligomycin which blocks the transport of H+ not only blocks the production of ATP but also leads to cessation of oxygen consumption

• This is called tight coupling

o Can be destroyed by allowing the protons to reenter the matrix via a pathway other than the ATPase

o Uncouplers are small molecules which can diffuse across the mitochondrial membrane and carry protons from outside the mitochondria into the mitochondria (e.g. dinitrophenol)

o By allowing protons to re-enter and by-pass the ATP syntase oxidation increases and ATP synthesis decreases to zero

o Figure 12 (IX-16)

▪ ADP/ATP exchange and energy charge

• Rate at which ATP is produced and hence at which O2 is consumed is limited by the availability ADP

• In the inner mitochondrial membrane there is a ATP/ADP Antiport

• Energy charge is defined as the ratio of the number of high energy phosphate bonds to the total number of adenosine nucleotides

• Phosphate is also required and it as an antiporter with hydroxyl ions as well as a phosphate/dicarboxylic acid exchange protein, which will export phosphate and bring in succinate

▪ Non-shivering thermogenesis

• Thermogenin – contained in brown fat, and facilitate an alternate route for the return of protons from the cytoplasm to the matrix – in this way ATP synthase can be bypassed and the energy can be dissipated as heat

o Glycolysis – 5 or 7 depending on the shuttle used (glycerol or malate)

o 25 ATP from TCA including Oxi phospho

o total of 30 or 32

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download