Biochemistry 1 Midterm 1 Content PDF

BIOCHEMISTRY 1 Content for midterm 1  TABLE OF CONTENTS Cell membrane………………………………………………………... 2 Transport across the membrane………………………………………. 4 Amino acids, proteins………………………………………………… 5 Structure proteins……………………………………………………... 7 Enzymes: proteins with catalytic power………………...

BIOCHEMISTRY 1 Content for midterm 1  TABLE OF CONTENTS Cell membrane………………………………………………………... 2 Transport across the membrane………………………………………. 4 Amino acids, proteins………………………………………………… 5 Structure proteins……………………………………………………... 7 Enzymes: proteins with catalytic power……………………………… 9 DNA and RNA molecules…………………………………………… 13 Transcription………………………………………………………….. 17 Translation…………………………………………………………….. 21 1  CELL MEMBRANES - Cells WITHOUT nucleus = prokaryotes (ex: bacteria, pathogens) - Cells WITH nucleus = eukaryotes (animals, plants, fungi) Eukaryotes Plasma membrane separates cell and environment. Internal membrane separates subcellular organelles of the cytoplasm. Components: - Lipids (bilayer: cholesterol and simples + composed lipids) - Proteins - Carbohydrates Lipids: - Phospholipids ➔ Major component of the cell membrane. ➔ The head is hydrophilic whole is: ➔ The tail is hydrophobic Amphiphilic Glycerolphosphatide: backbone of the phospholipid. o Give the structure of lecithin, cephalin and phosphatidic acid. Sphingolipids o Ceramide: sphingosine + 1 fatty acid o Sphingomyelin: ceramide + phosphate + choline o Cerebroside: ceramide + 1 sugar o Ganglioside: ceramide + Several sugars - Saturated fatty acid (FA): no double bond. - Monounsaturated: one double bond - Polyunsaturated: > 1 double bond Cholesterol o Provides rigidity of the cell-wall and contributes to atherosclerosis Movement of lipids in the membrane: - Rotation - Lateral diffusion - Transversal diffusion (flippase/floppase or scamblase) Proteins : - 60% of the membrane 2  - 50% of the dry weight - They have specific structure and function, composed of 20 amino-acids (AA) polymers. o Integral proteins = in the membrane. Have a hydrophobic and hydrophilic part. Function dynamic. o Peripheral proteins = on the membrane. Loosely bound, function static. Carbohydrates: - Branched oligosaccharides 50 = protein - Secondary: o α-helix (backbone coils). 3,6 AA/turn. α-keratin = hair, hoof, nails (24% of Cys) o β-sheet (backbone extended). Chains can be antiparallel or parallel. β-keratin = feather, beak, scale of reptiles and birds. Pleated sheet = cocoon, spider web. o Hydrogen bonds intra and inter-chains. o Disulfide bond between SH (Cys) o Ionic bonds: between COOH group of acidic-AA and NH2 group of basic-AA. o Dipol-dipol interactions o Van der Waals forces (hydrophobic), very weak. Between non-polar molecules. Gives a temporary partial negative charge. - Tertiary: o 3D structure with polar and apolar bonds. o Folding under temperature and pH conditions, active state. 5  Has a native conformation. o Defolding = denaturation = protein desactivation o Denaturation can be reversible (=> renaturation) or irreversible. o Denaturating agents: o ▪ Reducing-oxidizing agents (permanent waves) ▪ Hydrogen-bonding solvents (70% ethanol) ▪ Heavy metal ions (AgNO3) ▪ Salting-out ▪ Sodium duodecylsulphate, mercaptoethanol used in gel electrophoresis. - Quaternary: o Assembling of the tertiary structures into subunits. o Examples: haemoglobin made of 4 subunits, insulin made of inactive hexamer. Classification of proteins: - Globular o Spherical shape o Albumin (ovalbumin of the egg) / Globulin (γ-globulin in blood) o Enzymes, hormones o Water soluble. - Fibrous o Sheet-like structures o Collagen (skin, bone, tendon) /Elastin (arteries) /Keratin o Water insoluble - Other types, intermediate proteins o Rod-like structure o Myosin (muscle) / Fibrinogen (blood clotting) o Water soluble - Simple proteins: only AA - Conjugated proteins: simple protein + non-protein compound. Can be : o Glycoprotein (Na+/K+ pump) o Nucleoprotein (ribosomes, viruses) o Phosphoprotein (casein in milk) o Chromoprotein (haemoglobin) o Hemoproteins (cytochrom C and haemoglobin) o Flavoproteins (FMN, FAD) Protein functions : - Contractile (actin, myosin) - Transport (ion channels) - Enzymes 6  STRUCTURE PROTEINS - Intracellular = keratin - Extracellular = collagen / elastin / fibronectin, laminin, proteoglycans. KERATIN Wool structure - First: α-keratin - 3 α-helix => protofibril => microfibril => macrofibril - S-S bonds, H-bonds and ionic interactions. Keratinocytes: Keratin-containing cell. Epithelial cells: 30% of keratin. When the cell dies: 90%. Role: surrounds and hold the nucleus by forming a cytoskeleton. - Synthesis: pre-keratin in the not-yet-keratinized cells. From the stratum basale to the stratum corneum = stratification. - Degradation: cannot be degraded. Thioglycolates breaks S-S bonds. Moth larvae produce keratinase. COLLAGEN - ¼ of all protein in body - Triple helix: tropocollagen (¼ shifted, make cross-striations)(covalent cross links, disulfide and H-bond) => microfibrils => macrofibrils => fibers - Gly, Pro, OH-Gly and OH-Pro are very prominent! -OH groups blocks the chain in the conformation. - Types: o Collagen-I: biggest amount o Collagen-II-IV: big amount o Collagen-V-XII: small amount (tissue specific) o Today XXVIII!! - Similar AA composition, micro and macro structures. Synthesis: in the cytoplasm of fibroblasts. Transcription classic, but translation create preprocollagen. Every 3rd AA: Glycine. Then cut off the C-term and N-term by procollagen peptidase => tropocollagen. Cross-links are made by Lys oxidase => Type I collagen. Degradation: Collagenase: microbial or tissue collagenase. ELASTIN - Gly, Pro only - Many soluble tropoelastin molecules => ++ insoluble and durable. 7  - Desmosin and isodesmosin binds tropoelastin units to each other. - Aorta, lungs, elastic ligaments, skin, bladder, elastic cartilage. Synthesis: in the cytoplasm of fibrocytes from proelastin tropoelastin. Forms crosslinks between tropoelastin => elastin Degradation: Elastase from pancreas or leucocytes (granules). FIBRONECTIN, LAMININ, PROTEOGLYCANS - Fibronectin: 2 polypeptide chains. Cell-surface protein, and in blood-plasma. - Laminin: 3 polypeptide chain, adhesive glycoprotein. Binds epithelial cells to connective tissue. - Proteoglycans: Binds water and cations. Regulates movement of molecules in the extracellular matrix. 8  ENZYMES: PROTEINS WITH CATALYTIC POWER Enzymes are made to accelerate the reactions. Highly specialized. 1000-4000 enzymes/cell. History: - Early 1800s: the digestion of meat is made by stomach secretions - 1800s: Louis Pasteur = “ferments” existing only within living organisms. - 1878: Wilhelm Kühne = « enzyme » nonliving substance helping to digestion. - 1897: Eduard Büchner, Nobel price in Chemistry for “cell-free fermentation”. - 1946: Northrop and Stanley, Nobel price in Chemistry for demonstrating that enzymes = proteins. Role of enzymes: - Intermediary metabolism - Degradation of feedstuff - Pharmacotherapeutic agents acts on enzymes - Prodrugs are activated by enzymes - Helps the diagnosis Catalyst enzymes acts on substrates to create specific products. RNA enzyme is called ribozyme. Apoenzyme = only proteins (ex: ser-proteases) Holoenzyme + Cofactor = non-protein parts (ex: metal ions, water-soluble vit. derivates = coenzymes or prosthetic group) Coenzyme can dissociate (Ex: Transketolase: coenzyme TPP) Cofactor Prosthetic group cannot dissociate (Ex: FAD) Metal ion (Ex: Carbonic anhydrase: 4 (Zn) metal cofactor) Active site = catalytic site + substrate-binding site. Only a very small portion For small regulatory (3-4 AA) is directly molecules: they increase or involved in the catalysis decrease the enzyme activity. (like scissors) 9 Active site: where the substrate binds. Highly specific. Enzymes: - Unchanged by the reactions that they catalyze. - Can be used over and over again. Serine protease: - Chymotrypsin - Trypsin Mammalian pancreas - Elastase - Acetylcholine esterase - Thrombin Blood clotting factors - Plasmin Ser-195 is the catalytic site of each pancreatic Ser protease. DIPF (diisopropylphospho- fluoridate) inhibits Ser-195. BUT the substrates-binding sites are different for each pancreatic Ser-proteases. The enzyme action = lock and key model. It is specific. Induced-fit model: proteins structure is not as rigid, and substrate-binding site not exactly complementary to the substrate. Fluctuation theory: enzyme’s active site always changing conformation and substrate bound if the binding site is complementary. (best model) Michaelis-Menten theory: Need a first energy to bind enzyme + substrate, then need a second energy to generate a product. The first energy is reversible. Enzymes actions: - Speed up reactions - They lower activation energy - Exergonic reactions - Specific and selective: bond sp (α-amylase) < group sp (endopeptidase = pepsin, trypsin, chymotrypsin or exopeptidase = aminopeptidase, carboxypeptidase) dinucleotide 3-10 mononucleotide => oligonucleotide 11-100 => polynucleotide >100 mononucleotide => nucleic acid - Structure II: double-stranded structure. Antiparallel structure, H-bonds between complementary bases (AT = 2 H-bonds and GC =3 H-bonds). Chargaff’s rule: Number of purin bases = number of pyrimidine bases Denaturation: 70-80°C breaks the H-bonds Renaturation: cooling down Melting point: half are denatured, and half are linked. The higher number of GC bases the higher the melting point! (GC = 3 H-bonds so > AT = 2 H-bonds) - Structure III: double helix twist. - Structure IV: THE SUPERCOIL OF PROKARYOTES o Relaxed form: loose conformation o Positive supercoil: double helix is rolled up counterclockwise. Less active transcriptionally. o Negative supercoil: double helix is rolled up clockwise. Active transcription. Supercoiling is controlled by Topoisomerase. o Topoisomerase I: no ATP need, one DNA strand at a time. o II: need ATP. Both DNA strand at a time o III and IV: uncoil during transcription. Antibiotics inhibits them => cannot replicate! In prokaryotes DNA looks like a chromosome, only ONE. Still double-stranded, but circular. The coil of EUKARYOTES Made around histone proteins. Arg, Lys ++ composed, + charge. DNA is – charge. Love story. 14  Histone is an octamer of dimers: H2A, H2B, H3 and H4. And H1? Fix the structures together. Chromosome structure: DNA => Nucleosome => Chromatin => Chromosome RNA types: - mRNA: messenger - tRNA: transfer - rRNA: ribosomal - snRNA: small nuclear RNA - snoRNA: small nucleolar RNA - micRNA: mRNA inhibitory complementary RNA - siRNA: small interfering RNA Genetic code: - Gene = DNA region which is responsible to code of a protein or functional RNA - AA are base triplets, as they are 20 = 64 different combinations = redundant o CODE is DNA triplets info o CODON mRNA triplet in transcription o ANTICODON tRNA triplet in translation DNA replication: - Mitose: to form 2 identic cells - Meiose: double mitose. To form 4 daughter cells. Semi-conservative process. - 3 phases: Initiation, elongation, termination PROKARYOTE REPLICATION: - Initiation: o origin of replication recognized by DnaA. Then DnaB + DnaC. Opening of the 2 strands. o Free 3’-OH needed to “write the first letter” = Primer (primers are synthesized by the primase enzyme in the primosome). o SSB proteins: uncoil the coiling. - Elongation: o Leading strand: DNA polymerase III, 5’ -> 3’ direction ALWAYS! o Lagging strand: replication go 5’ -> 3’, go back, restart, go back… All the small fragments created are Okazaki fragments = DNA + primer. DNA Polymerase I replace the missing parts by DNA. DNA ligase ligates fragments. - Termination: the two parts joins. Two circles. 15 EUKARYOTE REPLICATION: Similar. Main differences: - Several origins of replications simultaneously - DNA-polymerase enzymes have other names: o DNA-polymerase α: DNA polymerase III but close to the primer o DNA polymerase β: DNA polymerase I o DNA polymerase γ: replication of the DNA in the mitochondria o DNA polymerase δ: DNA polymerase III but far from the primer (needs PCNA = proliferating cell nuclear antigen = tumor proliferation marker!!) - Telomere sequences: G-rich extra sequences on 3’ end = cap. Protects the end for shortening. Mismatch repair: - Prokaryotes: DNA Polymerase I and II - Eukaryotes: DNA polymerase β Cut off the bad part, put the correct plate. Non-repaired parts results to MUTATIONS. - Type A: spontaneous or induced by factors like UV - Type B: gametic or somatic, inheritance or tumor formation - Type C: gene mutation. Point mutation: o Substitution: sickle-cell anaemia!! o Insertion o Deletion o Inversion 16  TRANSCRIPTION RNA is a single stranded polynucleotide. RNA is synthesized from DNA. DNA strands have: - Coded strand - Non-coded strand = template for transcription. Genetic information is transcribed from DNA to RNA. The transcription unit: functional unit of DNA - Promoter: responsible for regulation of transcription. o TATA-box: where RNA polymerase binds 2/3 o GC-box: where RNA polymerase binds 1/3 o CAP-cAMP binding site - RNA coding region - Termination signal Transcription start site is composed of: - Upstream direction (toward promoter) - Promoter - Downstream direction (toward coding region) - UTR = untranslated region. Here, DNA is transcribed but do not code for anything. Gene structure: - Prokaryotes: polycistronic = one transcription for several genes. - Eukaryotes: monocistronic = one transcription for one gene. The RNA coding region: 17  - Introns (like unknown), will be cut out in mature mRNA - Exons: conserved part Transcription of mRNA: 1) Initiation: RNA polymerase is the one doing everything! Binds to CAP-cAMP complex to bind correctly to DNA strand. 2) Elongation: RNA polymerase builds nucleotides into mRNA chain. It uses is nucleoside triP (NTP) as substrates = ATP, GTP, CTP, UTP and nucleoside monoP (NMP) are built in mRNA chain. 3) Termination: starts at terminations signal: a. Rho(ρ)-factor independent termination: GC rich, dissociates. b. Rho(ρ)-factor dependent termination: GC rich, slow down synthesis Structure of mRNA in Prokaryotes: - Codons - Polycistronic - Shine-Dalgarno sequences (RBS ribosome binding site) between transcripts of genes - UTR region 18 Regulation of transcription in prokaryotes: - Operon model: o Lactose operon (Lac-operon): regulates transcription of genes of lactose degrading enzymes. o Tryptophan operon: inhibition of transcription. - Operator region: binding site for inhibitors The 2 MUST HAVE conditions for starting a prokaryote transcription: - No repressor on operator region - CAP-cAMP complex bound to promoter site. For eukaryotes: mRNA transports the genetic information from nucleus to cytoplasm for protein synthesis. 3 phases as usual. During transcription, pre-mRNA = cotranscriptional processing, mRNA need to maturate. RNA polymerases are distinct: - RNA poly I : most abundant, ribosomal - RNA poly II: mRNA and some snRNA, can be inhibited ++ by α-amanitin (death cap toxin) - RNA poly III: tRNA and some snRNA, can be inhibited by α-amanitin (death cap toxin) Transcription of eukaryotes: - Initiation: o Pre-initiation complex formation: TFIID binds to TATA-box => TFIIA and B binds => RNA poly II and TFIIF (helicase and protein kinase) => TFIIE and TFIIH o Helicase uncoils DNA o First few nucleotides are built in the mRNA chain o Protein kinase activate RNA poly II o Complex dissociates = elongation starts - Elongation: 19  o RNA poly II works. - Termination: o Cleavage sequence Maturation of eukaryote: - 5’ capping = elongation - 3’ tailing = polyadenylation (Poly-A chain added by poly A polymerase) - Splicing = termination, introns are removed by snRNA (ribozymes = U1, U2 etc), the process is named spliceosome. The ways of regulations in eukaryotes: - Modifications of chromatin = epigenetic regulation o Histones modifications: acetylation, methylation, phosphorylation o DNA methylation => gene silencing - Regulation of transcription factors o Cis-regulatory (promoter, enhancer, silencer) o Trans-regulatory (basal, activators, repressors) o Structures of transcription factors: Steroid and thyroid hormones are nuclear receptors. They activate the entrance into the nucleus (internalisation). Inside, receptors act as transcription factors. 20  TRANSLATION Processus of protein biosynthesis based on mRNA. Needed: mRNA, tRNA, Ribosomes (RNA + ~70 proteins) and some others. UAA, UAG and UGA code for STOP AUG code for Met, which is the universal START codon. tRNA: Has 3 loops: - DHU: binds aminoacyl tRNA synthetase - Anticodon: binds mRNA complementary codon - TφC: binds large subunit of ribosome Activation of AA: binding of adequate AA with 3’-CCA sequence of tRNA, with ester bond: - Aminoacyl tRNA synthetase, binds ATP + one AA - Becomes Aminoacyl-AMP + PPinorganic - Binds to DHU loop => aminoacyl-tRNA + AMP Structure of ribosomes: rRNA + proteins They are differentiated by a sedimentation coefficient (unit is Svedberg = S) Ribosomes have 3 binding sites on the large subunit: E, P and A. - A: aminoacyl tRNA - P : peptidyl tRNA - E: exit Small subunit: mRNA binding site! 21 Translation in eukaryotes: In RER (rough endoplasmic reticulum), from 5’ to 3’ (ALWAYS), from N to C. - Initiation: o Small subunit + eIF-2-GTP + further eIFs + tRNA Met! => pre-initiation complex o Kozak-scanning mechanism: consume 1 ATP/nucleotide until it finds Met. o Recognition of Met! => small subunit binds to it. o GTP is hydrolyzed => large subunit comes up. - Elongation: o Consist of cycles o Initiator Met-tRNA binds to P site = TφC loop => A site is free for a new nucleotide! o Aminoacyl-tRNA binds to A site thanks to EF-1α + GTP o Methione: => P→A site thanks to peptidyl transferase (no ATP needed) o Translocation: dipeptidyl-tRNA A→P site thanks to translocase (GTP is needed) o →E where it is dissociated from ribosome and bye bye o A site is free Termination in prokaryotes and eukaryotes: o STOP codon appears o PRF (protein releasing factor) binds to A site o Cleavage of peptide chain (GTP need) o Ribosome subunits dissociate Initiation in prokaryotes: o Only 3 IFs are needed instead of several. o Initiator amino acid: N-formylmethionine instead of Met-tRNA o NO Kozak scanning but Shine Dalgarno sequences of mRNA Elongation in prokaryotes: o Elongation factors are different: EF-1α → EF-Tu and EF-2 → EF-G o Transcription and translation are running simultaneously. o Polycistronic replication => polyribosome Regulation of translation: - Complementarity of 5’ end of mRNA - Preferred codon among tRNA specificities - micRNA, inhibitor of mRNA 22 Post transcriptional alteration of peptides: - Proteolytic cleavage - Phosphorylation - Hydroxylation = collagen Lys and Pro - Glycolisation : i.e different membrane proteins Post translational alteration of peptides: - Acetylation = epigenetic regulation - Oxidation = disulfide bridges Transport and folding of proteins: - The folding is induced by the native structure (after translation) - The transport is made by signal sequences on a special part of the protein, or by signal peptidase which turn-off when arrived. 23