Chapter 7 Notes - Biochemistry PDF

General observations for biosynthesis: ○ End products are more reduced than starting materials and reducing equivalents delivered by NADPH NAD+/NADH poised for energy rxn (more NAD+ than NADH) NADP+/NADPH poised for biosynthesis (more NADPH than NAD...

General observations for biosynthesis: ○ End products are more reduced than starting materials and reducing equivalents delivered by NADPH NAD+/NADH poised for energy rxn (more NAD+ than NADH) NADP+/NADPH poised for biosynthesis (more NADPH than NADP+) NADPH + NAD+ ←→ NADH + NADP+ (transhydrogenase: keeping reducing equivalents ready/poised) ○ Uses ATP ○ Key enzymes allosterically regulated (small regulator bound or not controls enzyme activity) Controlling ligand is often end product of biosynthesis rxn (feedback inhibition/negative feedback) ○ Made from 13 precursor metabolites to make everything for cell Transport for gram - cell (outer membrane as outermost layer) ○ Diffusion porins: small 600-700, hydrophilic ○ Facilitated diffusion: selective for solute (channel for certain compound) ○ Active transport: TonB dependent transporter uses H+ gradient (ATPase on inner membrane) Fe siderophore transport by TonB dependent transporters (most cells require Fe) Fe III is highly insoluble which makes it scarce in environment, in host Fe is not available Heme, ferritin, lactoferrin all bound in host ○ How do microbes solve iron problem Siderophores: small molecules that tightly bind to Fe Secreted by bacteria under Fe limitation Uptake Fe-siderophores using TBDT (TonB dependent transporters) Microbes can take up own and other siderophores  ○ ○ Cell wall transport has diffusion Cell membrane transport ○ 1) passive Diffusion Simple: can diffuse across membrane Facilitated: selective for solute ○ 2) active transport: requires energy, move against gradient Ion coupled: use H+/Na+ motive force Antiport: moves two different ions or solutes in opposite directions across a membrane ABC transporters: ATP hydrolysis drives transport (in gram + and -)  Periplasmic binding protein (gram + can have it but not in the OM like gram -) Transmembrane domain (substrate goes through it in cell membrane) ATP binding hydrolysis (in cytoplasm) Phosphotransferase system/group translocation: transport of sugars only Once inside, phosphorylates substrate as part of transport (moving down gradient since more glucose outside) P-sugar moves down concentration gradient Phosphoenolpyruvate (PEP) supplies energy Transport and phosphorylation in one step with 1 ATP which saves more ATP ○ Feeder pathways ○ Heterotrophs convert C substrate to central metabolite ○ Autotrophs fix CO2 to central metabolite ex calvin cycle  ○ Central metabolism: glycolysis, TCA, Pentose P pathway ○ Supplies 13 precursor metabolites ○ Core energy generating pathway in heterotrophs ○ Can work in forward and reverse directions depending on substrate and cell needs N assimilation ○ Sources: organic N, NH3, N2, NO^3- Ammonia NH3 ← → NH4+ in equilibrium (NH3 diffuses across membrane) ○ Sources converted to NH4+ ○ Glutamate and glutamine are carriers of ammonia ○ Organic N Uptake with transporters and use deaminase inside Excrete deaminase ○ Nitrate, NO^3- NO^3- → NO^2- by nitrate reductase NO^2- → NH3 by nitrite reductase ○ N2 fixation N2 → NH3 nitrogenase with FeMoCo cofactor Difficult to break triple bond Uses 16 ATP and 8 reducing equivalents Nitrogenase highly O2 sensitive and results in breakage Fixes sensitivity problem by separating cells that do oxygenic photorespiration and N2 fixation  Cyanobacteria that fix N2 Heterocysts: spatial separation Temporal separation (day time produce oxygen and night time fix N2) ○ Glutamate and glutamine synthesis ○ 1) glutamate dehydrogenase: high NH3 required More energy savable option 2oxoglutarate + NH3 + H+ + NADPH → glutamate + H2O + NADP+ ○ 2) glutamine synthetase: uses ATP Glutamate + NH3 + ATP → glutamine + Pi + ADP Glutamine + 2oxoglutarate + H+ + NADPH → (GOGAT) → 2 glutamate + NADP+ Make glutamine with ATP then make glutamate Glutamate-oxoglutarate amido transferase (GOGAT) makes glutamate from glutamine ○ Ex based on the following data, what growth condition has low levels of ammonia Growth Glutamate Glutamine GOGAT condition dehydrogena synthetase activity se activity activity A 34 162 114 B 126 150 12 A has low levels because glutamate more active with more ammonia  S assimilation ○ H2S ← → HS- ← → S^2- in equilibrium ○ Sources: H2S (only in anaerobic, can diffuse in), organic S, SO^4- (sulfate) ○ All forms go to H2S ○ H2S + O-acetely-L-serine → cysteine which is carrier of sulfur for cell ○ Sulfate expensive since needs ATP and reducing equivalents ○ P assimilation ○ Sources: PO4^3- (cant diffuse and needs transporter), organic P ○ PO4^3- converted to ATP (ATP is carrier and used for energy metabolism ○ No redox change ○ Assimilation sources Sources first Carrier in Does of converted to cell assimilation require redox  N N2 NH3 Glutamate no and glutamine NH3 no Organic N yes NO^3- yes S H2S H2S cysteine no Organic S Sulfate then yes H2S SO4^2- yes P PO4^3- PO4^3- ATP no Organic P no Quiz ○ Noncyclic photosynthesis requires e- donor ○ Can O2 be used as e- donor? No, in noncyclic photosynthesis rion can be e- donor ○ What is required for biosynthesis? NADPH/NADP+ > 1 ○ B→D→C→A regulated by A in B→D ○ Feeder pathway ex is calvin cycle ○ TonB dependent transporter powered by proton gradient across CM ○ There is hydrolysis of phosphate bond in ABC transporters What is the state where DNA replication begins? Origin of replication From the origin, what direction does replication proceed? Bidirectional Identify the leading and lagging strands? 5 → 3 direction is leading What bacterial enzyme relieves supercoiling? Gyrate What enzyme catalyzes strand elongation? Polymerase What are the DNA fragments formed on the lagging strand called? Okazaki fragments What enzyme joins Okazaki fragments? Ligate How does the cell prevent errors in replication? Proofreading and mismatch repair What is direction of DNA synthesis? 5 → 3 Can DNA polymerase start polymerization de novo? No  What is transcription? DNA to RNA What enzyme catalyzes transcription? RNA polymerase Where does RNA polymerase bind to DNA? Promoter What gives RNA polymerase specificity for binding? Sigma factor Are all RNA transcripts used to make proteins? No What does polycistronic mean? One mRNA codes for multiple peptides What is operon? Cluster of genes on DNA strand that are transcribed together (promoter, operator, structural genes) What catalyzes translation? ribosomes