Staniforth (Biosynthesis and metabolism) Flashcards
Why did evo of CO2 (decarboxylation) give strong thermodynamic pull to reactions?
- v stable
- easily escapes site of reaction (as gas/soluble bicarb)
- more products than reactants (-ΔG)
(R-COOH RH + CO2)
How does carbon enter metabolism?
- photosynthesis
- carboxylations
eg. pyruvate + CO2 –> oxaloacetate
How does hydrogen enter metabolism?
- H2O
- NH4
- H2 (g)
How does oxygen enter metabolism?
- H2O
- CO2
- molecular oxygen reactions
- Phe + O2 –> Tyr (+ΔG)
= biosynthetic reaction as uses NADPH, not NADP
Where is S found in cell?
- SH = high energy thioesters
- Fe-S proteins = redox centres
- SH groups important for protein folding
- energy store
Why is acetyl CoA high energy?
- great bond donor and easily separated to donate CoA group
Why are S proteins so common?
- pyrites used in primitive Earth instead of NADH –> NAD+
How does S enter metabolism?
- MOs/plants get from H2S
- higher organisms get from diet, eg. Met
What are the advantages of using S?
- S-S bonds strong but form and break under mild conditions (so more flexible)
- S binds to Fe
- thioesters have less resonance stabilisation than O esters –> carry more G
How is Ser used to get Cys?
- Ser activated by acetyl CoA
- captures S from H2S to give Cys
How does nitrogen enter metabolism?
- N2 (g) v stable = N fixation by nitrogenase
- NH3 quite stable = by glutamate deHase
- glutamate synthase
- glutamine synthetase
What is the importance of nitrogen in metabolism?
- forms H bonds and Schiff base links
How does P enter metabolism?
- naturally oxidises to phosphate under atmospheric conditions
- phosphate used directly by cell
What is the difference between 1° and 2°metabolic pathways?
1°
- basic housekeeping functions
- in essentially all cells
- largely constitutive
2°
- specialised functions
- in all differentiated cells
- inducible
What are the functions of glycolysis?
- ATP and NADH prod
- intermediates for biosynthesis
What is the location of glycolysis?
- cytosol
What is the overall reaction of glycolysis and pentose phosphate pathway?
- glucose –> pyruvate
When is glycolysis used, and when is phosphate pentose pathway used?
- glycolysis if cell needs energy
- PPP if cell needs biosynthesis
What are the functions of pentose phosphate pathway?
- gen C5 sugars and NADPH for biosynthesis
- breakdown route for C5 sugars
- other intermediates for biosynthesis
What is the location of pentose phosphate pathway?
- cytosol
What is the overall reaction of Link Reaction?
- pyruvate —-pyruvate deHase—> acetyl CoA + CO2 + NADH
Is Link reaction favourable, and why?
- v favourable
- decarboxylation
What are the functions of the Link Reaction?
- processes pyruvate for KC
- source of acetyl CoA
- NADH prod
What is the location of the Link Reaction?
- mito
How Krebs Cycle discovered to be a cycle?
- measured resp in muscle tissues
- added succinate and lots more C prod than what was added
- ∴ cycle w/ catalytic property
What are the functions of Krebs Cycle?
- NADH and GTP prod
- gen intermediates for biosynthesis
What is the location of the Krebs Cycle?
- mito
How does ATP prod by Krebs Cycle compare to glycolysis?
- 24 v 2
What is a top up (anaplerotic mechanism) for Krebs Cycle?
- pyruvate + CO2 + ATP + H2O –> oxaloacetate + ADP
Why is a cycle a good design for a precursor supply system?
- extra supply of any intermediate can top up cycle
What are the functions of mito e- transport system?
- gen ATP (via NADH and FADH2) and GTP
- maintaining redox balance (NADH–>NAD+)
What reaction occurs during β-ox of FAs?
- FAs –> acetyl CoA
- NADH and FADH2 prod
What are the functions of β-ox of FAs?
- extracting energy from lipid stores
- gen 2C units for biosynthesis
What is gluconeogenesis?
- opp of glycolysis
- biosynthesis of sugars from non-carb prescursors
What is the overall reaction of gluconeogenesis?
- pyruvate, AAs –> sugar
What is the function of gluconeogenesis?
- sugar supply when glucose scarce
What are the locations of gluconeogenesis?
- mito and cytosol
- mainly liver, some in kidney
What are the 3 methods for control of biosynthetic pathways?
- isoenzymes
- single enzyme cumulative control
- single enzyme concerted control
How can isoenzymes be used to control biosynthetic pathways?
- several enzymes doing same job, w/ small diff, so 1 can be inactivated but not others
What is an example of a pathway using isoenzymes?
- amino acid biosynthesis
- 3 diff asportkinases in E. Coli for conversion of aspartic acid to aspartyl phosphate
- same aspartokinase domain, but slightly diff regulatory domain
- 1 is not inhibited, 1 inhibited by Thr and 1 inhibited by Lys
How can a single enzyme with cumulative control be used to control biosynthetic pathways?
- eg. glutamine synthetase
- 1 product has inhibitory activity
- next has more until enzyme totally inhibited
- DIAGRAM*
How can a single enzyme with concerted control be used to control biosynthetic pathways?
- eg. lysine biosynthesis
- each product alone has no inhibitory activity
- combo of products gives inhibitory activity
- DIAGRAM*
What is the rate determining step?
- slowest step
What is the committed step?
- means reaction continues
- relax control and get end product
- don’t if heighten it
- not necessarily slowest
Why is distributive control of pathways a poss?
- prob over simplification to say cellular control of pathways occurs directly by variations in rate of indiv enzymes
What is the distributive control hypothesis?
- flux through a pathway is a system property rather than simply property of indiv control enzymes
What is flux?
- no. molecules being transformed per unit time
- not equal to conc
What is the equation to calc flux coefficient (C)?
- (steady state flux/steady state flux) / [enz]/[enz]
What diff values of flux coefficient (C) do enzymes have at diff points in pathway, and what does this contradict?
- early enzymes have low C values
- later enzymes have high C values
- contradicts traditional role of allosteric control
How do you do metabolic flux analysis?
- identify enzymes involved in pathway
- map levels of as many intermediates as poss (‘metabolome’)
- investigate effect of increase or decrease in amount/activity of specific enzymes
- work out contribution of each enzyme to overall flux
What is the metabolome?
- quantitative complement of all low mol weight molecules present in cell under given conditions
What is the greatest implication of distributive control hypothesis?
- for biotechnologists
- need to decide which enzymes to increase/improve to result in enhanced yields of useful products
In the pathway
A —enz 1–> B —enz 2–> C —enz 3–> D
what effect would increasing levels of enz 2 have?
- increase rate of B –> C
- decrease B
- increase C
- not as high and as an immediate effect as would expect, as enz 1 and 3 still have impact
How do you increase flux through pathway?
- increase amount of single enzyme rarely increases flux
- increasing several enzymes does
- nature usually increases all enzymes in pathway, eg. lac operon
Is pathway of biosynthesis distinct from pathway of degradation?
- usually
What is the basic principle of group carriers/donors?
- DIAGRAM*
- molecule of B loaded onto carrier and activates it
- carriers energetically unstable when loaded so -ΔG to donate group
- loading generally req energy
What are the major group carriers for biosynthesis?
- C1 = tetrahydrofolate
- C1-methyl = s-methyladenosine
- C1-carboxyl = biotin
- C2 = acetyl CoA
- C3 = PEP
- C5 = isopentenyl pyrophosphate
- amino = Gln/Glu
- sulfur = Cys
- sugar = nucleosidediphosphates
- complex (eg. C-C-N) = eg. Gly
Why is biotin a good carrier molecule?
- preferable for CO2 to leave as lots of Os close together
How does biotin donate its COO group?
- ATP + bicarb –> carboxyphosphate
- carboxyphosphate wants to lose phosphate, cat addition of COO group to biotin (forms carboxybiotin enzyme)
What is an example of COO addition via biotin?
- pyruvate –> oxaloacetate
- cat by pyruvate carboxylase
- biotin-COOH donates COOH
- KC anaplerotic reaction
What group does tetrahydrofolate contains many copies of?
- Glu
Can C1 be carried in a variety of oxidation states?
- yes
What is the role of FH4 in the biosynthesis of dTMP?
- donates methyl group (unusual)
- during donation, FH4 converted to FH2
- NADPH needed to reconvert FH4 (energy input)
- ultimate source of methyl group is Ser
What is the most common methyl group carrier?
- S-adenosyl methionine (SAM)
How does SAM donate its methyl group?
- activation req ATP
- CH3 activated by S+
- Ser ultimate source of methyl groups (via FH4)
Why is methyl group on SAM a good leaving group?
- S+ next to it tries to grab e-s
What is the outline of SAM structure?
- S+ w/ single bond to adenosine, CH3 and Met
What is an example of C2 donation?
- FA biosynthesis
Why does phosphoenolpyruvate have no carrier (C3 units)?
- in built energy source
Why is phosphoenolpyruvate (C3) a good donor?
- -ve groups attached to each other
- not good sterically so -ΔG to get rid of repulsion
How does isopentenyl pyrophosphate donate C5 group?
- mevalonic acid made up of 3 acetyl CoAs
- CO2 lost forming IPP, pulls reaction forward
- IPP v reactive, has high free energy of reaction
- reactions occur leading to highly reactive carbonium ion
How are NH2 groups donated?
- Gln –> Glu +NH2
- uses ATP
- no activated carrier
- only R group NH2 donated
What various types of C1 unit can be carried on FH4?
- methanol (CH3-OH)
- formaldehyde (H2C=O)
- formic acid (OH-HC=O)
What is the diff between purine and pyrimidine skeletons?
- purine 2 ring
* DIAGRAMS*
What is phosphoribosyl pyrophosphate important for?
- allosteric control and end product inhibition as control of biosynthesis often involves 1st/2nd step
What is phosphoribosyl pyrophosphate?
- high active form of ribose
What are the 2 phases of purine biosynthesis?
- activation of O by phosphorylation
- nucleophile attacks activated C
What kind of donor can Asp function as?
- NH2
What are the origin of all atoms in purine skeleton?
- N from Asp
- N from Gln
- NH from Gln
- CCN from Gly
- 2x CH from FH4
- CH from CO2
What is the core pathway in purine biosynthesis?
- ribose-5-P –> PRPP –> phosphoribosylamine –> IMP
What are the processing reactions in purine biosynthesis?
- IMP –> AMP
- IMP –> GMP
Why is GTP used in processing of IMP to other nucleotides?
- if making ATP, makes sense to use diff nt in its biosynthesis
Why is deoxyribose wanted in DNA instead of ribose?
- more stable
- so can survive longer than RNA
What is the overall control of purine nt biosynthesis?
- overall control/end product feedback
- balancing levels of ATP/GTP
- balancing levels of purines/pyrimidines
- balancing levels ribose vs deoxyribose
- not clear if cumulative or concerted but def not isoenzymes
Why is control of purine nt biosynthesis necessary?
- avoid wastage and holding anything back
How is the biosynthesis of purine nts balanced?
- end products (ATP/GTP) are co-reactants in opp branch for conversion of IMP
What is tetrahydrofolate used for, and why does it have such serious side effects?
- widely used drug in chemo
- side effects as affects DNA and RNA synthesis
What is Ki, and what do the values mean?
- inhibition constant = how tightly enzyme bound
- <1 means need hardly any and it binds, so good comp inhibitor
How can fluorouracil and methotrexate be used in cancer chemo?
- fluorouracil = fluorinated analog of dUMP, inhibitor
- only works w/ pyrimidine nts (not purine analogs)
- ring assembled, then ribose-P attached
- methotrexate kills all rapidly dividing cells (inc bone marrow, hair follicles) –> Ki < 1nM
How is chirality of Cα established?
- transamination reaction using pyridoxal phosphate
What are the 3 enzymes of amination?
- glutamate deHase
- glutamate synthase
- glutamine synthetase
Why is double bond formation important in AA formation?
- prevents rotation –> planar
What is Schiff base formation?
- NH2 + -CHO
Why is Schiff base formation important in establishing chirality?
- geometry of enzyme ensures H+ added from specific side
What are the metabolic families of AAs?
need to know 1 eg.
- oxaloacetate
- PEP
- ribose-5-P
- pyruvate
- 3-phosphoglycerate
- α-ketoglutarate
How are AAs classified into families?
- according to their biosynthetic precursor
What are the general principles of AA biosynthesis?
- intermediate of glycolysis/PPP/KC –> req side chain assembled on α-keto acid –> NH2 group added by transamination
What is the mechanism of glutamate deHase?
Transamination:
1) binding of PLP to enzyme
- binds reversibly
2) exchange
- Schiff base formation is reversible
3) transfer of -NH2 to PLP
- addition of water
4) 2nd substrate
- loss of water
- Schiff base formed
5) Schiff base linkage exchange w/ enzyme
- AA has effectively exchanged R group
How is transamination poss?
- exchange of amino groups freely reversible
- AAs form “pool” to freely interchange
- similar amount of each AA until 1 req
- not great for control, but good for supply
What are the contrasting roles of Glu and Gln in biosynthesis of NH2 containing molecules?
- Glu has COOH / Gln has CONH2
- α-amino group of most AAs from Glu
- Gln major donor of NH2 groups in biosynthesis
How is Ser biosynthesised?
- from 3-phosphoglycerate
- req NAD, Glu and H2O
How does KM control amination?
- α-oxoglutarate –> glutamate
- high KM at high [NH3] = use of NH3 and NADH
- low KM at low [NH3] = use of NH3, ATP and NADPH
What method of pathway control is used for end product inhibition on glutamine synthetase?
- cumulative inhibition
What are the 2 forms of glutamine synthetase interconverted at adenylyl transferase, in the control of glutamine synthetase?
- DIAGRAM*
- ATP used to add AMP group
- adenylyl transferase has dual specificity, specifically changed by protein
What are the 2 forms of P protein in the control of glutamine synthetase?
- adenylating form deadenylating form (addition of UMP)
- cat by uridyl transferase
- reverse reaction spontaneous
What is the role of deadenylating form of P protein?
- turns glutamine synthetase back to active form
How is uridyl transferase under allosteric control?
- glutamine –> uridyl transferase
What is the significance of control of uridyl transferase?
- reaction important threshold in N reactions, so has stringent control
- mini amplification cascade?
