11/18/2010Biochemistry: Metabolism II
General Metabolism II
Andy HowardIntroductory Biochemistry,
fall 201018 November 2010
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Metabolism:the core of biochem All of biology 402 will concern itself with the specific pathways of metabolism
Our purpose here is to arm you with the necessary weaponry
… but first, we need to explain the role of Ca2+ in muscle contraction
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What we’ll discuss
Metabolism Control Feedback
Flux Phosphorylation Other PTMs
Evolution Redox Tools for studying
Nutrition Macronutrients
Proteins Fats Carbohydrates
Vitamins Fat-soluble Water-soluble
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iClicker quiz question 1 An asymmetry between stage 1 of catabolism (C1) and the final stage of anabolism (A3) is (a) A3 always requires light energy; C1 doesn’t
(b) A3 never produces nucleotides;C1 can involve nucleotide breakdown
(c) A3 adds one building block at a time to the end of the growing polymer;C1 can involve hydrolysis in the middle of the polymer
(d) There are no asymmetries between A3 and C1
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iClicker quiz question 2
Could dAMP, derived from degradation of DNA, serve as a building block to make NADP? (a) Yes. (b) Probably not: the energetics wouldn’t allow it.
(c) Probably not: the missing 2’-OH would make it difficult to build NADP
(d) No: dAMP is never present in the cell
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Regulation Organisms respond to change
Fastest: small ions move in msec Metabolites: 0.1-5 sec Enzymes: minutes to days
Flow of metabolites is flux: steady state is like a leaky bucket
Addition of new material replaces the material that leaks out the bottom
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Metabolic flux, illustrated Courtesy Jeremy Zucker’s wiki
http://bio.freelogy.org/wiki/User:JeremyZucker#Metabolic_Engineering_tutorial
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Feedback and Feed-forward
Mechanisms by which the concentration of a metabolite that is involved in one reaction influences the rate of some other reaction in the same pathway
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Feedback realities Control usually exerted at first committed step (i.e., the first reaction that is unique to the pathway)
Controlling element is usually the last element in the path
Often the controlled reaction has a large negative Go’.
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Feed-forward
Early metabolite activates a reaction farther down the pathway
Has the potential for instabilities, just as in electrical feed-forward
Usually modulated by feedback
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Activation and inactivation by post-translational modification Most common:covalent phosphorylation of protein
usually S, T, Y, sometimes H Kinases add phosphateProtein-OH + ATP Protein-O-P + ADP… ATP is source of energy and Pi
Phosphatases hydrolyze phosphoester:Protein-O-P +H2O Protein-OH + Pi
… no external energy source required
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Phosphorylation’s effects
Phosphorylation of an enzyme can either activate it or deactivate it
Usually catabolic enzymes are activated by phosphorylation and anabolic enzymes are inactivated
Example:glycogen phosphorylase is activated by phosphorylation; it’s a catabolic enzyme
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Glycogen phosphorylase
Reaction: extracts 1 glucose unit from non-reducing end of glycogen & phosphorylates it:(glycogen)n + Pi (glycogen)n-1 + glucose-1-P
Activated by phosphorylationvia phosphorylase kinase
Deactivated by dephosphorylation byphosphorylase phosphatase
Muscle phosphorylaseEC 2.4.1.1192kDa dimermonomer shownPDB 2GJ4, 1.6Å
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Phosphorylation’s effects
Phosphorylation of an enzyme can either activate it or deactivate it
Usually catabolic enzymes are activated by phosphorylation and anabolic enzymes are inactivated
Example:glycogen phosphorylase is activated by phosphorylation; it’s a catabolic enzyme
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Amplification
Activation of a single molecule of a protein kinase can enable the activation (or inactivation) of many molecules per sec of target proteins
Thus a single activation event at the kinase level can trigger many events at the target level
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Other PTMs Are there other reversible post-translational modifications that regulate enzyme activity? Yes: Adenylation of Y ADP-ribosylation of R Uridylylation of Y Oxidation of cysteine pairs to cystine
Cis-trans isomerization of prolines
ADP-ribosylationof arginine; fig.courtesy RPI
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Metabolism and evolution Metabolic pathways have evolved over hundreds of millions of years to work efficiently and with appropriate controls
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Evolution of Pathways:How have new pathways evolved? Add a step to an existing pathway Evolve a branch on an existing pathway
Backward evolution Duplication of existing pathway to create related reactions
Reversing an entire pathway
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Adding a step
A B C D E P
• When the organism makes lots of E, there’s good reason to evolve an enzyme E5 to make P from E.
• This is how asn and gln pathways (from asp & glu) work
E1 E2 E3 E4 E5
Original pathway
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Evolving a branch Original pathway: D A B C X
Fully evolved pathway: D A B C X
E1 E2E3
E3a
E3b
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Backward evolution Original system has lots of E P
E gets depleted over time; need to make it from D, so we evolve enzyme E4 to do that.
Then D gets depleted; need to make it from C, so we evolve E3 to do that
And so on
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Duplicated pathways
Homologous enzymes catalyze related reactions;this is how trp and his biosynthesis enzymes seem to have evolved
Variant: recruit some enzymes from another pathway without duplicating the whole thing (example: ubiquitination)
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Reversing a pathway
We’d like to think that lots of pathways are fully reversible
Usually at least one step in any pathway is irreversible (Go’ < -15 kJ mol-1)
Say CD is irreversible so E3 only works in the forward direction
Then D + ATP C + ADP + Pi allows us to reverse that one step with help
The other steps can be in common This is how glycolysis evolved from gluconeogenesis
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Oxidation-reduction reactions and Energy Oxidation-reduction reactions involve transfer of electrons, often along with other things
Generally compounds with many C-H bonds are high in energy because the carbons can be oxidized (can lose electrons)
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Reduction potential Reduction potential is a measure of thermodynamic activity in the context of movement of electrons
Described in terms of half-reactions
Each half-reaction has an electrical potential, measured in volts, associated with it because we can (in principle) measure it in an electrochemical cell
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So what is voltage, anyway? Electrical potential is available energy per unit charge:
1 volt = 1 Joule per coulomb 1 coulomb = 6.24*1018 electrons Therefore energy is equal to the potential multiplied by the number of electrons
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Electrical potential and energy
This can be expressed thus:Go’ = -nFEo’
n is the number of electrons transferred
F = fancy way of writing # of Coulombs (which is how we measure charge) in a mole (which is how we calibrate our energies) = 96.48 kJ V-1mol-1
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Oh yeah? Yes. 1 mole of electrons = 6.022 * 1023 e-
1 coulomb = 6.24*1018 e-
1 mole = 9.648*104 Coulomb 1 V = 1 J / Coulomb=10-3 kJ / Coulomb
Therefore the energy per mole associated with one volt is10-3 kJ / C * 9.648*104 C = 96.48 kJ
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What can we do with that? The relevant voltage is the difference in standard reduction potential between two half-reactions
Eo’ = Eo’acceptor - Eo’donor
Combined with free energy calc, we seeEo’ = (RT/nF ) lnKeq andE = Eo’ - (RT/nF ) ln [products]/[reactants]
This is the Nernst equation
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Free energy from electron transfer
We can examine tables of electrochemical half-reactions to get an idea of the yield or requirement for energy in redox reactions
Example:NADH + (1/2)O2 + H+ -> NAD+ + H2O;
We can break that up into half-reactions to determine the energies
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Half-reactions and energy NAD+ + 2H+ + 2e- NADH + H+,Eo’ = -0.32V
(1/2)O2 + 2H+ + 2e- H2O, Eo’ = 0.82V
Reverse the first reaction and add:NADH + (1/2)O2 + H+ NAD+ + H2O,Eo’ = 0.82+0.32V = 1.14 V.
Go’ = -nFEo’ = -2*(96.48 kJ V-1mol-1)(1.14V) = -220 kJ mol-1; that’s a lot!
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How to detect NAD reactions
NAD+ and NADH(and NADP+ and NADPH)have extended aromatic systems
But the nicotinamide ring absorbs strongly at 340 only in the reduced(NADH, NADPH) forms
Spectrum is almost pH-independent, too!
So we can monitor NAD and NADP-dependent reactions by appearance or disappearance of absorption at 340 nm
NAD+
NADH
Absorbance
Wavelength
340 nm
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Classical metabolism studies
Add substrate to a prep and look for intermediates and end products
If substrate is radiolabeled (3H, 14C) it’s easier, but even nonradioactive isotopes can be used for mass spectrometry and NMR
NMR on protons, 13C, 15N, 31P Reproduce reactions using isolated substrates and enzymes
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Next level of sophistication… Look at metabolite concentrations in intact cell or organism under relevant physiological conditions
Note that Km is often ~ [S].If that isn’t true, maybe you’re looking at the non-physiological substrate!
Think about what’s really present in the cell.
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Mutations in single genes
If we observe or create a mutation in a single gene of an organism, we can find out what the effects on viability and metabolism are
In humans we can observe genetic diseases and tease out the defective gene and its protein or tRNA product
Sometimes there are compensating enzyme systems that take over when one enzyme is dead or operating incorrectly
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Deliberate manipulations Bacteria and yeast:
Irradiation or exposure to chemical mutagens
Site-directed mutagenesis Higher organisms:We can delete or nullify some genes;thus knockout mice
Introduce inhibitors to pathways and see what accumulates and what fails to be synthesized
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Nutrition Lots of nonsense,some sense on this subject
Skepticism among MDs as to its relevance
Fair view is that nutrition matters in many conditions, but it’s not the only determinant of health
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Macronutrients
Proteins Carbohydrates Lipids Fiber
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Protein as food
Source of essential amino acids Source of non-essential aa Fuel (often via interconversion to
-ketoacids and incorporation into TCA)
All of the essential amino acids must be supplied in adequate quantities
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Which amino acids are essential?
At one level, that’s an easy question to answer: they’re the ones for which we lack a biosynthetic pathway: KMTVLIFWH
That shifts the question to:why have some of those pathways survived and not all?
Answer: pathways that are complex or require more than ~30 ATP / aa are absent (except R,Y)
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The human list
AA molesessen- ATPtial?
Asp 21 noAsn 22-24 noLys 50-51 yesMet 44 yesThr 31 yesAla 20 noVal 39 yesLeu 47 yesIle 55
yesGlu 30 noGln 31 no
AA moles essen-ATP
tial?Arg 44 noPro 39 noSer 18 noGly 12 noCys 19 noPhe 65 yesTyr 62 no*Trp 78 yesHis 42 yes
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Carbohydrates as food
Generally recommended to be more than half of caloric intake
Complex carbohydrates are hydrolyzed to glucose-1-P and stored as glycogen or interconverted into other metabolites
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Lipids as food You’ll see in 402 that the energy content of a lipid is ~ 2x that of carbohydrates simply because they’re more reduced
They’re also more efficient food storage entities than carbs because they don’t require as much water around them
Certain fatty acids are not synthesizable; by convention we don’t call those vitamins
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Vitamins Vitamins are necessary micronutrients A molecule that is a vitamin in one organism isn’t necessarily a vitamin in another
E.coli can make all necessary metabolites given sources of water, nitrogen, and carbon
Most eukaryotic chemoautotrophs find it more efficient to rely on diet to make complex metabolites
We’ll discuss lipid vitamins first,then water-soluble vitamins
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Why wouldn’t organisms make everything?
Complex metabolites require energy for synthesis
Control of their synthesis is also metabolically expensive
Cheaper in the long run to derive these nutrients from diet
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Vitamins: broad classifications Water-soluble vitamins
Coenzymes or coenzyme precursors Non-coenzymic metabolites
Fat-soluble vitamins Antioxidants Other lipidic vitamins
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Are all nutrients that we can’t synthesize considered vitamins? No: If it’s required in large quantities,it’s not a vitamin
By convention, essential fatty acids like linoleate aren’t considered vitamins