12/1/2009Biochemistry: Metabolism I General Metabolism Principles; Nutrition Andy Howard...

12/1/2009Biochemistry: Metabolism I

General Metabolism Principles; Nutrition

Andy HowardIntroductory Biochemistry

1 December 2009


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Metabolism depends strongly on cofactors We’ll attend to the reality that a lot of the versatility of enzymes depends on their incorporation of cofactors; and most vitamins are precursors of cofactors


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What we’ll discuss Post-translational modification

Phosphorylation Other reversible PTMs

How pathways evolve Oxidation-Reduction Reactions: Quantitation

How we study metabolism (revisited)

Nutrition Macronutrients Micronutrients Specific cofactors and vitamins reconsidered

Fat soluble vitamins

Ascorbate


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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


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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 a-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 arachidonate aren’t considered vitamins


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Lipid vitamins Contain rings & long aliphatic sidechains

At least one polar group in each

Absorbed in intestine, carried via bile salts

Hard to study Most are formally built from isoprene units, as are steroids


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Vitamin A (retinol) 3 forms varying in terminal polar group

Involved in signaling and receptors -carotene is nonpolar dimer


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Vitamin A deficiency

Produces night blindness because the retina and cornea dry out

Most common cause: nursing infants whose mothers have vitamin A deficiency in their diet


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Vitamin D

Several related forms

Hormones involved in Ca2+ regulation

Figure courtesyCyberlipid

(cholecalciferol)


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Vitamin D deficiency Rickets in children:Bone disease, restlessness, slow growth

One form of vitamin D is actually synthesizable from cholesterol given adequate sunlight;

Therefore rickets is most common in densely settled urban environments


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Vitamin E (-tocopherol)

Phenol can undergo 1e- oxidation to moderately stable free radical

Antioxidant activity prevents damage to fatty acids in membranes

Fig. CourtesyUIC pharmacy program

phenol


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Vitamin K (phylloquinone)

Involved in synthesis of proteins involved in blood coagulation

Reduced form involved as reducing agent in carboxylation reaction on glu sidechainsFigure courtesy

Cyberlipid


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Vitamin overdoses?

It’s difficult to overdose on water-soluble vitamins: excess is simply excreted

Fat-soluble vitamins are stored in adipose tissue and can accumulate to high concentrations

May be toxic even dietarily Therefore: don’t eat polar bear liver

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.


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Ascorbate

The only common water-soluble vitamin that is not a coenzyme or coenzyme precursor

Vitamin in primates, some rodents Synthesizable in most other vertebrates

Involved in collagen Reduced form acts as reducing agent during hydroxylation of collagen

Deficiency gives rise to inadequate collagen - scurvy


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PTM role of ascorbate

Proline + O2 + -ketoglutarate + ascorbate 4-hydroxyproline + succinate + CO2 + dehydroascorbate

This is a post-translational modification that occurs to prolines within collagen

The hydroxylated prolines help stabilize the collagen triple helix

Hydroxylysine found in collagen too


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Dietary deficiency of ascorbate Primary sources of ascorbate are fruits, particularly citrus, and green vegetables

Ascorbate deficiency’s first symptom involves collagen degradation, leading to scurvy



Image courtesy U.Cincinnati Medical School


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Scurvy in history Shortage of green vegetables

in sailors’ diets meant scurvy was rampant on shipboard until the 18th century

Success of English navy over French 1760-1800 was partly due to the introduction of limes in English sailors’ diets 50 years before the French caught on




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Megadoses of ascorbate Linus Pauling (2-time Nobel laureate) became convinced late in his life that very high doses of ascorbate (> 1 g /day) were beneficial as a preventative

His assertions were met with skepticism from the established medical community

I would say the jury is still out!



Linus PaulingImage courtesy Oregon State U.

12/1/2009Biochemistry: Metabolism I General Metabolism Principles; Nutrition Andy Howard...

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