Biochemistry 432/832

February 21February 21

Chapters 27 and 28Chapters 27 and 28

Nucleic acid metabolism

Integration of metabolic pathways

Announcements:

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DNA DNA synthesissynthesis

Synthesis of Synthesis of deoxyribo-deoxyribo-nucleotides --- nucleotides --- reduction at the reduction at the 2’-position of 2’-position of the ribose ring the ribose ring of nucleoside of nucleoside diphosphatesdiphosphates

Deoxyribonucleotide Biosynthesis

• Reduction at 2’-position commits nucleotides to DNA synthesis

• Replacement of 2’-OH with hydride is catalyzed by ribonucleotide reductase

• An 22-type enzyme - subunits R1 (86 kDa) and R2 (43.5 kDa)

• R1 has two regulatory sites, a specificity site and an overall activity site

E.coli ribonucleotide reductaseE.coli ribonucleotide reductase

Regulation of deoxynucleotide synthesisRegulation of deoxynucleotide synthesis

Synthesis of dTMPSynthesis of dTMP

Synthesis of Thymine Nucleotides

• Thymine nucleotides are made from dUMP, which derives from dUDP, dCDP

• dUDPdUTPdUMPdTMP

• dCDPdCMPdUMPdTMP

• Thymidylate synthase methylates dUMP at 5-position to make dTMP

• N5,N10-methylene THF is 1-C donor

• Role of 5-FU in chemotherapy

The dCMP deaminase reactionThe dCMP deaminase reaction

The thymidylate synthase reactionThe thymidylate synthase reaction

Structure Structure of fluoro of fluoro compounds compounds - - thymine thymine analogsanalogs - -inhibitors inhibitors of DNA of DNA synthesissynthesis

Integration of metabolic pathways

Systems Analysis of MetabolismCatabolic and anabolic pathways, occurring

simultaneously, must act as a regulated, orderly, responsive whole

• catabolism, anabolism and macromolecular synthesis • Just a few intermediates connect major systems - sugar-

phosphates, alpha-keto acids, CoA derivatives, and PEP

• ATP & NADPH couple catabolism & anabolism

• Phototrophs also have photosynthesis and CO2 fixation

systems

Intermediary metabolism

28.2 Metabolic StoichiometryThree types of stoichiometry in biological systems

• Reaction stoichiometry - the number of each kind of atom in a reaction

• Obligate coupling stoichiometry - the required coupling of electron carriers

• Evolved coupling stoichiometry - the number of ATP molecules that pathways have evolved to consume or produce

The Significance of 38 ATPs

• Glucose oxidation

• If 38 ATP are produced, cellular G is -967 kJ/mol

• If G = 0, 58 ATP could be made

• So the number of 38 is a compromise

The ATP EquivalentWhat is the "coupling coefficient" for ATP produced or

consumed?

• Coupling coefficient is the moles of ATP produced or consumed per mole of substrate converted (or product formed)

• Cellular oxidation of glucose has a coupling coefficient of 30-38 (depending on cell type)

• Hexokinase has a coupling coefficient of -1

• Pyruvate kinase (in glycolysis) has a coupling coefficient of +1

The ATP Value of NADH vs NADPH

• The ATP value of NADH is 2.5-3

• The ATP value of NADPH is higher

• NADPH carries electrons from catabolic pathways to biosynthetic processes

• [NADPH]>[NADP+] so NADPH/NADP+ is a better e- donating system than NADH/NAD

• So NADPH is worth 3.5-4 ATP!

Nature of the ATP EquivalentA different perspective

G for ATP hydrolysis says that at equilibrium the concentrations of ADP and Pi should be vastly greater than that of ATP

• However, a cell where this is true is dead

• Kinetic controls over catabolic pathways ensure that the [ATP]/[ADP][Pi] ratio stays very high

• This allows ATP hydrolysis to serve as the driving force for nearly all biochemical processes

Substrate CyclesIf ATP c.c. for a reaction in one direction differs from c.c.

in the other, the reactions can form a substrate cycle

• The point is not that ATP can be consumed by cycling • But rather that the difference in c.c. permits both

reactions (pathways) to be thermodynamically favorable at all times

• Allosteric effectors can thus choose the direction and/or regulate flux in the pathway!

Substrate cycles

Unidirectionality of PathwaysA "secret" role of ATP in metabolism

• Metabolic pathways proceed in one direction• Either catabolic or anabolic, not both• Both directions of any pair of opposing pathways

must be favorable, so that allosteric effectors can control the direction effectively

• The ATP coupling coefficient for any such sequence has evolved so that the overall equilibrium for the conversion is highly favorable

ATP coupling coefficients for fatty acid oxidation and synthesis

‘Energy Charge’• Adenylates provide phosphoryl groups to drive

thermodynamically unfavorable reactions

• Energy charge is an index of how fully charged adenylates are with phosphoric anhydrides (number of phosphoric anhydrate bonds divided by total adenylate pool)

• E.C. = (2ATP+ADP) / 2 (ATP+ADP+AMP)

• If [ATP] is high, E.C.1.0

• If [ATP] is low, E.C. 0

Relative concentrations of AMP, ADP and ATP as a function of energy charge

Responses of regulatory enzymes to variation in energy charge

Catabolic

Anabolic

The oscillation of energy charge as a consequence of R and U processes

• Organs and tissues have metabolic profiles (specialized)

• Reflect metabolic function

• Brain - glucose uptake

• Muscle - Cori cycle (lactate)

• Adipose - storage of fat

• Liver - glucose synthesis

• Heart - prefers fatty acids as fuel (no storage)

• Differences: function, preferred fuel, whether or not fuel stored, what energy precursors they exploit

Metabolism in a multicellular organism

Fueling the Brain• Brain has very high metabolism but has no fuel

reserves

• This means brain needs a constant supply of glucose

• 120 g glucose and 20% of O2 consumes, mass of brain is 2%

• In fasting conditions, brain can use ketone bodies (from fatty acids)

• This allows brain to use fat as fuel!

Metabolism in a multicellular organism

Muscle• Muscles must be prepared for rapid provision of

energy

• Resting state: 30% of O2, exercise: 90% of O2

• Fuel source: glucose (exercise), fatty acids (resting state)

• Stored fuel: Glycogen (local) provides additional energy, releasing glucose for glycolysis

• No export of glucose (lactate is exported)

Muscle Protein Degradation

• During fasting or high activity, amino acids are degraded to pyruvate, which can be transaminated to alanine

• Alanine circulates to liver, where it is converted back to pyruvate - food for gluconeogenesis

• This is a fuel of last resort for the fasting or exhausted organism

Adipose tissue

• Function: storage depot for fatty acids

• release of f.a. into bloodstream

Liver• Function: main metabolic processing center

• Regulates glucose metabolism (blood G <-> liver G <-> glycogen)

• Regulates fat metabolism

• Fed conditions (synthesis of f.a. ->TAG -> storage)

• Fasting (srorage ->f.a.-> acetyl-CoA)

Control of metabolic pathways• Substrate/product activation/inhibition (product of a

pathway inhibits committed step; substrate activates the pathway)

• allosteric control (binding of an effector at one site affects enzyme activity at another site)

• covalent control (phosphorylation, adenylylation, redox, etc)

• gene expression– requires time (transcription - RNA synthesis, translation -

protein synthesis)

Metabolic conversion of glucose-6-phosphate in the liver

Common intermediates

Analyses of individual enzymes of pathways

Inhibitor analyses, radioisotopes, compartmentalization

Parallel analyses of thousands of enzymes or pathways

Bioinformatics, functional genomics, expression analyses, proteomics

Methods to study metabolism