Transcript Chapter 26 - s3.amazonaws.com

Biochemistry 432/832
February 21
Chapters 27 and 28
Nucleic acid metabolism
Integration of metabolic
pathways
Announcements:
-
DNA
synthesis
Synthesis of
deoxyribonucleotides --reduction at the
2’-position of
the ribose ring
of nucleoside
diphosphates
Deoxyribonucleotide
Biosynthesis
• Reduction at 2’-position commits
nucleotides to DNA synthesis
• Replacement of 2’-OH with hydride is
catalyzed by ribonucleotide reductase
• An 22-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 reductase
Regulation of deoxynucleotide synthesis
Synthesis of dTMP
Synthesis of Thymine
Nucleotides
• Thymine nucleotides are made from dUMP,
which derives from dUDP, dCDP
• dUDPdUTPdUMPdTMP
• dCDPdCMPdUMPdTMP
• 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 reaction
The thymidylate synthase reaction
Structure
of fluoro
compounds
- thymine
analogs inhibitors
of DNA
synthesis
Integration of metabolic
pathways
Systems Analysis of Metabolism
•
•
•
•
Catabolic 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 - sugarphosphates, 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 Stoichiometry
Three 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 Equivalent
What 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 Equivalent

•
•
•
A 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 Cycles
If 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 Pathways
•
•
•
•
A "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
Metabolism in a multicellular organism
• 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!
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)
Common
intermediates
Metabolic
conversion
of glucose-6phosphate in
the liver
Methods to study metabolism
Analyses of individual enzymes of
pathways
Inhibitor analyses, radioisotopes,
compartmentalization
Parallel analyses of thousands of enzymes
or pathways
Bioinformatics, functional genomics,
expression analyses, proteomics