Transcript phosphorylation

General Metabolism I
Andy Howard
Introductory Biochemistry
24 November 2009
Biochemistry: Metabolism I
11/24/2009
Metabolism:
the core of biochem
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All of biology 402 will concern itself with
the specific pathways of metabolism
Our purpose here is to arm you with the
necessary weaponry
Biochemistry: Metabolism I
11/24/2009
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What we’ll discuss
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Metabolism
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Definitions
Pathways
Control
Feedback
Phosphorylation
Thermodynamics
Kinetics
Biochemistry: Metabolism I
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Cofactors
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Tightly-bound metal
ions as cofactors
Activator ions as
cofactors
Cosubstrates
Prosthetic groups
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Metabolism
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Almost ready to start the specifics
(chapter 18)
Define it!
Metabolism is the network of chemical
reactions that occur in biological
systems, including the ways in which
they are controlled.
So it covers most of what we do here!
Biochemistry: Metabolism I
11/24/2009
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Intermediary Metabolism
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Metabolism involving small molecules
Describing it this way is a matter of
perspective:
Do the small molecules exist to give the
proteins something to do, or do the
proteins exist to get the metabolites
interconverted?
Biochemistry: Metabolism I
11/24/2009
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How similar are pathways in
various organisms?
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Enormous degree of similarity in the
general metabolic approaches all the way
from E.coli to elephants
Glycolysis arose prior to oxygenation of
the atmosphere
This is considered strong evidence that
all living organisms are derived from a
common ancestor
Biochemistry: Metabolism I
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Anabolism and catabolism
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Anabolism: synthesis of complex
molecules from simpler ones
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Generally energy-requiring
Involved in making small molecules and
macromolecules
Catabolism: degradation of large
molecules into simpler ones
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Generally energy-yielding
All the sources had to come from
somewhere
Biochemistry: Metabolism I
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Common metabolic themes
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Maintenance of internal concentrations
of ions, metabolites, & (? enzymes)
Extraction of energy from external
sources
Pathways specified genetically
Organisms & cells interact with their
environment
Constant degradation & synthesis of
metabolites and macromolecules to
produce steady state
Biochemistry: Metabolism I
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Metabolism and energy
Biochemistry: Metabolism I
11/24/2009
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Metabolic classifications
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Carbon sources
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Autotrophs vs. heterotrophs
Atmospheric CO2 as a C source vs.
otherwise-derived C sources
Energy sources
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Phototrophs vs. chemotrophs
(Sun)light as source of energy vs.
reduced organic compounds as a source
of energy
Biochemistry: Metabolism I
11/24/2009
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Fourway divisions (table 17.2)
Energy/Carbon
Phototrophs:
Energy from light
Chemotrophs:
Energy from
reduced organic
molecules
Autotrophs:
Carbon from
atmospheric CO2
Photoautotrophs:
Green plants,
cyanobacteria, …
Chemoautotrophs:
Nitrifying bacteria,
H, S, Fe bacteria
Heterotrophs:
Photoheterotrophs: Chemoheterotrophs:
Carbon from other Nonsulfur purple
Animals, many
[organic] sources bacteria
microorganisms, . . .
Biochemistry: Metabolism I
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Another distinction: the
organism and oxygen
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Aerobes: use O2 as the ultimate electron
acceptor in oxidation-reduction reactions
Anaerobes: don’t depend on O2
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Obligate: poisoned by O2
Facultative: can switch hit
Biochemistry: Metabolism I
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Flow of energy
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Sun is ultimate source of energy
Photoautotrophs drive synthesis of
[reduced] organic compounds from
atmospheric CO2 and water
Chemoheterotrophs use those
compounds as energy sources & carbon;
CO2 returned to atmosphere
Biochemistry: Metabolism I
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How to anabolism &
catabolism interact?
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Sometimes anabolism & catabolism
occur simultaneously.
How do cells avoid futile cycling?
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Just-in-time metabolism
Compartmentalization:
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Anabolism often cytosolic
Catabolism often mitochondrial
Biochemistry: Metabolism I
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Pathway
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A sequence of reactions such that the
product of one is the substrate for the next
Similar to an organic synthesis scheme
(but with better yields!)
May be:
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Unbranched
Branched
Circular
Biochemistry: Metabolism I
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Catabolism stages
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Stage 1: big nutrient macromolecules
hydrolyzed into their building blocks
Stage 2: Building blocks degraded into
limited set of simpler intermediates,
notably acetyl CoA
Stage 3: Simple intermediates are fed to
TCA cycle and oxidative phosphorylation
Biochemistry: Metabolism I
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Anabolism
stages
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Short list of
simple precursors
These are elaborated
in characteristic ways to build monomers
e.g.: transamination of -ketoacids to make
-amino acids
Those are then polymerized to form
proteins, polysaccharides, polynucleotides,
etc.
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Some intermediates play two
roles
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Some metabolites play roles in both
kinds of pathways
We describe them as amphibolic
Just recall that:
catabolism is many down to few,
anabolism is few up to many
Biochemistry: Metabolism I
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Differences between catabolic
and anabolic pathways
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Often they share many reactions, notably
the ones that are nearly isoergic (G ~ 0)
Reactions with G < -20 kJ mol-1 are not
reversible as is
Those must be replaced by (de)coupled
reactions so that the oppositely-signed
reactions aren’t unfeasible
Biochemistry: Metabolism I
11/24/2009
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Other differences
involve regulation
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Generally control mechanisms influence
catalysis in both directions
Therefore a controlling influence
(e.g. an allosteric effector)
will up- or down-regulate both directions
If that’s not what the cell needs, it will
need asymmetric pathways or pathways
involving different enzymes in the two
directions
Biochemistry: Metabolism I
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ATP’s role
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We’ve discussed
its significance as
an energy currency
It’s one of two energy-rich products of the
conversion of light energy into chemical
energy in phototrophs
ATP then provides drivers for almost
everything else other than redox
Biochemistry: Metabolism I
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NAD’s role
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QuickTime™ and a
decompressor
are needed to see this picture.
NAD acts as as
an electron
acceptor via net
Image courtesy
Michigan Tech
transfer of hydride ions,
Biological Sciences
H:-, in catabolic reactions
Reduced substrates get oxidized in the
process, and their reducing power ends up in
NADH
Energy implied by that is used to make ATP
(3.5 ATP/NAD) in oxidative phosphorylation
Biochemistry: Metabolism I
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NADPH’s
role
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Involved in
anabolic redox
reactions
Reducing power in NADPH  NADP
used to reduce some organic molecule
Involves hydride transfers again
NADPH regenerated in phototrophs via
light-dependent reactions that pull
electrons from water
Biochemistry: Metabolism I
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How do we study
pathways?
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Inhibitor studies
Mutagenesis
Isotopic traces (radio- or not)
NMR
Disruption of cells to examine which
reactions take place in which organelle
Biochemistry: Metabolism I
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Why multistep pathways?
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Limited reaction specificity of
enzymes
Control of energy input and output:
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Break big inputs into ATP-sized inputs
Break energy output into pieces that
can be readily used elsewhere
Biochemistry: Metabolism I
11/24/2009
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iClicker quiz question 1
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A reaction A+B  C+D proceeds from left
to right in the cytosol and from right to left
in the mitochondrion. As written, it is
probably
(a) a catabolic reaction
(b) an anabolic reaction
(c) an amphibolic reaction
(d) we don’t have enough information to
answer.
Biochemistry: Metabolism I
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iClicker quiz question 2
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An asymmetry between stage 1 of catabolism
(C1) and the final stage of anabolism (A3) is
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(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
Biochemistry: Metabolism I
11/24/2009
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iClicker quiz question 3
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Could dAMP, derived from degradation of
DNA, serve as a building block to make
NADP?
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(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
Biochemistry: Metabolism I
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Regulation
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Organisms respond to change
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Fastest: small ions move in msec
Metabolites: 0.1-5 sec
Enzymes: minutes to days
Flow of metabolites is flux:
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steady state is like a leaky bucket
Addition of new material replaces the
material that leaks out the bottom
Biochemistry: Metabolism I
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Metabolic flux, illustrated
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Courtesy Jeremy Zucker’s wiki
http://bio.freelogy.org/wiki/User:JeremyZucker#Metabolic_Engineering_tutorial
Biochemistry: Metabolism I
11/24/2009
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Feedback and
Feed-forward
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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
Biochemistry: Metabolism I
11/24/2009
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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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11/24/2009
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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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Biochemistry: Metabolism I
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Activation and inactivation by
post-translational modification
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Most common:
covalent phosphorylation of protein
usually S, T, Y, sometimes H
Kinases add phosphate
Protein-OH + ATP 
Protein-O-P + ADP
… ATP is source of energy and Pi
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Phosphatases hydrolyze phosphoester:
Protein-O-P +H2O Protein-OH + Pi
… no external energy source required
Biochemistry: Metabolism I
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Phosphorylation’s effects
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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
Biochemistry: Metabolism I
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Glycogen phosphorylase
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Reaction: extracts 1 glucose
unit from non-reducing end of
glycogen & phosphorylates it:
(glycogen)n + Pi 
(glycogen)n-1 + glucose-1-P
Activated by phosphorylation
via phosphorylase kinase
Deactivated by
dephosphorylation by
phosphorylase phosphatase
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Amplification
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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
Biochemistry: Metabolism I
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Other PTMs
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Are there other reversible posttranslational modifications that regulate
enzyme activity? Yes:
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Adenylation of Y
ADP-ribosylation of R
Uridylylation of Y
Oxidation of cysteine pairs to cystine
Cis-trans isomerization of prolines
Biochemistry: Metabolism I
11/24/2009
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Evolution of Pathways:
How have new pathways evolved?
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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
Biochemistry: Metabolism I
11/24/2009
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Adding a step
E1
E2
E3
E4
E5
ABCDEP
Original pathway
• 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
Biochemistry: Metabolism I
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Evolving a branch
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Original pathway:
D
E1 E2
A  B  C E3
X
Fully evolved pathway:
E3a D
ABC
E3b X
Biochemistry: Metabolism I
11/24/2009
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Backward evolution
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Original system has lots of E  P
E gets depleted over time;
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Then D gets depleted;
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need to make it from D,
so we evolve enzyme E4 to do that.
need to make it from C,
so we evolve E3 to do that
And so on
Biochemistry: Metabolism I
11/24/2009
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Duplicated pathways
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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)
Biochemistry: Metabolism I
11/24/2009
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Reversing a pathway
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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 CD 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
Biochemistry: Metabolism I
11/24/2009
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Many cofactors are
derived from vitamins
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We justify lumping these two
topics together because many
cofactors are vitamins or are
metabolites of vitamins.
Biochemistry: Metabolism I
11/24/2009
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Family tree of cofactors
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Cofactors, coenzymes, essential ions,
cosubstrates, prosthetic groups:
Cofactors
(apoenzyme + cofactor  holoenzyme)
Coenzymes
Essential ions
Activator ions
(loosely bound)
Ions in
metalloenzymes
Cosubstrates
(loosely bound)
Biochemistry: Metabolism I
11/24/2009
Prosthetic groups
(tightly bound)
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Metal-activated enzymes
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Absolute requirements for mobile ions
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Often require K+, Ca2+, Mg2+
Example: Kinases: Mg-ATP complex
Metalloenzymes: firmly bound metal
ions in active site
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Usually divalent or more
Sometimes 1e- redox changes in metal
Biochemistry: Metabolism I
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Coenzymes
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Organic moeities that enable enzymes to
perform their function: they supply
functionalities not available from amino
acid side chains
Cosubstrates
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Enter reaction, get altered, leave
Repeated recycling within cell or organelle
Prosthetic groups
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Remain bound to enzyme throughout
Change during one phase of reaction,
eventually get restored to starting state
Biochemistry: Metabolism I
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Major cosubstrates
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Facilitate group transfers, mostly small groups
Oxidation-reduction participants
Cosubstrate
ATP
S-adenosylMet
UDP-glucose
NAD,NADP
Coenzyme A
Tetrahydrofolate
Ubiquinone
Source
Function
Transfer P,Nucleotide
Methyl transfer
Glycosyl transfer
Niacin
2-electron redox
Pantothenate Acyl transfer
Folate
1Carbon transfer
Lipid-soluble e- carrier
Biochemistry: Metabolism I
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Major prosthetic groups
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Transfer of larger groups
One- or two-electron redox changes
Prosth.gp.
FMN, FAD
TPP
PLP
Biotin
Adenosylcobalamin
MeCobal.
Lipoamide
Retinal
Vitamin K
Source
Riboflavin
Thiamine
Pyridoxine
Biotin
Cobalamin
Function
1e- and 2e- redox transfers
2-Carbon transfers with C=O
Amino acid group transfers
Carboxylation, COO- transfer
Intramolec. rearrangements
Cobalamin
Methyl-group transfers
Transfer from TPP
Vision
Carboxylation of glu residues
Vitamin A
Vitamin K
Biochemistry: Metabolism I
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Adenosine triphosphate
Synthesizable in liver (chapter 18)
Building block for RNA
Participates in phosphoryl-group transfer
in kinases
Source of other coenzymes
Biochemistry: Metabolism I
11/24/2009
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S-adenosylmethionine
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Made from methionine and adenosine
Sulfonium group is highly reactive: can
donate methyl groups
Reaction diagram courtesy of
Eric Neeno-Eckwall, Hamline
University
Biochemistry: Metabolism I
11/24/2009
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UDP-glucose
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Most common donor of glucose
Formed via:
Glucose-1P + UTPUDP-glucose + PPi
Reaction driven to right by PPi hydrolysis
Structure courtesy of UIC
Pharmacy Program
Biochemistry: Metabolism I
11/24/2009
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NAD+ and NADP+
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Net charge isn’t really >0 ;
the + is just a reminder that the
nicotinamide ring is positively charged
Most important cosubstrates in oxidationreduction reactions in aerobic organisms
Structure courtesy of
Sergio Marchesini, U.
Brescia
Biochemistry: Metabolism I
11/24/2009
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Differences between them
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The chemical difference is in the
phosphorylation of the 2’ phosphate group of
the ribose moiety
The functional difference is that NAD is
usually associated with catabolic reactions
and NADP is usually associated with anabolic
reactions
Therefore often NAD+ and NADPH are
reactants and NADH and NADP+ are products
Exceptions: photosynthesis and ETC!
Biochemistry: Metabolism I
11/24/2009
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How do we get back to the
starting point?
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NADH is often oxidized back to NAD+ as
part of the electron-transport chain
Imbalances can be addressed via
NAD Kinase (S.Kawai et al (2005),
J.Biol.Chem. 280:39200) and NADP
phosphatase
Biochemistry: Metabolism I
11/24/2009
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iClicker quiz question 4
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Based on what you have learned, would
you expect glycogen synthase to be
activated or inhibited by phosphorylation?
(a) activated
(b) inhibited
(c) neither
(d) insufficient information to tell
Biochemistry: Metabolism I
11/24/2009
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iClicker quiz question 5
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What would you expect to be the
phosphate donor in the NAD kinase
reaction?
(a) free phosphate
(b) pyrophosphate
(c) ATP
(d) pyridoxal phosphate
Biochemistry: Metabolism I
11/24/2009
Page 58 of 75
Reduced forms of NAD(P)
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Reduction occurs on the
nicotinamide ring
Ring is no longer netpositive
Ring is still planar but
the two hydrogens on
the para carbon are not
Biochemistry: Metabolism I
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FAD and FMN
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Flavin group based on riboflavin
Alternate participants in redox reactions
Prosthetic groups: tightly but noncovalently
bound to their enzymes
That protects against wasteful reoxidation of
reduced forms
FADH2 is weaker reducing agent than NADH
These are capable of one-electron oxidations
and reductions
Biochemistry: Metabolism I
11/24/2009
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FAD and FMN structures
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FAD has an AMP attached P to P
Structure courtesy
Paisley University
Biochemistry: Metabolism I
11/24/2009
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FMN/FAD redox forms
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Two-electron version: H+ + :H- transferred
Reaction diagram courtesy of Eric
Neeno-Eckwall, Hamline University
Biochemistry: Metabolism I
11/24/2009
Page 62 of 75
(ADP-3’P)
Coenzyme A
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Reactive portion
is free sulfhydryl
at one end of
the molecule
Can form
thioester with
acetate, etc.
Pantoate +
b-alanine =
pantothenate
Biochemistry: Metabolism I
(Pantoate)
2-mercaptoethylamine)
b-alanine)
Structure courtesy of
MPB project, George
Washington University
11/24/2009
Page 63 of 75
Thiamine Pyrophosphate
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Based on thiamine, vitamin B1
Carboxylases and oxidative
decarboxylases use this coenzyme
So do transketolases (move 2 carbons
at a time between sugars with keto
groups)
Thiazolium ring is reactive center:
pKa drops from 15 in H2O to 6 in
enzyme
Biochemistry: Metabolism I
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TPP reactions
pyrimidine
thiazolium
Diagram courtesy of
Oklahoma State U.
Biochemistry program
Biochemistry: Metabolism I
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Pyridoxal
phosphate
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PLP is prosthetic group for many
amino-acid-related enzymes,
particularly transaminations
Carbonyl group of PLP bound as
a Schiff base (imine) to -amino
group of lysine at active site
First step is always formation of
external aldimine; goes through
gem-diamine intermediate to
internal aldimine
Biochemistry: Metabolism I
11/24/2009
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Biotin
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Rarity: vitamin is the prosthetic group
Used in reactions that transfer carboxyl
groups
… and in ATP-dependent carboxylations
Biochemistry: Metabolism I
11/24/2009
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Biotin reactivity
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Covalently bound to active-site lysines to
form species called biocytin
Pyruvate carboxylase is characteristic
reaction:
Diagram courtesy
University of Virginia Biochemistry
Biochemistry: Metabolism I
11/24/2009
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Tetrahydrofolate
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Primary donor of one-carbon units
(formyl, methylene, methyl)
Supplies methyl group for thymidylate
Dihydrofolate reductase (DHFR) is an
interesting drug target
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Methotrexate as cancer chemotherapeutic:
cancer needs more thymidylate than healthy cells
Trimethoprim as antibacterial:
Bacterial DHFR is somewhat different from
eucaryotic DHFR because bacteria derive DHF
from other sources; humans get it from folate
Biochemistry: Metabolism I
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THF structure and function
Figure courtesy
horticulture program,
Purdue
Biochemistry: Metabolism I
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Cobalamin
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Largest B vitamin
Structure related to heme but missing
one carbon in ring structure
Cobalt bound in core of ring system
Involved in enzymatic rearrangements
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Catabolism of odd-chain fatty acids
Methylation of homocysteine
Reductive dehalogenation
Biochemistry: Metabolism I
11/24/2009
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AdenosylCobalamin
Reactive
Co-C bond
“Missing” carbon
Diagram courtesy of
Swiss Food News
Biochemistry: Metabolism I
11/24/2009
Page 72 of 75
Lipoamide
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

Protein-bound form of lipoic acid
Contains five-membered disulfide ring
Covalently bound via amide to protein
lysine sidechain
Involved in swinging arm between active
sites in multienzyme complexes
Disulfides break periodically
Example: pyruvate dehydrogenase
complex
Biochemistry: Metabolism I
11/24/2009
Page 73 of 75
Lipoamide 2e- reduction

Cf. Scheme 7.6: thioester starting point
Fig. Courtesy Biochem
and Biophysics
program, Rensselaer
Biochemistry: Metabolism I
11/24/2009
Page 74 of 75
iClicker quiz question 6

Which coenzyme would you expect
would be required for the reaction
oxaloacetate + glutamate 
aspartate + -ketoglutarate?
(a) ascorbate
(b) PLP
( c) thiamine pyrophosphate
(d) NAD
(e) none of the above
Biochemistry: Metabolism I
11/24/2009
Page 75 of 75