Transcript Enzyme Mechanisms - Illinois Institute of Technology

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
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What we’ll discuss
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Metabolism
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Definitions
Pathways
Control
Biochemistry: Metabolism I
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Metabolism,
cont’d
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Feedback
Phosphorylation
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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!
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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?
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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
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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
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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
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Metabolism and energy
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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
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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, . . .
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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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