- Looking at a comparison between an animal that lives in low temperature
and an animal that lives in high temperatures. Low temp would have a low
rate function, and high temp would have a high rate function. (so graph
would be a straight line). The reason for that is kinetics, kinetics are
temperature sensitive. You can make an animal live at a different
temperature, and its rate function would just shift down the graph. If you
look at rate function (lets say metabolic rate) of goldfish at 25 degrees, and
look at the one that’s adapted to 10 degrees, and if you look at the Antarctic
fish (-1.86 degrees water), they all follow the horizontal line which means
organisms can adjust their metabolic rate within a wide range of
temperatures so basically its always the same. This is called rate
compensation. They have an ability to adjust; they can do that do that on a
seasonal basis, and an on evolutionary basis. Paper that Hochachka/Somero
wrote in 1968 called Adaptation of Enzymes to Temperature and proposed a
mechanism to explain this. They suggested that temperature (besides having
kinetic effect) also could modify activities, meaning it would directly modify
the enzyme and therefore the activity.
- Hard to define adaptation, because its overused and it occurs at all biological
levels. Have an idea of what adaptation is (exam). First definition: basically
increasing fitness in a certain environment. Second: there are certain things
that are important for an organism in the environment and if they don’t
respond to them they die.
- Key ideas are listed in slides. But there are problems with these definitions.
What is an environment? Where the organism lives or the ecological niche
which is controlled by biotic and abiotic factors. Adaptation has to be
considered as optimization process (can solve problems but only to a certain
extent) and there is no such thing as perfection. It is a continuous process
because the environment is constantly changing. If there is a change in the
amount of oxygen, for example a reduced amount in water, the fish increases
amount of water flow across its gills or it could start using anaerobic
metabolism. (different solution for same problem). 3 determinants to
adaptation: first is genome specified which means the genome of that
organism really determines the limits of its adaptational ability (certain
things organisms can do that others cant because they don’t have those
genes). The second thins is its environmentally induced, so environment is
responsible for bringing adaptation to surface. Developmentally defined
means that in certain stages of development you are more likely to adapt.
- Two components of natural selection. One of them is that organisms very and
the variation is inherited in part by offspring. The second component is that
organisms can produce more offspring than can possible survive (Atlantic
cod, produce millions of eggs but only 1 may survive).
- Differential survival is an accumulation of positive or favorable mutations
and those are the ones that will contribute to next generation. Time is
extremely important because evolution doesn’t happen quickly. - Must be variability in the organisms like phenotypic plasticity. Evolution
doesn’t start from zero, it relies on variation from the past generation.
Processes are random, thus different phenotypes are possible. No such thing
as the most advanced organisms, but some can be more highly evolved.
Selection is to existing environments, not one that happened before or will
- Central dogma- proteinB. 3 characteristics to any
enzyme. Catalysis meaning each one has a catalytic rate at concentration per
unit time. The second one is regulation, where they can have their
concentration regulated, rate etc… Third component is the structure or
confirmation of any enzyme. Any enzyme has a primary structure, secondary
(folds), tertiary (folding of the folded structure), quaternary (subunits that
come together). Most enzymes are part of metabolic pathways but there are
some that are in isolation. How do enzymes work> Boltzmendist distribution
is particular molecules that have a specific amount of energy. Uncatalyzed
reactions take a smaller amount of energy because they already have enough
energy to do it on its own. The catalytic process converts more of those
molecules to product, and moves the free energy line to the left.
- Generally an enzyme is large, however the site at which catalysis actually
occurs which is called the catalytic vacuole, or active site of the enzyme is
relatively small. Once LDH (enzyme) binds the substrates, the confirmation
of the enzyme changes (called induced fit) and there is strain and contortion
which breaks the substrates apart and create products. Size of big enzyme
puts more pressure on substrate to change. Site-directed mutagenesis
changes the protein by mutating certain amino acids in the active site. What determines the confirmation? The amino acid, and the environment of the
- Molten globule is undifferentiated amino acids linked together. It is an open
structure, starting point of final confirmation of the protein. Michael Smith,
Nobel prize. C Anfinsen also got a Nobel prize he started out with a protein
with a very specific confirmation and put it in the presence of urea and it
makes the protein linear (breaks down II and III structure of protein), then
he removed urea and got back to original structure and that indicated that
there is information within that primary sequence which directs
confirmation of that protein, and that tends to be the side chains of those
amino acids. Hydrophilic side chains interact with water and hydrophobic go
in the centre of the molecule.
- Smaller proteins can fold spontaneously but large proteins need assistance
with the presence of chaperones and one of the main ones is called HSP70.
- Summary of protein formation- all of the steps can be modified by the
- How the enzyme is regulated. 3 mechanisms- concentration or quantity, type
or quality, modulation. Concentration or quantity of the specific protein in
the cell is hard to determine. 70% of the total weight of the cell is proteins.
Western blotting will determine amount of specific protein in a cell, uses
antibodies. Another way is antibiody precipitation. Make antibodies of LDH,
precipitate LDH specific protein and quantify it. Direct relationship between
concentration and activity. Glutamate dehydrogenase is in the mitochondria
so we can determine more easily how much of it there is.
- What determines enzyme concentration. [Enzyme]= Rate of synthesis- rate of
degradation. By changing either rate we can either increase or decrease it.
First we start off with DNA, and copy number is extremely important.
Glucokinase is an enzyme that phosphorylates glucose and insulin controls it.
- Quantitative strategy is important but it takes time to synthesize a protein
but more importantly is that time= energy.
- Last time- enzymes have 3 general principles- catalysis, regulation,
structure. Conformation of enzyme is determined by amino acid sequence
and the microenvironment of the protein. If it’s the amino acids that are
important, then some fold spontaneously and some have assistance to fold.
How do we regulate the amount or the type of the enzyme that’s present?
- Type or qualitative strategy. Not talking about an evolutionary process but
how we can change the existing proteins or enzymes which are found in that
cell. First way to change type of enzyme is through process called alternative
splicing. Its estimated that 74% multi-exon genes can be spliced. Means that
some exons can be shuffled (1234->1324) or you can delete (1234->124).
The protein that’s formed has now changed. Splicosome is made up of RNA
and protein and has the ability to change these pre-mrna to make functional
mrna. The DNA might be polymorphic. Within the genome of an organism
there is a 1% change over a million years. What kinds of variation? Gene duplication events (the frequency is minor) but they become fixed within the
genome so they get transferred from one generation to the next. Allelic
variation- changes in nucleotide sequences, it is quite frequent but allelic can
be lost during reproduction.
- Evolution was associated with 3 whole genome duplications. Process of
differential duplication and also called segmental duplication. Its when a
species have two of the same genes. They are either individual genes or
clusters of genes. What is the outcome of these duplication events? Outcome
is going to be more genetic material, when you have more genetic material
you might get selection of it, different proteins which can be used
differentially. The outcome depends on the amount of divergence within
those genes. Partial divergence- genes only partially diverge from one
another. There has been a number of amino acids that have been changed
within the protein so the function is altered a bit, still binds same substrates.
Often call them isozymes or isoforms. Complete divergence is when the
duplicated locus is completely different than the original one, major changes
in amino acid sequences. It no longer recognizes the same substrate (7-TMD
dehydrogenases). What is a characteristic of a dehydrogenase? They are
redox reducers, oxidizers (NAD(H), NADP(H) binding site), lactate
- All adrenoreceptors. There is a large number of different kinds of receptors.
You can see the 3 different geno duplication evenets. 1R, 2R (tetrapod
divergence) and 3R.(occurred only in the fish, when you look at pink stars
only see them for alpha adrenoreceptors). Some ancestral gene duplicated
and formed two arms of a certain tree. All the proteins are homologous.
- Orthologs are proteins that come from a common ancestor and are found in a
number of different species and they usually have a very close function
between species. Paralogs are different genes found in the same organism
that arise from a gene duplication event (alpha mouse and beta mouse would
- Expression of variation. Start off with an original locus, X. Duplication occurs,
now you have two locus X. Both can generate the same gene products. One of
the loci can become silent, but it may also accumulate mutations. As long as
they are not massive, you can generate locus X’ and gene product X’. If there
is complete divergence we get a family of proteins, so they diverged enough
from each other to indicate they belong to one another but they don’t bind
same substrate. We can also have Allelic variation. If two alleles are the same,
it is considered homozygous x. However one of the alleles may go through
mutations in their nucleotides to form a different type of allele (slightly
- Allelic variation. Generally they are considered to be point mutations and
allozymes are products of the heterozygous locus. They don’t “breed true”
because one of the alleles goes into the mother and the other into the father
and you don’t know which one you’re going to get. So in the next generation
the allozymes may not remain there. - How do we detect variation. The easiest way is to look at the proteins by
electrophoresis. Isoelecrofocusing is more specific because it also uses a pH
gradient. The problems with using either one is that proteins don’t have a
huge difference in electric charge (only 5 charged amino acids).
- Tried to see the amount of variation between animals. Came up with average
heterozygosity (HET with line on top is the average # or loci that are
polymorphic). The end result from the population geneticists, when you look
at invertebrates you get 47% polymorphism. A lot more variation than what
people thought. This represents the raw material for natural selection. If they
are selected for it then they become a part of the population.
- Selectionists vs neutralists. Debate if it was important or not. Important
outcome was the molecular clock where it states that some proteins are
evolving quickly and some very slowly. Histone would be a very slow
evolving protein because it interacts with DNA. A rapidly evolving protein is
fibrin or thymin. The scientists looked at animals that lived in stable
environments vs animals that lived in changing environments. Higher
complexity = higher HET value. They looked at these differences and there
was no correlation what so ever. Paper came out last year and they got the
whole oyster genome and found that it is highly polymorphic or nucleotides
were quite different between individuals. They had a lot of the same
sequences, and were over represented with host-defense genes against biotic
and abiotic stress. There may be a variation at the nuclotide basis between
where the organism lives and HET (?).
- Hb is an honorary enzyme because it does everything enzymes do but it uses
oxygen. Myoglobin is one subunit and Hb is a 4-subunit protein. There are
difference between oxygen binding between the two. Myoglobin is found in
muscles and organs. Oxygen association curves between the two are
different. Why does myoglobin have such a high affinity for oxygen compared
to hemoglobin? Has to be on that side so it can steal oxygen from Hb.
Cytochrome oxidase would be on the far left because it takes oxygen from
myoglobin. // There are differences in hemoglobin’s, fetal vs adult. Fetal
hemoglobin binds oxygen easier because it steals oxygen from mothers
blood. There are also difference in interactions with modifiers. DPG is very
important when it comes to modulation in this particular protein.
- LDH. Isozyme story just like Hb but it is a different protein. Why do we have
LDH? Lactate dehydrogenase. Pyruvate is important because if there is no
oxygen, it will go to lactate, if there is oxygen it will go to acetyl CoA. Muscles
in legs is a white mucle, not much mitochondria so less oxygen, therefore
lactate is produced.
- Last time: Hemoglobin, LDH (multiple forms or isosymes) allows glycolysis
to continue with absence of oxygen. Modulation strategy: how we control
enzymes that exist in the cell. 4 ways we can do this: modulation by substrate
or small molecules. If you plot substrate concentration vs enzyme activity the curve will either be hyperbolic or sigmoidal. Vmax values depend upon the
concentration of the enzyme, so the only way you can change Vmax is if you
change the concentration of the enzyme. Km values are very specific to the
enzyme, it doesn’t matter how much you have but how it interacts with its
substrate. The higher the Km value, the lower the affinity for the substrate
the enzyme has. Hyperbolic ones have 1 or more subunit, 1 binding site per
subunit, and no interaction between subunit binding sites. For sigmodial or
allosteric, there are at least 4 subunits and they interact with one another
and they are either in the tense conformation or the relaxed conformatin..
- Hb cooperativity. Hb is made up of two subunits, alpha and beta. Without
oxygen, these subunits are in the tense state. In the presence of oxygen, the
subunits get pushed toward the relaxed state. Once oxygen binds one subunit
it binds to the others much more rapidly (progressive). There are a number
of modifiers of Hb: ATP, protons, DPG- very important modifiers of the ability
of oxygen to bind to Hb and they keep Hb in the tense state which does not
allow the subunits to go to the relaxed state. Puropose of the modifiers is so
that the oxygen can actually leave.
- Pyruvate Kinase exists at the bottom of glycolysis. Glucose--- PEP-> PYR-
>Lactate (via LDH). From PEP to PYR the enzyme is pyruvate kinase and it’s
the only enzyme in glycolysis that produces ATP. It shows sigmoidal kinetics
and it is a tetrometric enzyme just like Hb and have a PEP binding site. When
the first PEP binds (just like in case of Hb), it changes from a tense
confirmation to a relaxed confirmation and that binding site is filled with
PEP. These interactions are called homotropic interactions (same binding
sites on different subunits). There are also binding sights for negative and
positive modulators. Negative is Ala and positive is FBP. These binding sites
will interact with PEP binding sites and are called heterotropic interactions
(because they are different). When you ad Ala, it interacts with PEP binding
site in a negative state (away from relaxed state, to tense state). When you
add FBP, the PEP interacts positively with the binding site pushing the
enzyme to relaxed state. Generally the heterotropic modifiers do not affect
Vmax, but they interact within the Km values. FBP increases affinity of PEP
for that enzyme and Ala does the opposite. FBP also eliminates any
interaction between PEP binding sites (changes it to hyperbolic kinetics).
- (3 “lecture begins)
- Two general mechanisms. First one is an irreversible process. Pro-insulin is
made in the Beta cells of the pancreas but its useless. It has to be converted
to insulin. Pro-insulin is a prohormone and it is hydrolyzed, it removes
almost 30 amino acids to turn it into an active insulin and then can be
released and functional. Pro-insulin is not released, but the 29 amino acid
piece (c-peptide) is indicated for diabetes, so if you have a lot of it its good.
The other type of peptide-bond cleavage is trypsinogen. It gets released after
a meal by the pancreatic fluids and an enzyme called enterokinase cleaves a
few amino acids from trypsinogen to form trypsin (which is very active). By
keeping them in the inactive form (trypsinogen), then the pancreas isn’t over working itself but only after a meal, because enterokinase is only released
after a meal.
- Protein in centre and a huge number of posttranslational activity that can
occur. Phosphorylation occurs within enzymes to either activate the or
inhibit them. Phosphorylation/dephosphorilation are reversible processes.
This is important because of signaling.
- Glycogen phosphorylase. It contols glycogen metabolism. (glycogen is
storage of glucose). Glucose either goes through glycolysis or gets
metabolized. Glycogen phosphorylase had 2 forms, a (active) and b
(inactive). There are two enzymes to get from a to b and b to a. The
difference between the a and b form is that the subunits are phosphorylated.
They also realized that you could inhibit these processes by certain
compounds, for example, EDTA- take out the Mg- which keeps the GPase b
from phosphorylating. Or they could use Na/KF to inhibit the other enzyme.
- Get a signal (hormone) (E or glucagon) and are known to break down
glycogen. cAMP-PKA- kinase- GPase (active) and then phosphorylates
individual glucose molecules in glycogen.
- Cell signaling systems. Receptor-Effector system (2 messenger)-
Intermediate coupling system. Key functions for both systems are protein
phosphorylation and transcriptional control.
- A, B and C classes – A is the biggest class (rhodopsin and adrenergic).
- Adrenoreceptors. Alpha 1,2 Beta 1,2,3. All vertebrates have these receptors
and are highly tissue specific.
- See 7 transmembrane domains and see where the ligand binds and where
the g-protein binding site is. The ligand changes the conformation of the
structure and opens up the g-protein binding site.
- Modulation of existing enzymes. Shifting Km values, not Vmax. Not covalent
modification but post-translational modification. Phosphorylation is very
important for the signal transduction.
- G-proteins are heterotrimeric protins (a, b, y). In humans there is about 20
forms of the alpha subunits, 6 or 7 betas and 3 or 4 gammas. Alpha subunit
defines the g-protein. An example is rhodopsin. In its inactive state it consists
of 11-cis-retinol. After light hits it, there is movement of the protein so the
alpha subunit is able to bind and the receptor is activated. There are “hooks”
in the middle and they bind beta and gamma together and keep them
attached to the membrane. Important: Gs (stimulatory) Gi (inhibitory), and
Gq. The alpha subunit undergoes the g-protein cycle. At rest, a g-protein
binds guanine nucleotide and is most important in protein synthesis and in
the g-protein cycle. So at rest a, b, y are bound together with the GDP. Once a
ligand hits the receptor it allows for the g-protein to associate with that
receptor-ligand complex. Then there is an exchange process (GDP->GTP)
and that activates the alpha subunit of the g-protein. So you end up with
alpha subunit attached to GTP and the disassociation of b,y from a. The
stimulatory alpha subunit binds to AC to activate it which then takes ATP-> cAMP. So we have a complex: cAMP bound to alpha-GTP complex. The alpha-
GTP complex is actually GTPase. So ulitmatly you get reformation and GDP
attached to AC, and releases AC and once again gets high affinity for beta and
gamma and re-attaches (cycle begins again). Cholera (dehydration) is a toxin
that acts within the g-protein cycle. And also Pertussis toxin . These toxin
modify the g-protein cycle.
- ADP-ribosylation. Cholera toxin causes ADP-ribosylation. Alpha subunit
attached and blocks GTPase activitiy, [cAMP] increases. Cholera subunit
works on the Alpha(s) where as Pertussis works on Alpha(i). Pertussis blocks
exchange of GDP for GTP thus everything that is supposed to happen after
- Gs system. This receptor is the beta adrenoreceptor which binds NE, E or
adrenaline. It is extremely important to activate AC which in turn eventually
activates glycogen phosphorylase. There are other hormones that act the
same way, not necessarily beta adrenoreceptors but are linked to Gs
proteins, LH, FSH, TSH, ACTH. Gi is linked to Ai adrenoreceptor but it inhibits
cAMP (also Ach, opoids, CA). Third type of g-protein is Gq and prototype is
A1 adrenoreceptor. Adrenaline and noradrenalin can bind to this receptor. In
any cell in the body you may have all 3 types of these receptors. For each one
of these receptors there are antagonists so the agonists don’t work. We can
determine specificity of cells by adding antagonists to certain receptors.
GTpase increases glucose production and glycogen. So if certain receptors
are blocked there would be an increase or decrease of glucose and glycogen.
- G-protein assists in down-regulation process.
- Effector system-1. AC is made up of 2 -6transmamebran proteins and they
contain catalytic subunits and are responsible for binding of g-protein. There
are 8 potential isosymes of AC, 5 of which are sensitive to Calcium (3 are
positively affected and 2 are negatively affected). Forskolin activates AC and
we can use it to study the system (independent of any of the g-protein
receptors). cAMP activates PKA (kinases utilize ATP).
- PKA is tetromeric enzyme (2 catalytic subunits). Need to have more than one
cAMP because it releases the regulatory subunit of PKA so only the catalytic
is left which leaves it activated. Reversible dephosphorilation reaction that
happens with this specific signal transduction cascade.
- Effector system-2. PLC hydrolyzes PIP2 to DAG or IP3 which regulates
intracellular Ca2+ levels. Direct relationship between IP3 concentration and
Calicum (positive correlation). PKC works on the membrane and PKA works
inside the cell. These kinases phosphorylate proteins and once they are
phosphorylated are responsible for taking that signal to inside the cell.
- Tyrosine kinase receptors are for growth factors, important for long-term
and slow processes. They autophosphorylate. Important in protein synthesis,
gene expression, glucose uptake, and glycogen synthesis. Found on skeletal
muscles and adipose cells (fat cells). When insulin gets released, it goes to
skeletal muscle, adipose tissue, binds to tyrosine kinase, causes signal
transduction one of them is movement of glucose transporters to the membrane. Once they get to the membrane they get incorporated into the
membrane and act to get glucose into the cell.
- Phosphorylation is key to metabolic control. Mobilizing glycogen and
increasing glucose. Important for metabolism.
- Phosphorylation- key metabolic control. Phosphorylated proteins change the
biochemistry of the cell. GPase mobilizes glycogen (breaks it down) which
generates glucose. All these systems work with protein kinases, g-protein
coupled receptors (PKA, PKC, PKB, PKB) which cause phosphorylation of
- Pyruvate kinase. Glucose after many steps produces a product called PEP,
which in the presence of Pyruvate kinase, makes pyruvate. This is important
because it converts ADP to ATP. It shows homotropic interactions with PEP,
it is a tetramer. On each subunit there is a PEP binding site and these sites
interact with one another and they show alistery kinetics. This enzyme is
regulated by small molecules which are Ala and FBP (small molecule
effectors). Isolated liver cells are called hepatocytes and its important in
terms of glucose regulation. Glucagon also controls glucose levels in the
blood along with insulin and they are counter regulatory to one another
(insulin-reduces glucagon-increases). If you were to fast or not eat your
glucagon levels would jump in the liver. The glucagon would hit its receptor
(GluR) which is a GPCR but unlike the adrenoreceptor which is a member of
family A, this is a member of family B. If you take these hepatocytes in a little
beaker and add glucagon on to them, and then you isolate the enzyme
pyruvate kinase, the control (without glucagon) the curve looks like the one
on the left but with glucagon there is a shift to the right so substrate affinity
has decreased, or K0.5 value increased. SO the glucagon is doing something
to the enzyme which causes enzyme substrate affinity to plummet. GPCR is
linked to AC, meaning phosphorylation is occurring so we expect that
pyruvate kinase is getting phosphorylated. SO glucagon is pretty much
phosphorylating pyruvate kinase and that reduces its activity. In the absence
of glucagon Ala doesn’t inhibit the enzyme as much, but when glucagon is
added Ala basically kills it (prevents PEP from binding to the subunits). So
phosphorylation makes the subunits fit into a tense confirmation, and Ala fits
really well in this tight confirmation.
- PFK. This enzyme is at the beginning of glycolysis. It is also, along with
pyruvate kinase, very important in glycolysis they both control amount of
glucose that moves down. It takes F6p + ATP= FBP + ADP. This step is very
important because it utilizes ATP (breaks it down) it is an energy requiring
step, which is the opposite of pyruvate kinase (it makes ATP). Add glucagon
in one beaker and not in the other. WE see a shifting of the curve to the right
again with glucagon (just like what happened with pyruvate kinase),
however, it doesn’t make any physiological sense. In the presence of
glucagon, PFK is not phosphorylated. Lecture 8
- (Last time) Phosphorylation modifies homotropic and heterotrophic
interactions (pushes curves). Glucagon does the same thing to PFK as
pyruvate kinase but there is no change in phosphorylation-status. By binding
enzymes to cell structures you can affect pathway flux. Enzymes binding up
to z-line and its important in terms of controlling energy flux through muscle
cells- so location of enzymes is very important.
- Preparations used.
- Whole animal- very difficult to work with because of stress and that affects
metabolism. With the whole animal you can estimate O2 consumption and
CO2 production, and it gives us an indicator of aerobic metabolism, but some
animals don’t use that some use anaerobic metabolism. You can use heat
because head generation occurs as a result of metabolic processes. This is a
lot more accurate. RQ values used to determine the type of fuel that
particular organism is using. You can use NMR and MRI from whole animals.
- NMR. Mass ions, look at spinning rate. Put organism into a big magnet,
generate a current, probe that hits the structure you’re looking at and the
ions start to move again and you get frequency spectrum.
- Extracorporeal system. Removes blood through animal, put It through
electrodes and then put it back in the animal. Can measure pH, CO2, O2. Can
change external environment and observe how those factors change.
- Zebra fish are a really good model for growth and metabolism. Developped a
transgenic zebra fish and injected with a marker which affects a certain
enzyme so when the animal isn’t fed, the green marker becomes more and
more visible (PEPCK promoter enzyme).
- Isolated organs. How do you assess competency of these organs? Once you
remove them from the animal the tissue starts to die and changes start to
occur which don’t make it a good example. What kind of medium is used?
- Tissue slices. This is acceptable for the kidney, brain and some muscles (few
muscles that are homogeneous which means there is many fiber types). The
problem is that when you start slicing it starts to disintegrate right in front of
you, you’re activating death pathways. An advantage would be statistics. You
can get a large number of slices so you can compare them and come up with
an n value, average etc.
- Isolated cells. They isolate them using collagen and trypsin. This
disadvantage is that if you look at the border of the cell, it looks very smooth
and it should be that way. So you have probably cut away a lot of receptors,
destroyed proteins. The advantage is that you can get millions of cells from a
liver, for example. So statistics become much simpler. You can change the
intracellular medium. You can also do culture experiments (leave them
sitting for a period of time) and they tend to regain all of their functions for a
- Take tissue out and homogenize it carefully, but what happen is you start to
break apart metabolic pathways by adding fluids, diluting it. But still is
acceptable to look at protein synthesis by looking at the homogenized tissues. You can also use centrifugation and with that you can isolate nuclei,
- Crude to pure enzymes. No metabolism left, not physiological but all
biochemistry. Only proteins/enzymes left which you can purify and end up
with a particular protein in that medium which you want to look at. There
are a huge amount of methods that can be used to isolate certain enzyme
- Study of metabolism. We have to figure out which metabolic pathway we
want to study, we have to know what the components are and what the
various steps are. You also have to know how to measure flux or Jnet. If you
want to study glycolosys, you can study it down to pyruvate or lactate. So
either by the disappearance of glucose or the appearance of pyruvate or
lactate we can measure the amount of time or flux. Enzymes have a forward
reaction rate and a reverse reaction rate. Cells are open, so we must study
intermediates in the pathway and activities of enzymes within the pathway
and we can get a good idea of metabolism.
- Amounts of intermediates. Feed preparation with radioactive label and
freeze metabolism with perchloric acid , then you spin that stuff down
because it destroys all proteins and enzymes and leaves you with a clear
layer with all your metabolites. Then you measure the intermediates.
- Enzyme activites 1. There are 3 kinds of enzymes in metabolism. The first
one is substrate-saturated enzymes (GPase, lipases). There are large amounts
of substrates and the enzyme just sits there until it gets turned on by
phosphorylation. Second kind of enzyme is equilibrium or near-equilibrium.
They generally have high activities in relativity to the flux of that pathway.
Most enzymes within glycolysis are this kind (forward or reverse equally
well). So what they do is they transmit flux (allow substrates to go to
products, they don’t control things).
- The third type is non-equilibrium enzymes. They only go one direction, they
don’t come back because free energy to go backwards is too high (has to be
another enzyme to help reversible reaction). PyK, is rate limiting, or non-
equilibrium. How do we determine which enzyme is the controlling enzyme
- We have to determine activity in each one of the enzymes and compare it to
overall flux of that pathway or Jnet. Plot velocity of those enzymes vs Jnet. All
well above Jgly because you’re adding a lot of substrate to the enzymes
(saturate them) and getting maximum activity out of them so this flux ratio
gives you poor resolution. A better method is determining how far out of
equilibrium an enzyme is. If were able to do that we should be able to
determine whether or not that controls flux.
- Thermodynamics tells us how far out of equilibrium a certain reaction is. We
must determine what the free energy change is and that determines whether
or not the system is out of equilibrium. Biology keeps things out of
equilibrium, because if they go to equilibrium you’re “dead” and you can do
any work on them. The question is how far out of equilibrium are we. We
have to re-formulate the second law of thermodynamics (equation on the slide). Under the conditions that were doing the experiment we have to
determine the concentration of A B C and D. If p =1 (equilibrium equals mass
action ratio) means the system is at equilibrium.
- Disequilibrium ratios. Gpase is not important because it is a substrate
saturated enzyme and it doesn’t control the flux. Out of equilibrium is
hexokinase, pyruvate kinase and the one between (check name of enzyme,
didn’t hear). The values don’t tell us anything about rate, but how far out of
equilibrium they are.
- Metabolic flux estimates- define a preparation, define STEPS within pathway
to be assessed, must be able to estimate flux or rate of pathway. Substrate
saturated enzymes, equil- transmit flux (don’t control flux), non-equil
(control flux) and the last two are based on velocity forward versus velocity
reverse. Establish equilibrium by 2 law of thermodynamics. Mass action
ratio/Keq which gives us better discrimination which steps in the pathway
may be controlling the pathwa. The value gives us the rate limiting enzymes
which act as valves (increase enzyme, flux increase and vice versa). This
gives us nothing about rate but tells us concentration.
- Hexokinase takes glucose into metabolic pathway (first enzyme in
- Cross over analysis. A-F represents intermediates. We run a control
experiment meaning we don’t do anything unusual to it. We get control
values for each metabolite (A can be 100, C 30 etc). We set them all to 1 or
100 and we get the dotted line which represents the control conditions and
the relative amount of A-F (not absolute amount). Then we do an experiment
(add something, stimulate with a hormone), if that increases the flux we
would expect a positive crossover. The new values are relative to the control
experiment. If enzymes act as valves, we would expect that wherever the
valve sits, we would expect that before that valve to fall, and past that valve
to increase (because you are opening the valve a little bit). The flux is being
determined by the non-equilibrium enzyme (which is the one where it is
crossing over, so in this case its between B and C) has the ability to change
the flux by some mechanism and in so doing it changes the flux of the
pathway. Heart is notrmally aerobic, but if you make it anaerobic it has to
increase glycolysis to get ATP. So second graph is making rat heart anaerobic,
and then you take another heart, make it anaerobic and return it to aerobic
condition. The prediction is that the rate of flux for glycolysis will decrease
(because now heart has oxygen, utilizes lipds). So you would expect a
negative crossover. If you can show that one enzyme in that pathway shows
negative crossover then it’s that enzyme that controls the metabolic pathway
and in this graph it appears to be PFK.
- Glucaneogensis (backwards glycolysis) and liver is the primary organ to do
it. Cross over analysys II looks at this. Glucagon stimulates it to happen
(increases blood glucose) Insulin goes up, glucagon goes down and vice
versa. The bottom graph is looking at starting at the bottom of glycolysis all the way to the top. So the control is no glucagon at all, and then they killed
those cells and in another batch of cells they placed glucagon, left it and then
killed those cells aswell. Then they look at each intermediate at that time
after glucagon treatment compared to the control. We know glucagon
increase glucanogensis, so we are looking for positive crossovers (enzymes
that are opening the valve). We see two of those going up, so they appear to
be rate limiting (PEPCK and FBPase). This is the qualitative approach.
- On the last slide we were looking at concentration, not rate. First
experiments were done on PFK because PFK was known to control glycolysis
within yeast, so they increased its activity by 4 or 5 fold and the result was
nothing. So they increased the other ones and still no change. So this talks
against the rate limiting enzymes.
- Another experiment came out, made a transgenic mouse, for PEPCK. They
wanted to put it in skeletal muscle because there isn’t any, or very little so
they wanted to direct that enzyme into skeletal to see what would happen.
The enzyme had low activities under regular conditions but when they added
it they were a lot more active. There were no negative effects. This is
different than in yeast, you can do whatever you want to it and there is no
change. So the mouse suggests that there is something to this rate limiting
- There is a lot of evidence for MCA but the most obvious is that if you look at
metabolic pathways in different organisms, its not always the same enzymes
that are rate limiting. Pierce and Crawford looked at the Fundulis fish is that
there are 15 taxa in the Atlantic Ocean. When you compare metabolism
between organisms its like comparing apples and oranges but when you
compare the different Fundulus it’s the same type of animal. So they can
determine what controls glycolysis, like rate limiting enzymes. They expected
to see PFK, and pyruvate kinase. They found that the enzymes didn’t change
at all but with the enzymes that are usually equilibrium enzymes, so there is
more to it than just rate limiting enzymes controlling metabolism.
- Jnet is overall flux of the pathway. We must be able to manipulate that
pathway. Whether or no the enzymes are rate limiting, or if we belive in
metabolic control (all enzymes involved), bottom line is that something is
controlling metabolism. There are a few enzymes within the metabolic
pathway that control it more or less.
- Gsase builds glycogen up, Gpase breaks glycogen down. Adenylates= ATP,
- What is a significant affects of anenylates. Tells us how much energy the cell
has (ATP=a lot, AMP= less).
- Normal concentration of total amount of adenylate in any cell is about 5 mM.
because adenylate kinase is kept close to equilibrium, and because most are
in the form of ATP we can look to see what happens when you change ATP
concentration. Under normal conditions (5), and ATP concentrations are
around 4.9, then the equilibrium constant forces ADP concentration and AMP
to be what they are on the chart. When things start to happen, in the chart
there is a 10% decrease of ATP, but ADP rises by 5x, AMP rises from 20x. The basis of regulation is that relatively large changes on a compound have a
significant change on an enzyme. ATP doesn’t do a lot in terms of regulation,
because it doesn’t change very much.
- AMP Kinase 1. Major energy sensor in our cells- responsible for shifting
metabolism from anabolic, to catabolic. Based upon energy, low energy
sensor meaning it gets activated by LOW energy, or AMP. Trimeric enzyme
which is always around but kept inactivated because concentration of AMP is
low and ATP is high. However when you exercise, AMP rises, ATP decreases
by 10x. AMP kinase is a kinase so it phosphorylates. This enzyme is active by
AMP but increases its activity by 200x if it gets postranslationaly modified.
- Muscle contraction- increase of AMP, decrease CrP (phosphagen, CrP + ADP=
ATP + Cr, phosphate donor). This activates AMPKinase and decreases
anything that’s associated with anabolic processes. In each tissue it does
different things. Slide 27 end.
- Two cells beside each other can have different metabolisms. A lot of
enzymes are regulated by levels of AMP or ATP. AMPK- low energy sensor
that phosphorylates specific kinds of enzymes and those enzymes then direct
metabolism within that cell. It is trimeric which is activated by AMP, but
super activated by phosphorylation.
- 33- shows how AMP kinase sits in the middle of all the pathways. All of them
take a lot of energy and when that enzyme gets activated they all shut down
but the blue get amped up.
- PFK. Glucagon seems to do the same thing but there’s no indication that PFK
gets phosphorylated. But It does it a different way and it is regulated by F-2
- Most specific and active of the activators of glycolysis. It took them a while to
find it because it disappears in high pHs (when homogenizing the tissues).
We now know that it is extremely active at very low concentrations (micro
molar concentrations). It is a heterotropic activator of PFK. It overrides all
inhibition, it is synergistic with AMP but is more potent than AMP. In the
presence of F-2 6-p2 (left) the cure is a lot more to the right. PFK goes in one
direction, in the glycolitic direction and cant go backwards. This is just the
counter-enzyme to PFK (way out of equilibrium). Not only does it activate
PFK, it inhibits FBPase. It comes from a by-functional enzyme, so it is dimeric
which in its dephosphorylated state acts and PFK-II and in phosphorylated
acts as FBPase-II . it is bifunctional (can go either way) and it is controlled by
PKA (glucagon is involved and epinephrine) that’s why it is sensitive to both
glucagon and epinephrine.
- We’ve known that PFK is very slow and in all systems where its been looked
at it is the controlling enzyme of glycolysis. We know that to get back up it is
done by FBpase-1. PFK is strictly controlling glycolysis and FBPase-1 strictly
controls glucaneogenesis. If you add glucose, it will increase f6p and because
PFK is so slow it just sits there until PFK decides to convert it to f16p. while
its waiting, PFK-II takes it to f-2, 6-p and it activates the process very rapidly. This particular metabolite also inhibits FBPase-1. So if it inhibits FBPase, the
system has nowhere to go but down to pyruvate not back up in
gluceanogeneiss. We also know that F-2 6-P gets inhibited by FBPase-II and it
phosphorylates instead of dephosphorylates and it is done by PKA. So it
helps that activity but inhibits the other activity. PKA is activated by
glucagon/E, FBPase-II when its dephosphorylated it is called PFK-II but when
its phosphorylated it is FBPase-II and it phosphorylates and takes it back up.
- It is not found in mammalian blood cell but is found everywhere else.
- Nutrient sensors (not important) – SREBP and ChREBP and are transcription
factors which are important to activate certain enzymes of glycolysis or
lipigenesis. They can direct metabolism within the cell.
- Rate-limiting enzyme theory is out the window because of speed and instead
theyre looking at MCA.
Begin next lecture
- Temperature. In a biochemical system has 2 effects. First one is rate or
kinetic affect. We know if you decrease temperature, that output decreases
and vice versa. This is known as the laws of Arrhenius and was very
interested in understanding how rate processes were controlled. He used
thermodynamic properties to define how temperature would act on rate
constants. (equation on slide). Always done at maximum concentrations of
substrate giving max activities of enzyme. In negative sloped graph the low
temperatures are on the right of x axis and high on the left, so temp always
increases rate. We call Q10 values (difference in rate between a certain
temperature) and they can be done at any point and any concentration as
long as you have defined the conditions of the experiment. Ea only at
maximum because it is thermodynamic characteristic of the enzyme.
- These are equilibrium or weak-bond effects so temperature changes these
within biological materials. Negative means you save (for hydrogen) about 5
kcal/mol. It doesn’t matter at what temperature that they’re formed they will
always save the same amount of energy. The endothermic requires energy
and because you have to put it in you might make a perdiction that if you live
at 1c and suddenly you decide to live at 30c these endothermic interactions
will be very different (good at 30, not at 1) because it takes energy to form
- Km values help us quantify weak bond effects. Evidence? Enzymes
inactivated at low temperature (Pyruvate kinase). This suggest that
something is going on. Melting curves for DNA, RNA, if you determine the
melting temperature (temperature that 50% of DNA is unwound)you know
there is correlation between melting temp and G_C content (3 bonds where
as A+T form only 2).
- Endotherms (birds, mammals) protect body temperature so it doesn’t matter
what ambient temperature is. Body temp will remain constant. They do it
based upon fat, rapid metabolism, but the efficiency of metabolism is always
the same no matter which organism you are. 35% efficient, the rest of that goes out as heat. Compensation is how they do it because the deleterious
effects are overcome somehow. Falling temp= increase metabolism.
Immediate or instantaneous= reflex and it depends on what is already there
and the other possibility is seasonal changes that occur over a longer period
of time and they are much different because you can have total
reorganization of metabolism.
- Fry came up that not all fish compensate for temperature. Goldfish and trout
seemed to do it well. Lethal temp vs acclimation temp. Animal can adjust
until it reaches extremes so he took area of the “parallelograms” and if you
do that for different species you find that they are different. The diagrams
are indications are ability of a species to compensate to a temperature, so the
higher the value the better they are able to do it.
- Hibernators compensate somewhat because the change their biochemistry.
- How can temperature change enzymes so they work better? Active
metabolism vs standard metabolism have different Q10 values. If they
increased substrate to mitochondria it would follow the red line, and if it was
low it would follow blue line. So changing substrate can change temperature
characteristic of the mitochondria. King crab muscle PFK. If you look at this
activity curve for this enzyme (relative activity to concentration of substrate)
shows sigmoidal curve at 15 degrees. When we run it at 5 degrees the kinetic
curve follows blue line. Looks like Vmax values have changed and usually its
just Km. The rate effect is strictly a thermodynamic characteristic of the
enzyme, because thermodynamics tell you that under conditions you are
running the experiment at maximal there has to be a change. There is a rate
effect on Km but its not because of thermodynamics but weak bond
interactions (second effect of temp on biological systems). If you look at Km
of control (1.3) and 5degree curve is about .8, so by reducing Km or
increasing affinity the rates have changed at those concentrations (but not by
a lot but Vmx has changed by more than 2x). The rate effect is driven by Km
or affinity changes. So temperature is having normal kinetic affect at the top
but at the bottom also having an affect by changing weak bond interactions
so rates get compensated for by decreasing Km value. AMP is known to be a
positive modulator of PFK and even if you do it at a lower temperature it
works. So temperature acts as a positive modulator, mimicking AMP (or
other small molecules). Positive thermal modulation- by reducing the temp
Km values were falling, enzyme sub affinity values increasing therefore
enzyme activities being compensated for.
- 7-what kind of stragety does an organism have to overcome. They can adjust
through evolutionary adaptation, and compensation (ability of enzymes to
immediately do something to themselves). All of them are specific to that
organism. 3 ways it can occur: modulation strategy, concentration strategy
and qualitative strategy. Enzymes sitting in cells, they get exposed to
different temperatures and enzymes respond. They respond in terms of
kinetic effects. When you decrease temperature on an enzyme, Km values change. And by changing them we get rate compensation. Temperature can
be a modulator. When we add AMP to this enzyme it shifts from sigmoidal to
hyperbolic (F6P). With temperature increases the same thing happens. Low
temperature impacts this enzyme in a positive way. The blue curve has a rate
of about 0.6. the Q10 value is less than one, which shows compensation. By
increasing km, this enzyme can compensate for changes in temperature.
- Shifting Km values is what thermal modulation is (positive). LDH- Km value
as a function of temperature you get a sharp increase in Km values as
temperatures fall- positive thermal modulation. Positive thermal modulation
you look at concentration of substrate, as you decrease it, you deacrease the
Q10 value or the rate value between 5-15 degrees. At 1mM pyruvate its 1.5,
and it decreases as you go along. Activity at low temperatures are higher
than activity at low temperatures. Weak interactive effects are doing this,
something is going on in that active site of the enzyme so substrate binds
better at that temperature which increases rate of reaction.As substrate
concentration decrease, Q10 decreases (hallmark of positive thermal
- Do all enzymes show this? Or only in ectothermic organisms? Enzyme in
rainbow trout that shows + thermal modulation. Q10 is high for this enzyme.
You can see the same thing, decrease concentration decrease Q10. In pig, km
values don’t change very much, Q10 don’t change very much. Infrequenty
find positive thermal modulation in mammalian enzymes.
- What happens when you decrease temperature even further? For this
enzyme, it platoes. What happens when you go from 12 to 8 degrees? What
happens to the activity? The Km value doesn’t change, but the activity will
continue to drop. Its apparent that there is some lower limit of positive
thermal modulation. Suggest that there has to be a balance between
maintaining regulation (km values) and maintaining rates. Its apparents that
over these ranges, rates are let loose whats important is control and
- Something that can happen-maintain Km values, which means also Q10
values are maintained and stay the same (not showing compensation) this is
very low temperatures. The next is negative thermal modulation. Two things
are changing, the amount of energy in the system (lower and lower
temperatures, thermal energy decreases) and at the same time, Km values
start to rise. When they rise, the enzyme substrate affinity falls. It can get so
depressed that it can be biologically inactivated (opposite of positive thermal
modulation) Q10 values go very high, not good for enzyme.
- Appears to be a trade off between rate compensation and maintaining
regulation. If you look at muscle Pyk in a variety of organisms and you look at
Km PEP vs temperature you get different shaped curves. The trout is
acclimated to 10, you see positive thermal m , none, and negative thermal m.
They all have different binding characteristics in their substrate depending
on their species. The warm bodied like tuna or rat, the temperature curves
are flat meaning they don’t show major negative or positive thermal
modulation. Very possible that they have adjusted to maintain regulation but not rates, no compensation shown. Different patterns depending upon the
species. Different binding characteristics to their substrates with the same
enzyme. Warm body organism- km temp cuves are more flattened meaning
they don’t show major positive or negative thermal modulation, possible
they have adjusted to maintain regulation.
- 12- km of PEP shows the U shaped curve. Secondary substrate ADP- flat, no
change in Km which means its temperature independent. Why would this
happen? Where else is ADP used? ADP is in mitochondria, produces ATP.
This Km value may be independent from temperature because it maybe
changed. If it followed the same positive thermal modulation, might increase
its affinity so much that it might decrease its affinity for phosphorylation. So
for a substrate that has dual functions, producing ATP, it makes a little bit of
sense. Polymorphism – 5 or 6 different forms of a particular enzyme. You see
one is temperature independent where as the other one has strong positive
thermal modulation. Bat liver HL and NL- animals that are feeding a lot want
to keep pyruvate kinase open as long as possible so these animals might
show this kind of response so they can recompensate and push more and
more food into fat. If temperatures get really low, they basically shut down.
The hibernator does this at 4-6 degrees but they need to rapidly activate
their metabolism so by having the temperature independent response means
that its driven only by increases in response and arousal.
- Km vs temperature. First respsonse is positive TM- gives a Km value
decrease as temperature decrease. Increase in affinity leads to compensation
at those substrates. No thermal thermal modulation (line)- Km independent
temp response and at these conditions rates are normal, depends on enzyme-
maximal regulatory capacity meaning Km doesn’t change. Negative TM- Km
value rise, temperature falls, so affinity decreases and activity falls which can
lead to biological deactivation.
- Balance between rate and regulation. Appears to be an immediate response
to a declining or increasing habitat. Might be important for organisms that
are experiencing rapid fluctuations. Mytilus follows the green kinetic curve,
hyperbolic. When water goes away, they can go even higher than 25 degrees.
So what happens to the enzyme depends on whether there is positive TM or
not. We would expect a normal rate because were increasing amount of
thermal activity, it would increase activity of the enzyme. If there is postive
TM we would expect an increase of Km and activity. Without TM, the anial
eats itself up and allows for slowing of metabolism.
- Temperature has a rate effect and an equilibrium effect. Temperature
enhances/increases reaction rate. The point is that organisms cant
necessarily do that there has to be other mechanisms, they are in fact,
regulators. How temperature impacts the active site of amino acids is
important. Temperature modulates Km and Vmax value. But, Km values are
going to be differentially affected.
- pH changes with temperature. pH is homeostatically controlled in the human
body. Whats the pH inside cells? We know that there is a gradient of pH- mitochondrial membrane. How do we estimate it? NMR can be used,
- Weak acid/base indicators and sensitive and cheap and can get an estimate
of intracellular pH in various organisms. DMO is a weak acid which can also
be used when a c14 carbon is stuck to eat. Unionized form is permeable so it
can go through many membranes. After it is ionized or gone to equilibrium
you terminate the animal and can help determine pH by comparing the pH
without and with the DMO. In the liver different cells have different pHs, but
its better than nothing.
- Activities under t