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FUNdamentals 1
09/03/08 (Dr. Pritchard)
11:00 – 12:00
Introduction:
Today, I am going to speak about the opposite process of what Dr. Miller talked
about, which is making new glucose (gluconeogenesis). It is what it sounds like, the
synthesis of new glucose.
Slide 1 - Gluconeogenesis
 People require a lot of glucose (about 160 grams a day). That is a lot of sugar. It is
about a half full cup and everyone needs this much.
 Why does the brain need so much sugar? The answer is that the brain is filled will
billions of neurons that have to maintain electrical potentials and it takes a lot of
ATP to do that.
o The brain uses glucose to make the ATP and maintain these electrical
potentials in the neurons.
 We have a little glucose in our blood and other body fluids.
 If you haven’t been exercising, you have a fair amount of glucose in your muscle
and liver (180-200g)
 Bottom line: if you don’t get enough glucose in your diet, then you will have to
make your own, and that process is called gluconeogenesis
 IMPORTANT: 90% of gluconeogenesis occurs in the liver and kidneys
Slide 2 - Historical Perspective
 It turns out that seven of the same enzymes used in gluconeogenesis are also used
in glycolysis. The reactions will be catalyzed in the reverse direction though.
 Eduard Buchner was a biochemist (Buchner funnel) and he had a microbiologist
brother and they decided they were going to make a lot of money selling yeast
extract because it was believed to be healthy (and it is because it is filled with Bvitamins).
 The problem was how do you package it? They didn’t want to heat it up, sterilize
it and package it because they thought they would inactivate it.
 Storing processes were very limited during this time. A common way to preserve
things in those days was to salt it down. This doesn’t work with something that is
liquid because then you don’t have any way to remove the salt.
 The other way things were often preserved is they were dumped in with a lot of
sugar (sucrose).
o High osmolarity of the sugar keeps bacteria and mold from growing on it.
 They added sugar to the yeast and the whole thing began to smell like alcohol
because of anaerobic glycolysis.
 This was interesting to them because up until then people believed that
fermentation could only occur with living organisms.
Slide 3 - Historical Perspective (cont’d)
 Other people after them discovered that this yeast extract could be separated into
two fractions. One fraction would go through a porous filter, cozymase, and
another fraction would not go through the filter and that was called zymase.
 Also found that phosphate was required and became linked to sugar during the
process.
 Other people found the same enzymes in muscle. Remember, this study was done
in yeast.
o Virtually the same enzymes with a few exceptions.
 Muscle does not make alcohol.
 In the absence of O2, the end product of glycolysis is lactic acid
Slide 4 – Enzyme differences between glycolysis and gluconeogenesis
 If you read this diagram downwards it is glycolysis.
 If you read this diagram upwards it is gluconeogenesis.
 You should learn this table! Know the differences.
o Some are unique to glycolysis, some are unique to gluconeogenesis. Seven
are common.
Slide 5 - First Reaction of Gluconeogenesis
 First step: conversion of pyruvate to oxaloacetate
 This is the overall reaction, more detail will be presented in the next slide
 Overall, you have pyruvate (a simple, 3 carbon, keto-acid) and bicarbonate (buffer
in the blood…made by mixing carbon dioxide and water)
 This process requires ATP and you make oxaloacetate.
 You’ve added a CO2 to pyruvate
Slide 6 – Biotin & Avidin
 Reactions that add carbon dioxide almost always use a cofactor called Biotin,
which is often considered a vitamin.
 You rarely find deficiencies of Biotin, unless people are on a really weird diet.
Even the bacteria in our guts make Biotin. Someone’s diet must be really messed
up to have a lack of Biotin (e.g. eating lots of raw eggs)
 The egg white contains a protein called Avidin, which tightly binds Biotin. It is
almost a covalent binding, but not quite. VERY TIGHT binding.
o It makes the Biotin unavailable.
o And in this particular case people can actually get a Biotin deficiency.
 Note the structure of Biotin on slide
o 2 rings (one with a sulfur, one with two nitrogens)
o The carbon dioxide ends up being attached here (bottom right N-H bond in
the green shading)
o There is also a “flexible leash”…in the enzyme, Biotin is always
covalently bound to a imide bond, to a lysine…basically you have the
Biotin on a long leash and the way that this works is that it will pick up
carbon dioxide and then swivel a great distance to another part of the
enzyme for the next step, the transfer of the carbon dioxide
.
Slide 7 – Biotin & Oxaloacetate Involvement
 This reaction is in several steps, but it is catalyzed by the same enzyme
 First of all, you end up making carbonyl phosphate (a carbon dioxide linked to a
phosphate) so what you are doing is activating carbon dioxide
 Then, the carbon dioxide is transferred to the nitrogen of Biotin and you get a new
compound which reacts with pyruvate that has been deprotonated (pyruvate is at
the top right hand corner of slide) to make oxaloacetate
 Step 1: converting pyruvate to oxaloacetate
Slide 8 - Oxaloacetate
 This occurs in the mitochondria. The problem is there is no transporter molecule
that can move oxaloacetate from the inside of mitochondria to the outside.
o What happens is oxaloacetate is reduced (with NADH) to malate
 Malate CAN be transported out because there is a transporter for it.
 Once it is out, it is reoxidize to oxaloacetate again. This is called a shuttle.
Slide 9 - The PEP carboxykinase reaction.
 The second step in gluconeogenesis is converting oxaloacetate to PEP.
 Note GTP instead of ATP in slide (for regulatory reasons)
 Net effect is you make PEP
 CO2 is removed even though you go through all the trouble w/ Biotin to put it on
 The reason you do this is because PEP is a very high-energy compound.
 What you’ve done is you’re essentially using two ATPs because one ATP is not
enough to convert
 In the first step you are adding the CO2 and using an ATP
 In the second step you are using GTP (equivalent to an ATP) and in effect, you
are using two high-energy molecules to make this because one simply does not
have enough energy.
o The cleaving off of the CO2 is energetically favorable & helps drive the
reaction forward
 This is a common type of series of reactions seen in Biochemistry. You are
pushing a reaction uphill that would normally be energetically unfavorable, but
you do it by coupling it to the hydrolysis of two high-energy bonds of ATP or
ATP like molecules.
Slide 10 – PEP formation
 “This is the same thing.”
Slide 11 – Glycolysis Pathway
 Reading downward = glycolysis; upward = gluconeogenesis
 The 4 stars on the reaction are reactions that are unique to gluconeogenesis, but
most of the reactions here (with double arrows) are common with both glycolysis
and gluconeogenesis
Slide 12 - Enolase Reaction
 The third reaction is the conversion of PEP to 2-phosphoglycerate (2-PG)
 This is basically a hydration reaction where the water adds to the double bond
Slide 13 - The Phosphoglycerate Mutase Reaction
 Next reaction, you move the phosphate from the 2  3 position and the enzyme
that does that is phosphoglycerate mutase
Slide 14 - Isomerase, Kinase and Mutase
 A mutase is an enzyme that catalyzes the transfer of a functional group (such as
phosphates, sulfates, acetyl groups, etc ) from one position to another. This could
be a good exam question.
 Most of the time when you think of a kinase you think of an enzyme that
catalyzes the addition of a phosphate from an ATP to something. But it can also
catalyze the reverse reaction. You can get a phosphate from some other molecule
adding to ADP that will make an ATP. If the ATP is involved in phosphate
transfer (either getting or removing) that is called a kinase
 Almost always where we are talking about a carbohydrate enzyme and it has
“isomerase” in the title, you know it is talking about converting a ketose to an
aldose or vice versa
 Remember these terms because some of the enzymes we will encounter will have
these terms
Slide 15 – (1,3-BPG)  (3-PG)
 The next reaction of gluconeogenesis
 We now have the 3-PG and what happens is with ATP you add another phosphate
to the carboxylic acid group.
 This molecule (far left molecule: 1,3 – Bisphosphoglycerate) is a mixed
anhydride, but it is a carboxylic acid activated with phosphate.
Slide 16 - The glyceraldehyde-3-phosphate dehydrogenase reaction
 You’ve seen this enzyme before in glycolysis going the other direction
 In gluconeogenesis, it catalyzes the conversion of 1,3-BPG  G-3-P
 This NADH has reduced this carboxylic acid group to an aldehyde.
Slide 17 – DHAP  G-3-P
 In glycolysis, triose phosphate isomerase converts G-3-P  DHAP
 If DHAP didn’t have a phosphate group on it, then it would be dihyrdoxyacetone
(DHA) and it stains your skin yellow and is used in self tanning lotion
 In this slide, all this is showing is that these molecules can be readily converted
 This particular enzyme is considered a perfect enzyme. It is almost as if every
collision of the enzyme with G-3-P results in a conversion. It is a very efficient
enzyme.
Slide 18 – Glycolysis Pathway
 Where are we on this pathway? “This point” (at the position of G-3-P/DHAP
transition).
o Remember in glycolysis, these are the two molecules that are produced by
the breakdown of fructose 1,6-bisphosphate
 In gluconeogenesis these molecules are joined to make fructose 1,6-bisphosphate.
 The same enzyme does the job: aldolase
Slide 19 - Aldolase
 When you join these two small molecules (one a ketone and one an aldehyde) that
is called an aldol condensation. In glycolysis, it is called an aldol cleavage when
you break it down
 Remember, you see all these reactions in pathways. You may wonder why you
don’t go from glucose to pyruvate. Why all the steps? The problem is you are
trying to set the molecule up so that it will be in a form where you can join two
molecules together, or cleave them, depending on which direction you are going.
 The problem in Biochemistry is you have to stay at body pH…it is not like
Organic Chemistry when you can do things that can’t be done in a human cell.
(Dr. Pritchard elaborated a little bit on this concept, but just stressed that this
pathway occurs at body temperature and you are limited in your reactions)
Slide 20 – Aldol Condensation
 Shows you what I said earlier. By setting these things up the right way you can
either cleave them (glycolysis) or join them (gluconeogenesis)
Slide 21 – Diagram: Dihydroxyacetone phosphate & G-3-P
 People have done X-ray crystallography of aldolase and they can tell you exactly
how the molecule is oriented in space. Note the two molecules in the slide.
Slide 22 – Glycolysis Pathway
 Now we are at this point. We have made fructose 1,6-bisphosphate.
 Note the star, which means the next reaction is unique to gluconeogenesis
Slide 23 – Fructose 1,6-bisphosphate
 Look at the structure for a second
o The 6th position with the phosphate on it
o The 1st position with the phosphate on it
 What happens in this reaction is that the Fructose 1,6-bisphosphotase cleaves off
a phosphate. Remember from glycolysis that the opposite reaction requires an
ATP; there is no ATP involved here, you don’t generate an ATP
Slide 24 – Diagram: Glucose-6-phosphate  Fructose-6-phosphate


Fructose 6-phosphate is converted to Glucose 6-phosphate and the enzyme is
Phosphoglucose isomerase. Remember, when you see isomerase in an enzyme
name it means you are converting an aldose to a ketose or vice versa
Here you are converting a ketose to an aldose
Slide 25 – Transferring Glucose-6-phosphate into the cytoplasm
 When glucose is taken up by cells, almost all cells will phosphorylate it using an
ATP and hexokinase. Once this happens the glucose cannot get out. It has a
negative charge and is trapped within the cell.
Slide 26 - Glucose-6-phosphatase
 Most cells don’t have an enzyme that will take the phosphate off the glucose
again, except the liver and the kidneys (where gluconeogenesis occurs)
 The way this works is that G-6-P gets transported into the endoplasmic reticulum
and then a membrane bound enzyme (glucose 6-phosphatase) cleaves off the
phosphate, the glucose gets into the lumen of the ER, gets packaged into vesicles
that move to the plasma membrane of the cell where they fuse and the glucose is
released into the blood stream.
 This is a good exam question: Where does this reaction take place?”
Slide 27 - The Cori Cycle
 Husband and wife biochemist team that won The Nobel Prize
 They noticed that when animals do vigorous exercise you don’t have enough
oxygen to do it all aerobically, so you end up doing anaerobic glycolysis and that
has an effect of producing lactic acid.
 Remember why you are making this lactic acid. When you do glycolysis you
convert your NAD to NADH. You rapidly start running out of NAD and they way
that it is normally regenerated is the electron transport chain in the mitochondria
and that requires oxygen (oxidative phosphorylation).
 If you can’t do that because you don’t get enough oxygen, then you have real
problems with not enough NAD. They way you regenerate it is you turn it into
lactic acid and that regenerates your NAD as depicted in the slide.
 What these people found out is that this lactic acid from muscles gets into the
bloodstream, travels to the liver where the lactic acid is convereted back to
pyruvate and then that pyruvate (starting material for gluconeogenesis) goes on to
make more glucose and that glucose gets put into the bloodstream where it is
carried to the muscle and broken down again to get lactate…and so on and so on
o This is the so-called “Cori Cycle”
Slide 28 - Regulation of Gluconeogenesis
 At one particular stage “substrate level control” is present where: a lot of G6P
builds up in the liver or kidney, then it activates the glucose-6-phosphatase
(enzyme in the ER membrane) that cleaves the phosphate off to get glucose.
 Glycolysis and gluconeogenesis are reciprocally regulated
 Something that will stimulate glucose breakdown (glycolysis) almost always will
inhibit gluconeogenesis or vice versa

You don’t want both going on at the same time because you will waste ATP,
sometimes this occurs in newborn babies to keep them warm
Slide 29 – Fructose 2,6-bisphosphate
 This molecule looks similar to a molecule seen before (Fructose 1,6bisphosphate…the intermediate in gluconeogenesis and glycolysis), but this
molecule has a phosphate in the second position, not the first
 This is involved in the pathway as a regulatory molecule (only small amounts are
made, but has a powerful effect)
 The small portions of this molecule activates phosphofructokinase (the key
enzyme in glycolysis), in other words, if you want to breakdown glucose fast, you
need a little of this regulatory molecule in your bloodstream
 At the same time, it inhibits fructose 1,6-bisphosphate which is a key enzyme in
gluconeogenesis
o If you’re breaking down glucose, you don’t want to waste energy and ATP
to make more of it
 The enzyme that makes this and breaks it down are joined together
o Same bifunctional enzyme
Slide 30 - Fructose-2,6-bisphosphate
 The breakdown fructose 2,6-bisphosphate requires the cleaving off of a phosphate
and that takes a special phosphatase
 To make fructose 2,6-bisphosphate you cleave an ATP to add a phosphate and
that takes a special kinase
Slide 31 - 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
 People have observed the crystal structure of these thing and discovered there are
two major domains and a minor one
o Phosphatase domain: involved in breaking down the regulatory molecule
o Kinase domain: involved in adding the phosphate
o Regulatory region
Slide 32 - 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
 When blood-glucose levels get low a hormone called glucagon is produced and
gets into the blood
 The net effect of this is to cause increased phosphorylation of a lot of molecules
 Glucagon tends to have the opposite effect of insulin
 Insulin triggers an increase in the phosphatase activity (takes phosphate off)
 When you have low glucose, it increases the phosphorylation and the net effect is
it inactivates the kinase (makes the regulatory molecule) and activates the
phosphatase (breaks the regulatory molecule down)
 The idea is if you have low glucose, the last thing you want to do is break down
that little bit of glucose you have by glycolysis…you want to shut it down
 You want to make glucose as fast as you can by gluconeogenesis


You can memorize these concepts, or just think about them. They make sense!
o Low glucose = no breakdown of it, make more of it
o High glucose = use it, break it down
You can rationalize these regulatory steps
Slide 33 – Diagram: Regulating Gluconeogenesis and Glycolysis
 You can rationalize almost all of these concepts on this slide
 Why carry out glycolysis in the first place? To break down glucose & make ATP
o If you have lots of ATP, there is no need for glycolysis
 Note the plus and minus symbols on the slide
o Minus = negative regulator, or inhibitor of the enzyme
 Example: this enzyme is Phosphofructokinase is a key enzyme in
glycolysis, so lots of ATP will inhibit this enzyme that is involved
in making more ATP
 If you have lots of ATP, you should start making more glucose by
gluconeogenesis; you can afford it, you have enough ATP
o Plus = activates enzyme
 Low ATP values means AMP levels are high
 AMP positively activates this enzyme
 AMP will negatively affect the other enzyme (Fructose – 1,6-bisphosphatase)
 Again, you can rationalize these concepts.
Slide 34 - Substrates and Non-substrates for Gluconeogenesis:
 What substrates do we use for making glucose
o Pyruvate
o Lactate – you can always convert lactate to pyruvate
o TCA cycle intermediates
o Most amino acids
 In the breakdown of amino acids, you will see that many of them
are converted to keto-acids (e.g. alpha-ketoglutarate) that are TCA
cycle intermediates
 Notice what is not a substrate:
o Acetyl-CoA
o Fatty acids
o Lysine & Leucine (all other amino acids can be)
 Nice exam question
 Basic idea here is something that you will likely get on your board exams in one
form or another. The idea is that animals are unable to convert fat into glucose.
That is the key point.
 Loads of fat can provide your energy needs for months. That is not good enough
if you are starving because you can’t convert that fat into glucose. Your brain and
kidneys need glucose. You need this glucose and you have to make it if you are
starving and you need to use gluconeogenesis for that
 If you starve you are going to lose protein in your muscles and that protein is
broken down to amino acids, which in turn are broken down into TCA cycle
intermediates



Eventually you get oxaloacetate, which is used for making sugar
Starvation is a terrible way to diet! You looks your muscles.
Low carb diets still have a lot of protein and you use that protein to make sugar
because your brain needs it!
Slide 35 – Diagram: Citric Acid Cycle
 Why is it that you can’t convert fat to glucose?
o All you need to do is somehow get it converted to oxaloacetate (on
pathway to make more glucose), then you could make sugar out of it
 When you break down fat, you get Acetyl-CoA (cofactor that carries acetyl group
that has two carbons in it)
 In the Citric Acid Cycle you split off two CO2
o No matter how much Acetyl-CoA you put in, you are not going to get any
more oxaloacetate
o You have loads of energy (in ATP form), but no more oxaloacetate,
therefore no more sugar
 However, breaking down proteins into amino acids can be converted to alphaketoglutarate; this works and makes more oxaloacetate
 Amino acids/Proteins can be used to make sugar
 Main point: Acetyl-CoA coming from fat is not good enough to make sugar for an
animal
Slide 36 - Plants and bacteria can make glucose from acetate.
 However, plants can do it
 They use the Glyoxylate Cycle
 Notice there is no carbon dioxide split off
 Two types of enzymes here that animals don’t have
o Isocitrate lyase
 Cleaves the isocitrate (instead of splitting a CO2)
 Cleaves into molecules called glyoxylate and succinate (in Citric
Acid Cycle)
o Malate synthase
 Can add an Acteyl-CoA to glyoxylate to make malate (in Citric
Acid Cycle)
 There is a shunt that avoids the two steps that split off the carbon dioxide
 This just reinforces the idea that animals cant turn their fat into glucose
Slide 37 – Comparison of the TCA Cycle and Glyoxylate Cycle
 Comparison of the two cycles here
Slide 38 – Pyruvate  Glucose
 Another illustration of gluconeogenesis
Slide 39 – Enzymatic differences in glycolysis and gluconeogenesis
 Remember this table!
Slide 40 – Blank Slide