You’ve gotta spend (energy) money to make (energy) money! The first steps of breaking down blood sugar for energy (glycolysis) may seem counterintuitive if you just try to memorize them (it starts with spending energy in an “investment phase”!), but learning about METABOLISM need not bring pessimism! 

When you hear the word METABOLISM it’s usually referring to CATABOLISM – that’s where you break down bigger molecules into smaller molecules “for parts” and for energy. But the flip side of catabolism is ANABOLISM, where you build bigger molecules from smaller molecules (you can remember this by thinking of anabolic steroids, which some athletes illegally and dangerously use to build muscle). And a lot of times those smaller molecules are breakdown products of other bigger molecules. And a lot of the same metabolic pathways can go either way and use the same workers. You can think of it kinda like LEGOs – you can take apart and rebuild the same thing over and over or you can use pieces you break off from one thing to make another thing, etc. 

Say you have 2 LEGOs – a blue one (B) and a red one (R). You can stick them together to get BR, so B + R -> BR. And you can take them apart B + R <- BR. When a step can go in either direction we call it reversible, and we draw a double arrow facing both ways, like

B + R ⇆ BR

In theory, *all* steps are reversible – but we call some of them irreversible because it’s so unfavorable to go the other way. Unlike some steps, which are easily reversible – kinda like loose LEGO pieces – the “irreversible” steps are more like those pieces that stick together so tightly you’re likely to take off a fingernail before you get the piece to come off! 

Often such steps are accompanied by the addition of ATP – you can think of it as energy money being spent as a “downpayment” to “show commitment” to a process and prevent the molecules from “backing out” 

A great example of this is in GLYCOLYSIS, which starts with an “investment phase” in which energy is spent followed by a “payout phase” in which energy is produced. 

In GLYCOLYSIS (a catabolic process) you split the sugar glucose (which has 6 carbons (6C)) into 2 molecules of pyruvate, each of which have 3 carbons (3C). This nets you 2 ATP (energy “arcade tokens”) and 2 NADH (energy “IOUs”)(more below), and those 2 pyruvate, which can be further broken down in the citric acid cycle (aka Kreb’s cycle).

If you reverse glycolysis to its anabolic counterpart, so that instead of breaking glucose down, you’re making it, the process is called GLUCONEOGENESIS (literally, birth of new glucose – yes, kids, this is where glucose babies come from)

As you’d expect, gluconeogenesis requires energy. But, what you might not have expected – glycolysis requires energy 2 – in terms of ATP, it takes 2. It then makes 4, so you have a net gain of 2, but this energy-maker starts out as an energy-taker! 

To understand why, let’s first look at what I mean by “energy” and then we’ll look at glycolysis & gluconeogenesis in a molecular story time.

You might not stop to think about it often but, even when you’re sleeping, there’s tons of stuff happening in each of the billions of cells in your body – molecules running all around in there doing stuff. And all of that running around and stuff-doing takes energy. So your body has to have ways to take energy from foods we eat, store it, transfer it, and use it where and when you need it.

Your body’s main form of energy storage and transfer is a molecule called ATP – Adenosine TriPhosphate. Much more on it here: http://bit.ly/2WiPOpgbut basically it has 3 phosphate groups (phosphorus surrounding by 4 oxygens) stuck together – and each of those phosphates is negatively charged so, since opposites charges repel, they want to get apart from each other so, like clamping together a stiff spring, it takes energy to keep them bonded together (we call this chemical potential energy)- if you let one go (split ATP into ADP + Pi) you release energy, and you can use that energy to do things (like build proteins and stuff) 

Your body can take all sorts of different fuel sources (sugars, fats, proteins) and – like using different currencies and denominations to purchase arcade tokens –  generate ATP from them, which can be “spent” for all sorts of things. These energy-producing processes are often multi-stepped. Some of the steps are clearly useful because they generate ATP (usually by transferring an inorganic phosphate (a free-floating phosphate group that’s not hooked onto anything) to ADP (adenosine Diphosphosphate). Or they generate NADH by reducing (passing electrons to) NAD+. NADH is a bit like an “energy IOU” – much more on it in yesterday’s post: http://bit.ly/31Kb5sU 

but basically, in a process called OXIDATIVE PHOSPHORYLATION, which takes place in special double-membrane-bound compartments in the cell called mitochondria, NADH can transfer those electrons to another molecule that wants them more which passes them to another molecule that wants them even more . . . until it reaches oxygen which wants them the most. And each of those pass-offs releases a little energy and that energy is used to pump protons (H+) out of the inner room of the mitochondria (mitochondrial matrix) into the outer room of the mitochondria (inter membrane space), creating a proton gradient. Only one way back in is provided – through a molecule called ATP synthase that, like a hydraulic dam, uses the incoming rush of protons to turn a molecular motor and make ATP from ADP & Pi. For each NADH that goes in, ~10 protons get pumped out, and it takes 4 protons coming in for 1 ATP to be made. 10/4= 2.5, so you get ~2 and a half ATP per NADH.

Unlike the ATP & NADH-generating steps, some of the steps of metabolic processes like glycolysis don’t seem helpful at first glance… But if you geek out and don’t just glance, but instead “think like a molecule” you can see that what’s going on is “forward planning” that not just makes biochemical sense, but is biochemically beautiful!

Glycolysis has 10 main steps, and you can follow along in the figures

One thing you’ll notice is that each of these steps is catalyzed (sped-up) by things ending in “-ase” – when you see “-ase” think ENZYME! Enzymes are usually proteins (sometimes protein/RNA complexes (like the case with ribosomes) or just RNA (we often call such enzymes ribozymes) and they mediate and speed up reactions by doing things holding the molecules together in the right positions for whatever they need to do and providing an optimal environment for the reaction to take place. 

Think back to the LEGOs. Since LEGOs have matching bumps (which are apparently officially called “studs” or “knobs”) and indents (which are called “anti-studs” or “stud receptacles” or “tubes”) if you were to put a couple LEGOs in a bag and shake them a little you might, by chance, get them to meet just the right way that studs & anti-studs alight and stick. But this is much more likely to happen if you grab them with your fingers in the right orientation. This is kinda like what enzymes do.

The bricks still have to choose to stick (form new bonds) but the enzyme makes it much easier to do so. But they also make it easier to “undo so” – basically they just give the pieces more chances to choose.

(i.e. if 2 molecules don’t want to join you can hold them together all you want and they won’t form a new bond – but if 2 molecules do want to join and you hold them together they’re more likely to form a new bond – and they’re more likely to form a new bond if they’re brought together with the help of enzymes than if they have to find each other on their own). So the same enzyme can help out a reaction in both directions (and so sometimes the names might seem confusing because they’re written in terms of the “opposite direction”)

So, back to glycolysis (which literally means sweet (glyk) dissolution (lysis):

ENERGY INVESTMENT PHASE

STEP 1: enzyme: hexokinase; reaction: glucose + ATP -> glucose-6-phosphate (G6P) 

  • a kinase is a phosphate adder, and in this step phosphate is added to the “6th” of glucose’s 6 carbons
  • this is the first of the “irreversible” steps – you start by putting down an energy-money downpayment
  • it also helps with the committing because the charge makes it “impossible” for glucose to get out of the cell – what with the cell’s fatty membrane and all – so the glucose gets trapped

STEP 2: enzyme: phosphoglucose isomerase; reaction: glucose-6-phosphate ⇆ fructose-6-phosphate

  • isomers are different arrangements of the same atoms so, as the “isomerase” name suggests, what goes on in this step is just some rearrangement – specifically carbonyl (C=O) that glucose ends with gets shifted over, so that there’s an OH on the end. This might seem weird but just wait – it’s gonna be important for…

STEP 3: enzyme: phosphofructokinase; reaction: fructose-6-phosphate + ATP -> Fructose-1,6-bisphosphate

  • this is the second “irreversible step” – and it involves another phosphate adding – this time to the 1st carbon which you graciously made available in step 3
  • why the second “downpayment”? if you look at the structures you’ll see that you now have a phosphate on both ends, so when you split it in half each half gets one
  • the kinase involved is highly regulated to help regulate glycolysis as a whole

STEP 4: enzyme: fructose bisphosphate aldolase; reaction: fructose-1,6-bisphosphate ⇆ dihydroxyacetone phosphate (DHAP) + glyceraldehyde-3-phosphate (G-3-P)

  • this is the splitting I was talking about – you take a 6C thing and split it into two 3C things
  • the “halves” aren’t identical in the beginning (they have carbonyls in different spots) but, that’ll get sorted out with the help of another isomerase in…

STEP 5: enzyme: triose phosphate isomerase; reaction: DHAP ⇆ G-3-P

  • those unidentical halves have the same atoms just arranged a little different – they’re isomers – so, with the help of an isomerase, you can shift between them
  • only G-3-P can be directly used in the next step so, even though the reaction’s easily reversible, since you keep taking away the G-3-P, if you want to convert something, you’ve gotta convert the DHAP since there’s “no” G-3-P available. so eventually all the DHAP gets turned into G-3-P so you have 2 identical G-3-P’s entering the….

PAYOFF  PHASE

This is where you finally start making energy money (ATP) & energy IOUs (NADH). And the important thing to remember is that you’re going in with 2 copies of the G-3-P so each of the reactions gets “multiplied by 2”

STEP 6: enzyme: glyceraldehyde-3-phosphate dehydrogenase; reaction: glyceraldehyde-3-phosphate + NAD+ + Pi ⇆ 1,3-bisphosphoglycerate + NADH + H+

  • this is a kinda “weird” step because it adds a phosphate but a free-floating one (so it doesn’t cost any ATP) – the phosphate gets added to the “other end” so you now have one on each end again
  • the energy for the adding comes from the coupled redox reaction of NADH reduction & glyceraldehyde-3-phosphate oxidation, which is exergonic (energy-releasing) because the NAD+ wants the electrons more
  • this is your first IOU payment – it reduces NAD+ to NADH which can then be “cashed in” for ATP later in oxidative phosphorylation

STEP 7: enzyme: phosphoglycerate kinase; reaction: 1,3-bisphosphoglycerate + ADP ⇆ 3-phosphoglycerate + ATP

  • our first real payout!
  • don’t get confused by the enzyme name – before we had kinases *using* ATP to *add* phosphates, but here we’re adding a phosphate to ADP – and we’re getting that phosphate by taking off the one we just added in step 6 (the name refers to the reverse reaction)

STEP 8: enzyme: phosphoglycerate mutase; reaction: 3-phosphoglycerate ⇆ 2-phosphoglycerate

  • this is another isomerase, but I guess they thought “mutase” sounded cooler or something – but “all” you’re doing here is shifting the phosphate to the middle C

STEP 9: enzyme: enolase; reaction: 2-phosphoglycerate ⇆ phosphoenolpyruvate (PEP) + H2O

  • making PEP is a kind of “prep” – when you kick out that water you make a double bond between 2 of the carbons – making it a really awkward situation for that middle carbon – this molecule is really unstable
  • so the phosphate is now attached to a carbon that doesn’t really want it and can better survive without it – so that phosphate can more easily get removed in…

STEP 10: enzyme: pyruvate kinase; reaction: PEP + pyruvate kinase + ADP -> pyruvate

  • the last step & final payout of glycolysis
  • involves another kinase named for the reverse reaction
  • because PEP is so unstable, it’ll easily give up that phosphate to an ADP that pyruvate kinase helps it meet
  • and because of how unstable PEP is, although you’re not spending energy money, the reaction’s really unlikely to go backwards.

So you do all that for each copy of G-3-P you got from the investment phase. So, you end up with 2 X NADH (from step 6), 2 X ATP from step 7 and 2 x ATP from step 8. And you used 2 ATP in the investment phase. So, on net you have: 2 NADH + [(-2) + 2 + 2 = 4] ATP. And you also have those 2 pyruvates which can get further processed for more energy.

Gluconeogenesis is the “reverse” of glycolysis but it’s not a direct reverse because it has to “reroute” around the irreversible steps using different enzymes.  It reverses step 10 (the de-pep-ing) in 2 or 3 steps – it’s a bit “steppy” because that pyruvate that got made in glycolysis gets shipped into the mitochondria. In the mitochondria, carboxylase converts pyruvate to oxaloacetate (at the cost of 1 ATP) & then phosphoenol-pyruvate carboxykinase converts that oxaloacetate. That can’t go through the mitochondrial membranes, so it first gets made into malate by malate dehydrogenase, then that malate goes into the cytoplasm where another malate dehydrogenase turns it back to oxaloacetate which can then be turned into to phosphoenolpyruvate (PEP) (at the cost of 1 GTP) by PEP carboxykinase in the cytoplasm. Alternatively, the PEP can be made in the mitochondria in some animals and transported out like that

It uses fructose 1,6-bisphosphatase to reverse step 3 (remove the second phosphate that was added to go from fructose 1,6-bisphosphate to fructose 6-phosphate.

And to reverse the 1st step of glycolysis (initial phosphate-adding), glucose-6-phosphatase removes the phosphate to form glucose. This happens in the lumen of the endoplasmic reticulum (another membrane-bound compartment that’s often used for protein-modifying) and shipped out into the cytoplasm by glucose transporters. 

I learned some cool things about glycolysis in this book “For the Love of Enzymes” by Arthur Kornberg that president-elect of the @IUBMB, Dr. Alexandra Newton, gave me. For example, the discovery of glycolysis came with the birth of “biochemistry” – and it came by accident – in a story not so well-known as the Pasteur and the Petri dish one about the discovery of the antibiotic penicillin being made by mold on a contaminated bacteria culture plate.

Basically, a couple of guys in the late 1900s (1897 to be exact) – Hans & Eduard Buchner – thought there could be medicinal value to the stuff inside of yeast cells (cell-free yeast extracts). So they ground up & squished a bunch of yeast cells, filtered it and, to preserve it, added some sugar (hey – it works for jams right?!) Well, they found that the yeast extract started getting all bubbly – it was fermenting (fermentation is a process you can learn more about in yesterday’s post whereby NAD+ can be regenerated from NADH without going through oxidative phosphorylation)

This was really surprising because, before this, it was thought that complex processes like this could only take place INSIDE OF LIVING CELLS – but this was just the cell extract! Further excitement came when studies with muscle extracts showed that lactic acid fermentation used a lot of the same reactions as the alcoholic fermentation that the yeast were doing. In fact, glycolysis is a nearly universal process in all sorts of organisms. Lots of pioneering biochemists joined in on the fun and, by 1940, the glycolytic pathway was figured out! Nowadays, we study reactions with purified proteins outside of cells all the time because it gives us better control 

Why the obsession with LEGOs today? I’ve been really stressing out lately with my thesis committee meeting coming up and my mom insisted I do something fun & relaxing – and send her picture proof – so today I did some LEGOs – which – as a bonus make a mini AKTA (protein purification helper machine)

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