Glycolysis

  1. Glycolysis:
  2. Here is the overall scheme:
Glycolysis2.svg
I will outline all the steps below.
The key points are:
  • Takes place in the cytoplasm
  • Separate into “investment phase” and “payoff phase” (spend 2, get back 4, net yield 2)
  • Payoff is in the form of "substrate-level phosphorylation." We saw one of these in the Citric Acid Cycle. It's just an enzymatic reaction where a phosphate is taken off a substrate and added to ADP to make ATP…the reverse of what we usually expect.
  • Input is glucose or similar simple sugar
  • Output molecules are: Pyruvate, which can go to the mitochondria for further processing; NADH, which can be used in other reactions including to feed electrons into oxidative phosphorylation; 4 ATP…but only 2 net gain
  • Has an important regulated enzyme: phosphofructokinase (PFK). This enzyme is target for feedback to either increase or decrease glycolysis.
  • Investment phase results in fructose (6-carbon sugar) with a phosphate on both the first and sixth carbon (Fructose 1,6 bisphosphate).
  • Payoff phase occurs after this is broken into two 3-carbon sugars, each with a phosphate on them. The most important one of these is glyceraldehyde-3 phosphate (G3P)

Feedback:


The idea of feedback was addressed in the Enzyme chapter and it is an important concept. This is just one really interesting example we will do in more detail.
If there is an excess of ATP available in the cell and the citric acid cycle is running at full capacity, ATP will bind to an
allosteric site on PFK (allosteric means it binds someplace else on the protein other than the active site…this would be a non-competitive inhibitor). ATP is also a substrate for PFK. This is not a contradiction because there are two different sites. The active site, where phosphate is transferred to the fructose 6-phosphate, binds with high affinity (That means it binds even if there is a low concentration of ATP). The separate, allosteric site binds with low affinity. So, it is only occupied if there is high concentration of ATP around. Binding of ATP to the allosteric site inhibits the enzyme.
There is also an inhibitory site for citrate. So, higher concentrations of citrate inhibit this enzyme. Finally, it is stimulated by AMP (adenosine monophosphate), which would only be around if levels of ATP were really low.

Gluconeogenesis (this is not in the book, but is on the scary chart)


Here’s a challenging idea for you:
Like all enzyme pathways, glycolysis can be run backwards or forwards. In your liver “gluconeogenesis” (literally: making new glucose), can use
all but one of the enzymes of glycolysis to form glucose from intermediates “siphoned” off the citric acid cycle, if energy production needs to be reduced. Also, lactic acid can be used to build glucose. Again, all the enzymes are the same as in glycolysis except one. The role of that enzyme has to be carried out by a different protein. What do you think is that one enzyme is? It’s phosphofructokinase.
It’s not that the enzyme cannot work in reverse. Have any idea why PFK cannot be used, practically, in gluconeogenesis?
Here’s a hint: Gluconeogenesis would only occur if the cell had excess ATP.

Details


Glycolysis,

all the steps (you are not supposed to memorize these. The important stuff is in bold):
  1. Hexose Kinase (1st step), product is glucose 6-phosphate, keeps glucose inside cell and keeps gradient “downhill” into the cell. This uses an ATP, but is not regulated. This is the first “investment.”
  2. 2nd step rearranges it to fructose 6-phosphate. That is, the hydroxyl of position 2 oxidizes the carbonyl on position 1. Position 1 gets the H to make a hydroxyl there and position 2 becomes a carbonyl. The enzyme can be called “hexose-phosphate isomerase” or, as it is on the diagram, phosphoglucose isomerase. Like most steps, this is freely reversible.
  3. This is the rate-limiting and tightly regulated step. Phosphofructokinase (3rd step). This adds a second phosphate to fructose 6-phosphate, making fructose 1,6-bisphosphate. The regulation is via an allosteric site that binds ATP. Now, you have to know two things here: the enzyme has 2 sites that bind ATP, one in the active where it is necessary as a substrate for the reaction and a second that inhibits the reaction; the first site binds with higher affinity than the second. So, if the concentration of ATP is low to average in the cytoplasm, only the active site binds ATP. However, if the concentration of ATP gets high, indicating that more ATP is really not needed, the second, inhibitory site becomes occupied and shuts the enzyme off.
  4. Aldolase (4, clips the six-carbon sugar diphosphate into 2 Triose phosphates called dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (G3P). G3P is the more useful one and the two are freely interconverted by triode isomerase. Since there are two, G3P made, all the subsequent steps can be done twice for each glucose.
  5. The next step is carried out by Triose Phosphate Dehydrogenase (takes hydrogen off a triose phosphate). If something is “dehydrogenated” it is the same as being oxidized. Note, it’s not taking off an H+, it is an H with its electron. The thing that is reduced is NAD+. If you are interested in the chemistry here, it’s kind of interesting. Attacks position 1, the carbonyl. The exchange results a carboxyl with a phosphate on it. That’s a really high-energy phosphate. Hydrogen from the carbonyl carbon is transferred to NAD+, reducing it. This is the first energy payoff in that it creates two NADH + H+ (one for each of the G3P), which can be used in oxidative phosphorylation or as a high-energy cofactor in certain other reactions.
  6. The first "Payoff." Substrate-level phosphorylation. The phosphate on position 1 doesn’t stay long, as it is such a high-energy phosphate. It is transferred to ADP to make ATP in the next step. This process is called “substrate-level phosphorylation,” and is carried out in this case by the enzyme phosphoglycerate kinase. I know what you are thinking: “Wait…shouldn’t that name mean it adds a phosphate to 3-phosphoglycerate?” How clever of you to notice. The enzyme is named for the reverse reaction to the one we are talking about as part of glycolysis. There are several key enzymes for which this is the case. Important concept: It turns out that if you purify it away from everything else, it seems to favor the reverse reaction. It can drive it either way. The reason the reaction goes primarily toward producing phosphoglycerate is because there IS a next step. You see, as soon as 3-phosphoglycerate is formed, it gets pulled into that step.
  7. Next is a simple rearrangement where the phosphate gets moved from position 3 to position 2.
  8. 2-phosphoglycerate then undergoes a step carried out by a fairly famous enzyme: Enolase. This enzyme pulls a water molecule out but makes the most unstable, highest energy molecule in the series, Phosphoenolpyruvate (PEP). Enolase is essential in every cell that does glycolysis. It is rather famously inhibited by fluoride ions. This is why we put fluoride in toothpaste (and, in less conspiracy-minded communities, in the water). The fluoride kills the bacteria in your mouth. Anyway, that phosphate next to the double bond has to go.
  9. Second Payoff Step. The last step takes that phosphate and transfers it onto ADP to make one more ATP. The product is pyruvate, which goes off to the mitochondria for the TCA cycle. Again, this enzyme has a backwards name.

Here is a link to a
glycolysis rap…it’s accurate and not too painful to watch…just a little creepy.