Anaerobic Glycolysis and Cellular Effects of Acid/Base

I. Overview of Anaerobic Glycolysis: A. Anaerobic metabolism is defined as the breakdown of 6 C sugar (glucose) to yield a usable form of energy without using oxygen in the process. B. Likely to be well-developed1 in cells that: 1. Cannot make effective use of whatever oxygen might be present. This would be true of any cell having few mitochondria (e.g., type IIb fibers). 2. Also may be well developed in cells that might occasionally not have enough oxygen -- oxygen is present, perhaps even in relatively large amounts but it is still not enough. This is the case in type I fibers -- compared to type IIb fibers, the pathway is not well developed but compared to other cells in the body, anaerobic glycolysis is well developed and can be even more developed by specific training regimes. C. Fuels. Glycolysis only uses 6 C sugars and to use them must get them in a certain form (see below). These sugars can be: 1. glucose or fructose (both C6 H12 O6) . Muscles have some glucose and there is additional glucose available from the blood, but glucose per se is not the normal fuel for glycolysis. 2. Glycogen: a starch that is a branched polymer of glucose molecules. This is the major store of glucose in the muscle cell. Glycogen exists as granules in the sarcoplasm; there are many times more molecules of glucose in glycogen than there are free molecules of glucose in a muscle. The other main area in the body where glycogen is found is in the liver. D. Preparatory Steps: 1. If using glucose as fuel, the molecule must first be activated by adding ATP. This gives a 6 C compound with a phosphate attached. Thus, the molecule can be abbreviated 6 C -P where the -P means that the covalently attached phosphate is held by a bond that has relatively low energy stored in it. 2. If using glycogen as the fuel, phosphate (PI) reacts with the glycogen to break off a molecule of the same 6C - P just described (see D #1). However, notice that in this case, no ATP is used to make the 6 C - P. More about this later. 3. The removal of 6C -P by using Pi is catalyzed by the enzyme glycogen phosphorylase. This very important enzyme (know its name and what it does) regulates the availability of glucose to the cell. It is an allosteric

Copyright ()2003 by KN Prestwich, Department of Biology, College of the Holy Cross, Worcester, MA 01610 USA. kprestwi@holycross.edu. Non-commercial free use of these materials is encouraged 1 Well-developed means that there are many copies of each type of enzyme molecule used in the glycolysis pathway. We will see in a few more classes that reaction rate is proportional to the number of copies of the enzyme needed for a reaction or pathway -- the more, the faster the reaction or pathway can go (assuming plenty of substrate).

enzyme and it has both an activator and inhibitor site in addition to the active site. Glycogen phosphorylase is: (a) activated by higher than normal [ADP] in the sarcoplasm. (b) inhibited by higher than normal [ATP] in the sarcoplasm. 4. In either case, the 6C - P compound reacts with ATP which gives it a second low-energy covalently bonded phosphate P - 6C - P. This reaction is catalyzed by our old friend PFK. Recall that PFK tends to control the overall rate of glycolysis by changing the shape of its active site and becoming a better or worse catalyst. Recall that PFK is: (a) activated by higher than normal [ADP] in the sarcoplasm. (b) inhibited by higher than normal [ATP] in the sarcoplasm. Just like glycogen phosphorylase. Here is a summary of the preparatory steps: Glycogen phosphate
PFK

Glucose

6C - P

ATP ADP

P - 6C - P E. Glycolysis proper 1. Glycolysis means the breakdown ("lysis") of glucose. Appropriately, the next step then is to break the compound made by PFK, P-- 6C --P into two (eventually) identical 3C--P compounds2. 2. The next thing that happens is the 3C-P reacts (again using an enzyme) with a molecule of something called nicotinamide dinucleotide -- we'll call it NAD= . Here are a number of very important things to know about NAD=. (a) It is an example of what we call a coenzyme . Coenzymes are substances that are required for certain enzymes to function. Unlike enzymes they are not proteins and they often do participate and often are changed by the reaction. This is certainly the case in NAD= as we will now see:

Note for people who already know more about glycolysis: If you happen to know a bit more about glycolysis than is important for this class, you'll realize that actually the two 3C--P compounds are not identical (glyceraldehyde-3-phosphate and dihydroxyacetone phosphate). But one of them is quickly converted to the other and so the result is two identical 3C -- P compounds.

(b) NAD='s role is to transfer energy that is carried on electrons from one place to another. It typically picks up two electrons (and the energy they are carrying) in a reaction and transfers them to some other compound (along with their energy). Thus, we also say that NAD= is an electron carrier. (c) When NAD= picks up electrons it is converted into NADH -- it always picks up a proton (hydrogen nucleus) at the same time. Thus, the reaction for this coenzyme is: appropriate enzyme = electron donor + NAD --------------------------------> NADH (d) Thus, NADH is the high energy version and NAD+ the low energy version. This is just like the ATP/ADP pair. (e) NAD= can take electrons from any compound where the electrons have high potential energy and NADH can give the electrons to any compound where they would have somewhat lower potential energy (again assuming the correct enzyme is present to facilitate the reaction). However, it will always need a protein enzyme to help accomplish this transfer. (f) As with ATP/ADP there is only a limited amount of this stuff. It is possible for a cell to quickly turn all of its NAD= into NADH or vice versa. Either situation is trouble for the cell (as we'll see in a moment) and so just like ATP/ADP there must be a mechanism to maintain the relative amounts of NAD= and NADH. (g) Finally, notice that the step that requires NAD+ occurs before any of the ATP-yielding reactions. If the cell runs out of NAD+ glycolysis will stop before any ATP is made! More about this problem in a bit. (h) One last note -- NAD= is made from the important B vitamin, niacin, 3. Assuming that there is enough NAD= , the next steps of glycolysis involve making 2 molecules of ATP from ADP and the energy in the 3C--P compound. 4. When this is done, the initial waste product of glycolysis is a 3C molecule called pyruvic acid. On the top of the next page is a summary starting with the split of P--6C--P and ending with pyruvic acid:

P - 6C - P

NADH 3C - P NAD+ ATP ADP ATP ADP 3C - P NADH NAD+

ATP

ATP ADP

ADP 3C (pyruvic acid) 3C (pyruvic acid)

knp

5. Notice that the gross yield of ATP from a single 6C compound is: (a) 4 molecules of ATP (from ADP and Pi). (b) Also notice that there were two 3C waste product molecules. 6. Let's return to the problem we mentioned dealing with = NAD . It should be obvious that if there is only a small limited amount of NAD= in your muscle cells that unless there is someway to re-generate it from NADH, you'll run out of NAD= and glycolysis will quickly stop. To give you an indication of how fast this will happen -- if you run full speed, and if there is no way to make NAD= from NADH, you'll run out of the NAD= that you need for exercise in about 1 second!. (a) Running out would cause glycolysis to stop immediatel and so you would no longer be making ATP from ADP. (b) as you know, the result would be that your muscles would go into tetany. Obviously this does not happen normally. So how is NAD+ regenerated? (c) the answer is that NADH transfers its electrons and hydrogen to pyruvic acid. The result is a molecule of lactic acid and NAD= :

Lactic dehydrogenase (LDH) Pyruvic acid + NADH ---------------------------------------> lactic acid + NAD+ (b) This reaction is catalyzed by the enzyme lactic dehydrogenase, LDH (know the initials) . (c) We say that pyruvic acid is the electron acceptor for NADH. (d) Here are diagrams of the structures of the two molecules; this shows you that the only obvious difference in the molecules is that lactic acid has two hydrogens that pyruvic acid lacks. With those hydrogens come the high energy electrons from NADH:
pyruvic acid CH3 lactic acid CH3

C == O

H - C - OH
C

C O

==

(e) So, we make lactic acid from pyruvic acid in order to have enough NAD= to continue glycolysis. We will see later that this step is only necessary when there is either not enough oxygen(type I fiber) or when the cell has trouble using oxygen (type IIb). (f) Notice that for each 6 C carbohydrate that entered glycolysis that there were 2 molecules of lactic acid created - more about this in a second. In this box below and on the next page, I give an Illustration of why it is the enzymes that determine the types of reactions that occur in cells. Different organisms regenerate NAD+ using different means. Here's what happens in yeast (for example when making beer): (next page)

==
OH

OH

pyruvic acid CH3

acetaldehyde CH3

ethanol (alcohol) CH3 + NADH

PDC
C

ADH
OH--- C --H + NAD+

==

C == O

O C O

H (the alcohol)

+
OH

CO2
(the fizz)

Two last points about lactic acid. Although lactic acid is a sort of a waste product, at least for muscles, it is in fact high in energy -- it contains more potential energy than pyruvate (WHY??). Other cells can and will use it. It should also be obvious that lactic acid is not a poison. It actually contains more energy than pyruvic acid and it is a common part of many foods. Perhaps the best know food is yogurt -- bacteria are added to milk. These use the sugars in milk and run them through glycolysis to get energy and they make lactic acid. The lactic acid curdles the milk -- remember that much of milk is protein and the acid causes the proteins to change shape. It also gives yogurt its sour tange. So, let's summarize the energy yielding parts of glycolysis and show what happens with NADH (next page):

==

P - 6C - P

NADH 3C - P NAD+ ATP ADP ATP ADP 3C - P NADH NAD+

ATP

ATP ADP

ADP 3C (pyruvic acid) 3C (pyruvic acid)

knp

3C Lactic Acid

3C Lactic Acid

And now let's summarize the entire process starting with a 6 carbon sugar:
glycogen phosphorylase

Glycogen phosphate

Glucose

6C - P
PFK

ATP ADP

P - 6C - P

NADH 3C - P NAD+ ATP ADP ATP ADP 3C - P NADH NAD+

ATP

ATP ADP

ADP 3C (pyruvic acid) 3C (pyruvic acid)

knp

3C Lactic Acid

3C Lactic Acid

Summary
1. Either 1 (starting with glycogen) or 2 (starting with glucose) ATP were required to start the process. 2. Glycolysis always produces 4 ATP per 6C that enters the pathway.

3. So, the net yield of ATP per 6C during exercise is: (a) if starting with glucose, 2 ATP (4 - 2 to start) (b) if starting with glycogen, 3 ATP (4 - 1 to start). 4. The yield of waste product, lactic acid, is: (a) 2 lactic acid per 6C that enters glycolysis (b) this means about 1 lactic acid per ATP

II. Acid-base
A. Definitions: 1. An acid: any substance that will release H+ into a solution. 2. A base: any substance that will combine with H+ and remove them from solution. 3. pH: a measure of acid base. Numerically it is equal to: pH = - log[H+] (negative log of the concentration of H+ ions). Here is a few quick solutions to this equation: if [H+] = 0.000001 mols/liter of water (i.e., 10-7 mols of H+ per liter), log[10-7] = -7 since pH = -log[H+] , then pH = - (-7) = 7. Notice that even though this seems like a small concentration of hydrogen ions, it corresponds to 1016 Hydrogen ions per liter of water. Three more examples: if [H+] = 0.0001 mols/liter of water (i.e., 10-5 mols of H+ per liter), log[10-5] = -5 since pH = -log[H+] , then pH = - (-5) = 5. if [H+] = 0.1 mols/liter of water (i.e., 10-1 mols of H+ per liter), log[10-1] = -1 since pH = -log[H+] , then pH = - (-1) = 1. if [H+] = 0.000000001 mols/liter of water (i.e., 10-10 mols of H+ per liter), log[10-10] = -10 since pH = -log[H+] , then pH = - (-10) = 10. Notice that as the pH gets lower, the [H+] gets larger. Thus, low pH values are associated with stronger acids and lots of hydrogen ions. B. About the pH scale: 1. pH = 7 is "neutral" - there are still plenty of H+ ions around (see box above). The solutions in our bodies are normally quite close to pH 7 -- they range between about pH 7.4 and slightly more acidic values down in pH 6 -these lowest (most acid values) tend to be found in active type IIb muscles. 2. pH 7 down to pH 0 -- increasingly acidic (see example above).

3. pH 7 up to pH 14 -- increasingly more basic or alkaline -- there are fewer and fewer H+ since something must be removing them. C. Why is pH so important? 1. The reason is simply that proteins are designed to work properly over a very narrow range of pH. When we look at protein structure, after we finish metabolism, we will see that small highly charged particles such H+ ions are particularly good at causing proteins to shift shape. Eventually such shape changes must result in a decrease in function. More about this later. 2. Incidentally, it is important to keep in mind that to varying degrees, changes in pH affect every protein in a cell. So decreases in pH have a general negative affect on cellular function. D. What happens to pH in anaerobic glycolysis? 1. Notice again that anaerobic glycolysis produces roughly one molecule of lactic acid for each ATP produced. 2. In heavy exercise, lots of ATP is needed and so lots of lactic acid may be produced -- especially in type IIb fibers. 3. The pH goes down rapidly. 4. Every protein is affected -- contractile proteins, pumps, and even the enzymes of the glycolytic pathway are all increasingly compromised by the increases in [H=] that accompany such exercise. 5. This leads to an increasing difficulty in synthesizing more ATP, more and more trouble in forming and breaking crossbridges etc. Performance decreases. 6. The subjective experience of the low pH is a burning sensation in the affected muscles and the measurable affect is a decrease in performance. This is lactate (pH) -induced fatigue.

Getting Rid of Lactic Acid and Restoring Correct Acid/Base -- this is a very important topic in exercise physiology. We will visit in more detail after we learn about aerobic metabolism and additionally when we consider blood and breathing during exercise and recovery.
Important Note: As I mentioned in class, the important things to learn here are: Gross and net yields of ATP What the fuels are for anaerobic glycolysis What the role of NAD= and NADH are. Why lactic acid is made. Why so much lactic acid can be made for so little ATP. Why lactic acid causes trouble. The role of enzymes in controlling rate and paths of reactions. A basic understanding of pH and Osmosis. Don't spend time memorizing the structures of molecules or the pathway.

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