“Hey bro, do you have this thing called G6PD? What is this G6PD?” What do the title and the preceding sentence have in common? These are perhaps the words of full-time national servicemen, both 30 years ago and now respectively, to their fellow soldiers as they are left perplexed by the medical questionnaires they are tasked to complete.
Whilst serving in the SAF as a full-time national serviceman, I had the opportunity to go on overseas military training exercises. Before embarking on these overseas training trips, servicemen would need to ensure that they are certified to be medically fit for the relevant training. This would often entail getting the required vaccinations at the camp, declaring one’s vaccination history, and declaring any medical conditions to the senior medics or the medical officers at the medical centre of the army camp, either verbally or through the completion of forms.
I remember filling in some of these questionnaires and one of the points on these questionnaires would be:
“2. G6PD: Yes/No”.
I’m not even kidding. It was just this, word for word. Allow me to explain why such wording can technically be misinterpreted. Servicemen have to fill in these forms individually. A few weeks prior to leaving for training in Australia, my entire company was given such a form to fill in. In the company office, someone from another platoon asked one of the platoon commanders, “Sir, how do we fill this in? I’m not sure what G6PD is”. The lieutenant replied, “If you are not sure what it is, you probably don’t have it.”
On the surface, this seems to be a reasonable position to adopt. After all, if you have gotten through life this far without doctors ever having diagnosed you with G6PD (glucose 6-phosphate dehydrogenase) deficiency, you probably do not have the said condition. Here is an image of a patch that a soldier can put on his ILBV to indicate his G6PD deficiency:
Most students who have learnt of G6P (glucose 6-phosphate) first studied it in the context of glycolysis, gluconeogenesis, and glycogenolysis. However, the G6PD enzyme is relevant in neither of these pathways. Instead, it is an enzyme that is relevant to the pentose phosphate pathway/HMP (hexose monophosphate) shunt, which occurs in the cytosol. Now, this HMP shunt is quite important because it provides a source of NADPH from the available G6P. NADPH is used in reductive reactions such as glutathione reduction inside RBCs (red blood cells), fatty acid and cholesterol synthesis. Additionally, the shunt also yields ribose for nucleotide (those ATCGs in DNA) synthesis and glycolytic intermediates (fructose 6-phosphate and glyceraldehyde 3-phosphate). This is particularly important in RBCs because NADPH is needed to keep glutathione reduced, which in turn detoxifies free radicals and peroxides.
In other words, here are the pairs of redox reactions. Firstly, G6PD catalyses the irreversible oxidation of G6P to 6-phosphogluconolactone, which reduces NADP+ to NADPH.
NB: 6PGL (6-phosphogluconolactonase) then catalyses the oxidative hydrolysis of 6-phosphogluconolactone to give 6-phosphogluconate.
Next, NADPH is oxidised back to NADP+ when glutathione reductase catalyses the reduction of GSSG (glutathione disulfide) to 2 GSH (glutathione). NB: The ‘S’ and ‘H’ in GSSG and GSH represent sulfur and hydrogen atoms respectively. Lastly, 2 GSH is oxidised back to GSSG when glutathione peroxidase catalyses the reduction of H2O2 to 2 H2O.
I’m sure you can now clearly see the problem with having G6PD deficiency. When there is deficiency in G6PD, all the above reactions are inhibited. There won’t be as much NADPH produced, because there isn’t as much dehydrogenation from G6P to 6-phosphogluconolactone. Consequently, there would be hemolytic anaemia due to poor RBC defence against oxidising agents (remember the last step in the chain in the above paragraph where H2O2 is reduced to water; that now doesn’t occur as much). H2O2 is a reactive oxygen species that can be converted to hydroxyl radicals, which can attack the proteins in the cell.
Consider this quote from reference [4]:
“Protein oxidation is defined as the covalent modification of a protein induced either by the direct reactions with reactive oxygen species (ROS) or indirect reactions with secondary by-products of oxidative stress. ROS can cause oxidation in both amino acid side chains and protein backbones, resulting in protein fragmentation or protein-protein cross-linkages. Although all amino acids can be modified by ROS, cysteine, and methionine that are the most susceptible to oxidative changes due to high reaction susceptibility of the sulfur group in those amino acids. Oxidative modifications of proteins can change their physical and chemical properties, including conformation, structure, solubility, susceptibility to proteolysis, and enzyme activities.“
As the quote above says, amino acids with sulfur-containing R-groups are most susceptible to oxidative attack due to the relatively high reactivity of sulfur. Hence, it isn’t a surprise to learn that the hydroxyl radicals can attack haemoglobin in RBCs, causing a cross-linking of sulfhydryl groups on globin chains, which in turn causes haemoglobin to be denatured and precipitate. And what happens when oxidised haemoglobin precipitates?
Heinz bodies.
Look at this image above. That damaged haemoglobin coloured in blue represents a Heinz body. That pink Pac-man thing represents a splenic macrophage, which comes and “eats” the Heinz body. Bite cells, represented by the cell below the cell with the damaged haemoglobin, thus result from the phagocytic removal of Heinz bodies by splenic macrophages. Such original and creative naming. Lol.
G6PD deficiency is an X-linked recessive disorder, and is the most common human enzyme deficiency.
If we were to be quite pedantic, then the construct “2. G6PD: Yes/No” is, strictly speaking, problematic because it is people who are deficient in the enzyme G6PD that have the condition known as G6PD deficiency. In other words, people who havesufficient G6PD are fine and do not have G6PD deficiency. So, since normal people have G6PD, would it be wrong for people to circle “Yes”? On the other hand, the form is probably asking people whether they have the deficiency.
My recommendation to the SAF is to unambiguously alter the construct in the medical questionnaire to “G6PD deficiency: Yes/No”.
Personally, I wouldn’t be surprised if some people think that the “D” in G6PD means “deficiency”. After all, it doesn’t help that there is a practice where people repeat the full word of the last letter of acronyms (e.g. POP parade, IPPT test, PSLE exam, GUI interface, PIN number, ATM machine, D-Day, HIV virus, DC Comics, LCD display, OPEC countries).
Ironically, such a practice is known as RAS syndrome, where RAS stands for “redundant acronym syndrome”. What a term! For all who design invitation cards. please take note. Please don’t put “Please RSVP” on invitations!
The world’s tiniest motor, ATP synthase, or F1FO-ATPase, stands at about 20 nm in height and 10 nm in diameter.
Look at the above artist’s impression of ATP synthase. What a beautiful image. This protein complex is something that many biology, biochemistry, health sciences students are required to learn about. Today, I wish to talk about ATP synthase as I find it quite fascinating.
To begin, let us resolve some issues regarding its nomenclature. ATP synthase belongs to a broader group known as ATPases. This might leave you wondering, as it did for me: “why does ATP synthase fall under this category? From its name alone, don’t ATPases couple the hydrolysis of ATP with endergonic reactions? In other words, don’t ATPases catalyse the dephosphorylation from ATP to ADP which would generate energy that would be harnessed by the ATPase to drive reactions which would otherwise not occur? ATP synthase isn’t hydrolysing ATP; it’s making ATP” It turns out that for most ATPases, ATP is hydrolysed; however, ATPases also include the inverse reaction to ATP hydrolysis, to wit, the phosphorylation of ADP with inorganic phosphate to give ATP.
It’s just like how the oxidation of succinyl-CoA to succinate is catalysed by succinyl-CoA synthetase. Even though succinyl-CoA isn’t the product in the forward reaction, the same enzyme is being used in both the forward and reverse reaction.
Under the umbrella term of ATPases, there are sub-categories: F-, V-, A-, P-, and E-ATPases. I will focus on F-ATPases (aka F1FO-ATPases), the most notable of which is ATP synthase. Unlike most of the other sub-categories of ATPases, F-ATPases catalyse the synthesis of ATP. A-ATPases work like F-ATPases too; it’s just that A-ATPases are found in Archaea. ATP synthase is sometimes known as Complex V.
“Nicholas, why is the F-ATPase called F1FO-ATPase? Shouldn’t it be named F1F0-ATPase? After all, isn’t the preceding number to 1 is obviously 0?” Let me explain. Oligomycin is an antibiotic that inhibits ATP synthase by blocking its proton channel through the Fo subunit. In my opinion, F0 has become commonly adopted by many authoritative textbooks and peer-reviewed articles for the following reasons:
1) The letter ‘o’ is visually similar to ‘0’. In classrooms or lecture theatres, where teachers and professors write about the Fo sub-unit by hand on the whiteboard, it might be impossible to determine if an ‘o’ or ‘0’ was written.
2) This is probably the biggest factor: ‘0’ precedes ‘1’. Meanwhile, ‘o’ is a letter while ‘1’ is a number.
3) There are many more symbols where ‘0’ is the subscript instead of ‘o’.
4) Many authoritative texts and teachers use F0 instead of Fo and when other people read or listen to them, they also do the same and adopt the convention. As such, the use of F0 is rapidly adopted.
5) There is little mention about this historical development in various resources.
This two-paragraph extract from a paper by McCarty (1992) elaborates on the situation.
Also of note is the fact that some older texts refer to ATP synthase as ATP synthetase.
According to the JCBN (Joint Commission on Biochemical Nomenclature)/NCIUBMB (Nomenclature Committee of the International Union of Biochemistry and Molecular Biology) newsletter in 1984, the word synthase can now be added to the name of any substance to signify an enzyme that catalyses a reaction producing that substance, i.e. its range is extended to include enzymes catalysing reactions that involve hydrolysis of a nucleoside triphosphate, which previously could not be called synthases. In addition, “synthetase” is now synonymous with “ligase”.
In view of this update, it is thus more precise to refer to Complex V as ATP synthase instead of ATP synthetase. I also want to talk about the theoretical or experimentally-determined values regarding how many ATPs are produced for each molecule of NADH or FADH2 that enters the mitochondrial electron transport chain. The oldest values are: each NADH and FADH2 molecule leads to the production of 3 ATP and 2 ATP respectively.
However, we also see values such as 2.5 ATP and 1.5 ATP for each molecule of NADH or FADH2 respectively. Some modern texts even state that each NADH leads to 2.7 ATP being produced and 1.6 ATP/FADH2. Which is it?
To understand how we got into this mess, we must understand the relevant historical development. In the 1960s, ATP was known to be the energy currency of life, but the mechanism by which ATP was generated in the mitochondrial matrix was assumed to be substrate-level phosphorylation. After all, substrate-level phosphorylation was seen in glycolysis, and the Krebs cycle where succinyl-CoA is oxidised to succinate, while dephosphorylating GTP to GDP, which in turn phosphorylates ADP to ATP.
A scientist named Peter D. Mitchell realised that the movement of ions across an electrochemical potential difference could provide the energy needed to produce ATP. His hypothesis was actually derived from information that was already well known in the 1960s. He knew that living cells had a membrane potential; interior negative to the environment. The movement of charged ions across a membrane is thus affected by the electrical forces (the attraction of positive to negative charges). Their movement is also affected by the chemical potential, where substances diffuse from regions of higher concentration. He went on to show that ATP synthesis was coupled to this electrochemical gradient.
But of course, as with many great scientists who came up with seemingly absurd hypotheses, I’m sure he was mocked and ridiculed for this. Here is a quote from a biography of Mitchell: “It is a striking fact that, at the time Mitchell proposed his theory, there was not a shred of experimental evidence in its favour.” When Mitchell died in 1992, the following obituaries were published:
FromThe Times, a British daily national newspaper, on 15 April 1992: “His hypothesis met, initially, with an almost derisory reception. Few of the workers in the field had the necessary background knowledge of electrochemistry to appreciate the proposals and they spent many years searching for a chemical intermediate, which Mitchell argued did not exist.”
From The Independent, established in 1986 as a politically independent national morning newspaper published in London, on 16 April 1992: ‘For 20 years Mitchell was ridiculed, and Jennifer Moyle was his only professional supporter’.
Mitchell’s contemporaries have been criticised for their hostility towards Mitchell’s chemiosmotic hypothesis. Such criticism may be misplaced, though. For example, the elegance of the hypothesis was immediately recognised by some. In a review published in 1962, Lehninger (yes, this is the same Lehninger as the one who authored the renowned biochemistry textbook) gave a sympathetic summary of the chemiosmotic hypothesis, and Mitchell was invited by his peers to present his hypothesis in seminars at various European universities.
Anyway, the chemiosmotic hypothesis eventually became the leading hypothesis. The brilliance of the chemiosmotic coupling was that it allows nonintegral stoichiometries of oxygen consumption and ATP synthesis. Let me explain.
Before the chemiosmotic model for oxidative phosphorylation was accepted, the assumption was that the overall reaction equation would take the following form because everyone thought that it would be substrate-level phosphorylation:
where the value of ‘x’ would be an integer. When intact mitochondria are suspended in solution with a oxidisable substrate such as NADH or succinate, along with addition of oxygen, ATP synthesis is readily measurable, as is the decrease in oxygen. In theory, this should allow ‘x’ to be calculated. However, measurement of ‘x’ is complicated by the fact that 1) in intact mitochondria, any ATP that is produced is used by the mitochondria in other reactions in the matrix. Also, 2) oxygen is also used in reactions unrelated to oxidative phosphorylation. Hence the discrepancy due to these two factors need to be taken into account. Most experiments, after the necessary corrections, have determined x to be between 2 and 3 when 1 mole of NADH is used as the oxidisable substrate.
Given the assumption that x was to be an integer, most experimenters agreed that the x should be 3 and 2 for NADH and succinate respectively. Just like that, these values have been used for years in peer-reviewed articles and textbooks.
With the chemiosmotic model involving the coupling of ATP synthesis (involving phosphorylation) and electron transfer (involving oxidation), there was no longer a theoretical requirement for x to be an integer. The measurement of proton fluxes (how many protons are pumped through the various complexes) is quite complicated but basically, the experimental consensus is that for each pair of electrons, 10 protons and 6 protons are pumped into the intermembrane space for one molecule of NADH and succinate respectively. For flow back into the matrix from the IMS, the most widely accepted value is that 4 protons are required to drive the synthesis of 1 ATP.
Since 1 NADH leads to 10 protons being pumped from the matrix into the IMS, and 4 protons are required for synthesis of 1 ATP, it follows that x = 10/4 = 2.5 for NADH. Thus, 1 NADH leads to the production of 2.5 ATP. It also follows that since 1 succinate molecule leads to 6 protons being pumped from the matrix into the IMS, then x = 6/4 = 1.5 for succinate. Thus, 1 succinate leads to the production of 1.5 ATP.
Since 1 NADH leads to 10 protons being pumped from the matrix into the IMS, and 4 protons are required for synthesis of 1 ATP, it follows that x = 10/4 = 2.5 for NADH. Thus, 1 NADH leads to the production of 2.5 ATP. It also follows that since 1 succinate molecule leads to 6 protons being pumped from the matrix into the IMS, then x = 6/4 = 1.5 for succinate. Thus, 1 succinate leads to the production of 1.5 ATP.
You might have noticed that I talked so much about succinate. What happened to FADH2? Here’s the brilliance of the mitochondrion. The TCA Cycle takes place in the matrix, and recall that one of the steps of the TCA involves the oxidation of succinate to fumarate. Now, this oxidation actually occurs at Complex II of the mETC. The oxidation of succinate to fumarate simultaneously reduces FAD to FADH2. FAD is a prosthetic group, a covalently-attached cofactor that is found within Complex II. FADH2 is then oxidised back to FAD when it reduces Q to QH2. QH2 then shuttles to Complex III. This is how electrons are carried from the TCA, through II to III, etc! Amazing, isn’t it? Thus, it could be alternatively said that 1 FADH2 leads to 1.5 ATP.
We could use the same logic and apply it to NADH, but then NADH could come from so many places. Just imagine. 1 isocitrate produces 2.5 ATP; 1 α-ketoglutarate produces 2.5 ATP; 1 malate produces 2.5 ATP. This isn’t practical at all. From the TCA cycle alone, NADH is produced in 3 instances:
1) the oxidation of isocitrate to α-ketoglutarate simultaneously reduces NAD+ to NADH 2) the oxidation of α-ketoglutarate to succinyl-CoA simultaneously reduces NAD+ to NADH 3) the oxidation of malate to oxaloacetate simultaneously reduces NAD+ to NADH
NADH can also come from another reaction within the mitochondrial matrix which is not, strictly speaking, part of the TCA. Firstly, consider the earlier oxidation of α-ketoglutarate to succinyl-CoA, where this reaction is catalysed by α-ketoglutarate dehydrogenase complex. α-ketoglutarate (5 carbons) is oxidised to succinyl-CoA (4 carbons). What happened to that one carbon? Well, it escaped as CO2. Look at the product of the reaction too. A “CoA” is added. Also, an NADH is produced. The enzyme is a dehydrogenase complex.
Does this ring a bell?
If you thought of the oxidation of pyruvate to Acetyl-CoA, then CONGRATULATIONS, you got it!
The thing you must understand is this: α-ketoglutarate dehydrogenase complex is virtually identical to pyruvate dehydrogenase complex. Within the matrix, the oxidation of pyruvate (3 carbons) to Acetyl-CoA (2 carbons) produce CO2, brings about the addition of a “CoA”, and lastly, reduces NAD+ toNADH.
In addition, NADH can be shuttled from the cytosol into the matrix to be used as substrate at Complex I.
Let’s go back and talk about the proton flux where protons move from the IMS into the matrix. How does it work? ATP synthase consists of two parts: Fo and F1. Fo has a number of sub-units and the number of sub-units is equal to the number of protons that move through Fo. In animal mitochondria, n = 8 (it varies, depending on the organism; in E. coli, n = 10). This means that 8 protons move through Fo in one revolution of Fo. Also consider the F1 portion, where one revolution of F1 produces 3 ATP. Since the synthesis of each ATP molecule requires the transfer of 1 Pi into the matrix, at a cost of 1 proton for each Pi transfer, that means that 3 additional protons are required for the synthesis of 3 ATP. This means that 8 + 3 = 11 protons are needed for the synthesis of 3 ATP. Remember that this is animal mitochondria that we are talking about. 11/3 gives 3.7 protons/ATP for ATP synthesis in animals. When we use the values of 10 protons/NADH and 6 protons/succinate or FADH2, we get: 10/3.7 = 2.7 ATP/NADH and 6/3.7 = 1.6 ATP/succinate or FADH2 in animals.
Then you might be wondering: how does this relate to 2.5 ATP/NADH and 1.5 ATP/NADH respectively? Recall that the experimental consensus was 4 protons/ATP. Now, what organisms were used to obtain that value? That is the crux of the matter. In the previous paragraph, I mentioned that the number of Fo sub-units vary depending on the organism. This totally changes everything. For example, in the case of E. coli andyeast, where n = 10, the values you get will be 2.3 ATP/NADH and 1.4 ATP/succinate or FADH2, if you follow the same calculation process in the previous paragraph.
These above calculated values of x (aka P/O ratio) define a range that includes the experimental values of 2.5 ATP/NADH and 1.5 ATP/FADH2. Thus, 2.5 and 1.5 are ubiquitous in biochemistry textbooks. Yet, for simplicity, some still choose to use 3 ATP/NADH and 2 ATP/FADH2.
I hope this blog post has perhaps allow you to understand why I find ATP synthase so fascinating. It is a pity that a lot of the above background information which includes highly interesting historical development isn’t discussed enough in textbooks, classrooms, and lecture theatres because of a lack of time, resources, etc.
