Human Genes for the Pyruvate Dehydrogenase Complex

 
The pyruvate dehydrogenase complex (PDC) catalyzes a very important metabolic reaction: the conversion of pyruvate to acetyl-CoA [Pyruvate Dehydrogenase Reaction]. The complex consists of three components: E₁ a dimer of E₁α and E₁β polypeptides; E₂, and E₃ [The Structure of the Pyruvate Dehydrogenase Complex].

Each of them are encoded by separate genes so there are four human genes required. We'll see shortly that there are two additional genes for a total of six. The E₃ subunit is shared with two other enzymes: 2-oxoglutarate dehydrogenase (a citric acid cycle enzyme) and 2-oxo acid dehydrogenase (a enzyme required for amino acid degradation) [Pyruvate Dehydrogenase Evolution].

The gene for E₁α: is called PDHA1 and it's located on the X chromosome at p22.2-p22.1 [Entrez Gene = 5160]. There are more than three dozen alleles that give rise to symptoms ranging from mild lactic acidosis to developmental defects. The accumulation of lactate is due to the fact that it can't be converted to pyruvate because the defect in pyruvate dehydrogenase causes buildup of pyruvate in the cell [Pyruvate]. Males often die at an early age. (Note that males are homozygous for mutant alleles because the gene is on the X chromosome) [OMIM 300502]. Females are also affected because only one X chromosome is active and if it happens to be the one carrying the mutations the entire cell is affected [Calico Cats].

A testis specific copy of the E₁α: gene is called PDHA2 and it's located on chromosome 4 (q22-q23) [Entrez Gene = 5161].

The gene for E₁β: is called PDHB on chromosome 3 near p21.1-p14.2 [Entrez Gene = 5162]. There are only two known alleles that cause a problem. Both are homozygous lethals but only after birth. The infants have severe problems and fail to develop normally [OMIM 179060]. Death usually occurs within a year of birth. It's likely that other mutations are embryonic lethals so we never see them as genetic diseases [Most Metabolic Diseases Affect Unimportant Genes].

The gene for the E₂ subunit is called DLAT (dihydrolipoamide s-acetyltransferase). It is located on the chromosome 11 at q23.1 [Entrez Gene = 1737 ]. Two alleles are known to cause problems but the patients respond well to dietary treatment [OMIM 608770]. It's very likely that more severe genetic defects are embryonic lethals.

The gene for the E₃ subunit is called DLD [Entrez Gene = 1738]. It is located on chromosome 7 at q31-q32. There are many alleles of this gene and some of them cause genetic diseases. The phenotype results from a defect in amino acid metabolism and not from a defect in pyruvate dehydrogenase. Recall that the E₃ subunit of PDC is shared with 2-oxo acid dehydrogenase, an enzyme required for the breakdown of branched chain amino acids. Deficiencies in the enzyme activity lead to accumulation of breakdown products that are secreted in the urine. This gives rise to a characteristic odor resembling maple syrup [OMIM 238331]. The particular genetic disease associated with the DLD genes is called maple syrup urine disease type III

There is one other minor component of the pyruvate dehydrogenase complex in humans. Protein X binds to E₃. It is encoded by the PDHX gene on chromosome 11 (p13). There are no known alleles in the OMIM database.

Pyruvate Dehydrogenase Evolution

Before discussing the origin of the pyruvate dehydrogenase complex (PDC) we need a little background information. There are three different reactions catalyzed by enzyme complexes resembling the pyruvate dehydrogenase complex. For example, one of the reactions of the citric acid cycle is the conversion of 2-oxoglutarate (α-ketoglutarate) to succinyl-CoA. As you can see from the reaction below it is very similar to the pyruvate dehydrogenase reaction. The main difference is that the substrate, 2-oxoglutarate, has five carbons while pyruvate only has three. The part of the molecule that reacts is the top part with a carboxyl (-COO^-) that is lost as CO₂ and a keto (-C=O) that ends up being attached to coenzyme A via a sulfhydryl linkage.


It should come as no surprise that this reaction is catalyzed by an enzyme called 2-oxoglutarate dehydrogenase (OGDH, also known by its old name: α-ketoglutarate dehydrogenase) (EC 1.1.4.2) that's almost identical to pyruvate dehydrogenase. In fact, both PDC and OGDH evolved from a common ancestral enzyme. We know that the citric acid cycle enzyme is a late comer because many species of bacteria don't have it. Indeed, they don't even have a citric acid cycle.

So we need to look elsewhere if we are going to find the source of PDC. The most primitive enzymatic reaction is almost certainly one that's required in amino acid metabolism.¹ In this case it's a reaction involved in the degradation of the branched chain amino acids; leucine, valine, and isoleucine. Look at the pathway below.

The first step in the degradation is the removal of the amino group (-NH₃⁺) and its replacement with an oxygen to form a keto (-C=O) group. This creates three similar 2-oxo acids (α-keto acids) all of which resemble 2-oxoglutarate and pyruvate. All three of the 2-oxo (α-keto) acids are acted upon by the same enzyme called branched chain 2-oxoacid dehygrogenase (OADH, BCOADH, α-ketoacid dehydrogenase) (EC 1.2.4.4) to create an acyl-CoA product. This is the same reaction as that catalyze by the pyruvate dehydrogenase complex except that the R group in pyruvate is -CH₃ while in the case of the branched chain dehydrogenase it's a three, four, or five carbon branched structure.

BCOADH is found in all species. It is the most "primitive" enzyme. Like PDC it has a complex structure with three different subunits. E₁ catalyzes the decarboxylation reaction. E₂ catalyzes the formation of acyl-CoA—it has the lipoamide swinging arm. E₃ catalyzes the oxidation of the lipoamide and the reduction of NAD⁺.

It looks like the "primitive" BCOADH could also catalyze the oxidative decarboxylation of pyruvate. In fact some of the modern enzymes still have residual activity towards the other substrates. Over time, the genes for some of the subunits duplicated and the two enzymes (PDC and BCOADH) diverged as they became more specialized for their modern substrates.

We can see the result if we look at the phylogenetic tree for the E₂ subunit (below). This figure is from a paper by Scharrenberger & Martin (2002). They use a slightly different nomenclature (PDH=pyruvate dehydrogenase complex). This is an unrooted tree so you can't really tell which enzyme came first but, as I explained above, there is good reason to believe that the E₂ from PDC and the E₂ from OGDH evolved from the E₂ gene for BCOADH via successive duplications.


Recall that the E₂ subunits form the core of the complex (left). They contain the lipoamide swinging arm that carries substrate to three different active sites. The E₃ subunits of the three enzymes are identical. There is only one E₃ gene and it supplies the dihydrolipoamide dehydrogenase activity for BCOADH, PDC, and OADH.

The situation with the E₁ subunit is more complicated. This is the part of the enzyme that recognizes the different types of substrate (e.g. pyruvate, 2-oxo acids, 2-oxoglutarate) so it makes sense that the three enzymes have different E₁ subunits. All the eukaryotic versions of the PDC E₁ subunit are related to the E₁ subunit from BOADH. So are most of the bacterial versions. Other bacterial versions of the PDC E₁ subunit are not related to those of the other enzymes (Schreiner et al. 2005).

The conclusion from the molecular data is that the pyruvate dehydrogenase complex evolved from the branched chain 2-oxo acid complex about 2 billion years ago. Subsequently, in some bacterial lineages a different E₁ subunit replaced the one that's homologous to the BCOADH subunit. The α-proteobacteria and cyanobacteria lineages that gave rise to mitochondria and chloroplast respectively, retained the PDC E₁ subunit that is related to BCOADH enzymes. This explains the eukaryotic versions of PDC.

1. This is a common theme in the evolution of metabolic enzymes. The evidence suggests strongly that amino acid metabolism is more ancient than most carbohydrate metabolism.

Schreiner, M.E., Fiur, D., Holatko, J., Patek, M. and Eikmanns, B.J. (2005) E1 enzyme of the pyruvate dehydrogenase complex in Corynebacterium glutamicum: molecular analysis of the gene and phylogenetic aspects. J Bacteriol. 187:6005-18.

Schnarrenberger, C. and Martin, W.. (2002) Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer. Eur J Biochem. 269:868-83.

Some Bacteria Don't Need Pyruvate Dehydrogenase

Recall that the pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA. This is an important reaction in all living cells because acetyl-CoA is required for fatty acid synthesis. The reaction is important in animals because acetyl-CoA enters the citric acid cycle where it is broken down to carbon dioxide and the energy is captured by the mitochondrial electron transport system in the form of ATP. This step isn't so important in most bacteria because they don't have a citric acid cycle. Most species can also save the two carbon atoms of the acetyl group in acetyl-CoA and use them to build carbohydrates such as glucose. Animals can't do this.

You would think that the pyruvate dehydrogenase complex (PDC) must be ubiquitous since it catalyzes such an important reaction. Not so. PDC is the only enzyme in eukaryotes but some bacteria have another enzyme that can make acetyl-CoA. As you might expect, the bacteria that gave rise to mitochondria do have a PDC that's related to the eukaryotic enzyme. This is because the genes were transferred from those bacteria to their eukaryotic hosts when the endosymbiotic event occurred about two billion years ago.

Lots of different kinds of bacteria have a similar PDC but some have a completely different enzyme called pyruvate:ferredoxin oxidoreducatase (PFOR) (E.C. 1.2.7.1) (Chabrière et al. 2001). This enzyme catalyzes a very similar reaction where pyruvate undergoes an oxidative decarboxylation yielding CO₂ and acetyl-CoA. The difference is that instead of having a complicated electron transport chain where electrons are passed to lipoamide, FAD⁺, and finally NAD⁺ [Pyruvate Dehydrogenase Reaction], the PFOR reaction is much simpler. Here electrons are transferred to ferredoxin, a small iron-containing protein.

The structure of pyruvate:ferredoxin oxidoreductase has been worked out from a combination of X-ray diffraction data and electron microscopy, just as we saw with the pyruvate dehydrogenase complex [The Structure of the Pyruvate Dehydrogenase Complex]. The structure of one such enzyme, from the bacterium Desulfovibrio vulgaris, is shown in the figure above. This complex consists of eight copies of the enzyme (Garczarek et al. 2007). In other species a simple two-copy complex suffices.

Ferredoxin is a cofactor in many biochemical reactions. As a general rule, enzymes that use ferredoxin are more ancient than enzymes that involve NAD⁺ as a cofactor. Ferredoxin metabolism doesn't need oxygen and the available evidence suggests that oxygen wasn't present in the ancient atmosphere. Modern bacteria that use pyruvate:ferredoxin oxidoreductase (PFOR) instead of the pyruvate dehydrogenase complex (PDC) are capable of anaerobic growth (without oxygen).

The structure of many ferredoxins have been solved. The one shown on the left is from Pseudomonas aeruginosa. It's a typical example (Giastas et al. 2006). The protein is quite small and most ferredoxins contain two iron-suflur (Fe-S) complexes. These are box-like structures formed from iron molecules (red) and sulfur molecules (yellow). They are bound to the protein through the sulfhydyl groups of the amino acid cysteine. Electrons are carried by the iron ions.

Fe³⁺ + e^- → Fe²⁺

There's another important reason why PFOR is important in some bacteria. Look at the PDC reaction shown above. The arrow points in one direction indicating that this reaction is essentially irreversible. It can't be used to fix carbon dioxide by combining it with the acetyl group to make pyruvate. That's not true of the much simpler PFOR reaction. In fact, the reverse reaction is the main CO₂ fixing reaction in many photosynthetic bacteria and in methanogens (bacteria that use methane as a carbon source).

But we're getting distracted. The point is that the pyruvate dehydrogenase complex probably arose late in evolution after photosynthetic bacteria had transformed the atmosphere into one that contained significant levels of oxygen. Where did such a complicated protein complex come from?

Chabriere, E., Vernede, X., Guigliarelli, B., Charon, M.H., Hatchikian, E.C. and Fontecilla-Camps, J.C. (2001) Crystal structure of the free radical intermediate of pyruvate:ferredoxin oxidoreductase. Science 294:2559-63.

Garczarek, F., Dong, M., Typke, D., Witkowska, H.E., Hazen, T.C., Nogales, E., Biggin, M.D., and Glaeser, R.M..(2007) Octomeric pyruvate-ferredoxin oxidoreductase from Desulfovibrio vulgaris. J Struct Biol. 2007 Feb 17; [Epub ahead of print] .

Giastas, P., Pinotsis, N., Efthymiou, G., Wilmanns, M., Kyritsis, P., Moulis, J.M., and Mavridis, I.M..(2006) The structure of the 2[4Fe-4S] ferredoxin from Pseudomonas aeruginosa at 1.32-Å resolution: comparison with other high-resolution structures of ferredoxins and contributing structural features to reduction potential values. J. Biol. Inorg. Chem. 11:445-58.

Killer Cellphones Destroy Bees

 
Friday's Urban Legend: Probably FALSE

An article in our local newspaper (The Toronto Star) suggests a link between the mass kill off of bees and cellular phones [Cellular phone uses linked to bee deaths]. A similar report appeared in The Independent in the UK [ Are mobile phones wiping out our bees?].

Here's the problem. There are reports in Canada and the United States of disappearing honey bees. Apparently, entire colonies are being abandoned. The phenomenon is somewhat localized. In Canada, for example, excessive bee loss is only reported in central British Columbia and the Niagara peninsula in Ontario. The phenomenon is called colony collapse disorder.

If the bee disappeared off the surface of the globe, then man would only have four years of life left.
.... Albert Einstein

This quote appears in several newspaper articles and on many blogs. Snopes is on to it and so far there's no proof that Einstein ever said this [Einstein on bees].We are told that "German researchers" have linked cellphone radiation to the disappearance of bees. The business reporter checked with Martin Weatherall to see if this is correct. Who is Martin Weatherall, you might ask?

Weatherall, a retired Toronto police officer who was forced out of his Woodstock, Ont., home after high levels of radio waves from nearby hydro-electric poles and cellphone towers made him electro-hypersensitive, is better able than most to understand the German study, which shows that bees refuse to return to their hive when cellphones are placed nearby.

Near the end of the story in the Toronto Star the reporter also checks with Ernesto Guzman, an expert on bees at the University of Guelph in Ontarion, Canada. Guelph is one of the top schools in veterinary medicine and agriculture.

Despite the new German research, bee researchers remain skeptical of the impact of radio waves on bees. They claim it is just one of several theories that include global warming and genetically modified crops.

"All of these are speculation. They deserve to be investigated. They are good hypotheses, some of them. Others are out of reality, in my opinion," said Ernesto Guzman, associate professor with the University of Guelph's department of environmental biology.

Guzman, a specialist in bee research, says he believes stress is the major factor in the situation south of the border while in Canada a combination of poor weather on fall food supply levels and an influx of mites is the likely cause.

Personally, I will take Guzman's word over that of a retired police officer suffering from "electro-sensitivity." If I were writing the headline it would be "Cellphone link to bee deaths discredited by expert." I guess it all depends on how you want to frame spin the article.

Orzel Is Confused

 
On his blog Uncertain Principles, Chad Orzel posted on "framing" [The Final Word on Framing. He said,

That's a thought, but I think the answer is much simpler: PZ and Larry Moran are not primarily interested in promoting science.

"That's crazy," you say. But here it is from the horse's mouth, Larry Moran in Chris Mooney's comments:

I think religion is the problem and I'll continue to make the case against religion and superstition. One of the many ways where you and Nisbet go wrong is to assume that people like PZ, Dawkins, and me are primarily fighting for evolution. That's why you argue that in the fight to save evolution it's "wrong" (e.g., not part of your frame) to attack religion.

When are you going to realize that our primary goal in many cases is to combat the worst faults of religion? Asking us to stop criticizing religion is like asking us to give up fighting for something we really care about. That's not "framing," it's surrender.

That's the beginning and end of the problem. The entire problem with "framing" is that Nisbet and Mooney are looking for the best way to promote science, while PZ and Larry are looking for the best way to smash religion. The goals are not the same, and the appropriate methods are not the same-- in particular, Nisbet and Mooney argue that the best way to promote science would be to show a little tact when dealing with religious people, and that runs directly counter to the real goals of PZ and Larry.

It's not a simple as that Chad. I try to do both things. I try to write about science with the hope that I'm telling people something they don't know. If you would take the time to look at my blog I think you might see the occasional posting on science-related topics. Religion isn't mentioned.

On the other hand, there are times when I post about the conflict between rationality and superstition. I think there's a problem there and religion is a big part of it. Quite frankly, I don't find the arguments of the Theistic Evolutionists the least bit effective. Why should I ignore them when they spout their silliness?

Telling me that I should not criticize religion because it's not helping science education is just nonsense. It's like telling me to abandon something I feel very strongly about just because you don't like it. If that's what framing is all about then I'm not interested.

The problem with the framers is that they get terribly confused about issues. For some reason they think I'm a one dimensional person who's only interest is science education. They think that when I criticize superstitious nonsense I must always be wearing my science education hat. That's why they tell me and Dawkins and PZ not to criticize religion if we're trying to educate people about science.

Well, I got news for them. We are involved in several issues. One of them is teaching science. One of them is fighting superstition. There are others. Don't tell me not to fight superstition because I should only be concerned about science education. I'm concerned about both. In an ideal world people would understand science and reject superstition. I'd like to work toward that goal.

New Ways of Looking at Evolution

 
John Logsdon over at Sex, Genes, and Evolution recommends a new book, The Origins of Genome Architecture by Michael Lynch. John also points us to a review article by Lynch [The Origins of Eukaryotic Gene Structure]. I second both recommendations. Read the article. Buy the book.

Here's a quotation from the article by Lynch,

Despite the enormous progress in molecular genetics over the past 50 years, no general theory for the evolution of the basic architectural features of genes has been formulated. Many attempts have been made to explain the features of genes, genomes, and genetic networks in the context of putatively adaptive cellular and/or developmental features, but few of these efforts have been accompanied by a formal evolutionary analysis. Because evolution is a population-level process, any theory for the origins of the genetic machinery must ultimately be consistent with basic population-genetic mechanisms. However, because natural selection is just one of several forces contributing to the evolutionary process, an uncritical reliance on adaptive Darwinian mechanisms to explain all aspects of organismal diversity is not greatly different than invoking an intelligent designer.

Some of you will probably see why I like this guy! He warns against "uncritical reliance on adaptive Darwinian mechanisms."

This paper represents a first step toward the formal development of a general theory for the evolution of the gene that incorporates the universal properties of random genetic drift and mutation pressure. Although the ideas presented are unlikely to be correct in every detail, at a minimum they serve as a null model. For if verbal adaptive arguments are to provide confident explanations for any aspect of gene or genomic structure, something must be known about patterns expected in the absence of selection. This is a significant challenge because at this point it is difficult to reject the hypothesis that the basic embellishments of the eukaryotic gene originated largely as a consequence of nonadaptive processes operating contrary to the expected direction of natural selection. A significant area of future research will be to take these observations on gene and genome complexity to the next level, to evaluate whether natural selection is a necessary and/or sufficient force to explain the evolution of the cellular and developmental complexities of eukaryotes.

Everyone needs to start paying attention. Random genetic drift is just as important for evolution as natural selection. That's not speculation. As far as I'm concerned, it's hard incontrovertible fact.

One of the "new ways" of looking at evolution is to consider mutation pressure, loosely defined as differences in the frequency of mutation. I'm not a big fan of this but it does emphasize that modern evolutionary theorists are thinking outside the Darwinian box—not surprising since Darwin died 125 years ago (today). I prefer mutationism, which is a way of emphasizing the imprtant role of mutation in directing evolution. Mutationism and mutation pressure are not the same thing.

Haldane's Dilemma

This is very interesting. Dembski has teamed up with Walter ReMine, demonstrating once again that the old addage "opposites attract" does not apply to kooks.

ReMine has an article on Uncommon Descent where he pushes his usual whine about evil scientists and how their world-wide conspiracy has kept him from revealing the fatal flaw in evolution [Evolutionist withholds evidence on Haldane’s Dilemma]. I can see how similar this is to Intelligent Design Creationism.

Some of you may not be familiar with the so-called "dilemma" of Haldane. Fortunately, ReMine provides a nice short summary.

For many years I have publicly claimed Haldane’s Dilemma is a major unsolved problem for evolution. A problem so severe it threatens macroevolution as a “fact” and evolutionary genetics as an empirical science. The problem, briefly, is that evolutionary geneticist, J.B.S. Haldane (1957), discovered an important argument that limits the speed of evolution. Under his calculations, an ape-human-like population, given a generous ten million years, could substitute no more than 1,667 beneficial mutations — which, according to evolutionary geneticists, are each typically a single nucleotide. All the human adaptations within that time would have to be explained with this small number of substitutions. For more information, see here: Haldane's Dilemma.

That's it. Fifty years ago J.B.S. Haldane did a quick calculation suggesting that if you make certain assumptions (now shown to be inaccurate) then you could only fix 1,667 beneficial human mutations in 10 million years. Apparently ReMine thinks this is way too little evolving, even if all it has to do is produce the likes of him and Dembski.

We don't need to thrash out why ReMine is wrong. That's been done many times. What's interesting about this is the Wikipedia article that ReMine wrote. Go there right now and take a look because it won't look like that for long now that ReMine has let the cat out of the bag. Oops! It's not on Wikipedia it's on another Wiki called ResearchID.org. My goof—thanks to Torbjörn Larsson for pointing this out. The real Wikipedia article [Haldane's dilemma] is pretty good. So now the only reason for taking note of this is the fact that ReMine is being promoted by Dembski. That's hardly news. Move along. There's nothing to see here.

For all you talk.origins fans, I found a little bit of history when I did the research for this article. Follow this link to a message from Saint Andrew (you know who that is). It's about a 1995 post from Ted Holden (yes, the famous Ted Holden who coined the term "Howler monkeys") defending Walter ReMine. Proving once again that kooks will recognize each other.

The picture of ReMine is from the video of a lecture on his book The Biotic Message. You can only watch a few minutes but that's enough. He does a fine job of spining "framing" his message for an audience of true believers.

[If you mention his name, he will come.]

Charles Darwin Died in 1882

 
In honor of Charles Darwin, who died on this day, I'm posting the opening paragraphs of a manuscript that might eventually be a book called Evolution by Accident.

I approached Westminster Abbey from the south side, crossing Abingdon Street in front of the Houses of Parliament. There was a long line of tourists in front of the ticket window and, not wanting to waste a beautiful Spring day, I decided to do a bit of exploring before joining the queue.

An old three story building caught my eye. It was the Jewel Tower, built 650 years ago to house the treasures of King Edward III. The Jewel Tower is all that remains of the medieval Palace of Westminster that was mostly destroyed by fire in 1834. The Houses of Parliament and Big Ben off to my left were built to replace the original palace—they look old but they have "only" been there for 175 years.

Going behind the Jewel Tower I spot the remains of the old moat and walls that used to surround Westminster Palace. They don’t serve any purpose now since they are well below ground level and, besides, Abingdon Street cuts right through the place where the wall and moat used to protect the old palace buildings.

I cross the street by Victoria Tower at the south-west corner of the Houses of Parliament and enter Victoria Tower Gardens. According to the medieval map in the Jewel Tower, this used to be in the middle of the Thames and there was a quay for loading and unloading boats along the edge of the palace where Victoria Tower now stands. The park is quiet and peaceful at this time of day. I imagine it gets more traffic at lunch time. The Thames is also quiet, but muddy. I watch a family of ducks swim by.

The object of my pilgrimage was inside Westminster Abbey and it was time to return to the entrance. Fortunately, the long line had dissipated and I was able to purchase my ticket (£2) after a short wait. The designated route takes you through the Great North Door where you enter the Transept. Turning left, I follow the other tourists as we are herded around the back of the Abbey through the rooms behind the alter. We pass the tombs of Queen Mary the First (1516-1558), Queen Elizabeth the First (1533-1603), and Mary, Queen of Scots (1542-1587) in the Lady Chapel. We stop to admire the shrine of Saint Edward the Confessor (1002-1066).

I’m getting impatient but I can’t move any faster because of the crowd of tourists. Eventually we wind around the Monastery and finally enter the Nave. Ignoring the monument to Winston Churchill (1874-1965) and hardly bothering to look up and admire the high ceiling, I head for the back left corner where I can see the statue of Isaac Newton (1643-1727). This is the same statue that plays such an important role in the Da Vinci Code but today I’m not interested in Newton or his orb. It takes me only a few seconds to find the marked stone on the floor. I’m standing on the grave of Charles Robert Darwin.

I can picture the scene on Wednesday, April 26, 1882—a grand funeral attended by all of London’s high society and the leading intellectuals of the most powerful nation in the world. Darwin would not have been pleased. He wanted to be buried quietly in the Downe cemetery with his brother Erasmus and two of his children. Darwin's family was persuaded by his friends Galton, Hooker, Huxley and the President of the Royal Society, William Spottiswoode, that, for the sake of England, Darwin should be laid to rest in Westminster Abbey. As Janet Browne writes in her biography of Charles Darwin, "Dying was the most political thing Darwin could have done."²

Looking around I can see the tombs of two of the scientists who were Darwin’s pallbearers, Joseph Hooker and Alfred Wallace. (Another pallbearer, Thomas Henry Huxley, is buried elsewhere.) Nearby are the final resting places of a host of famous scientists; Kelvin, Joule, Clerk-Maxwell, Faraday, Herschell, and Sir Charles Lyell. (Lyell was Darwin’s hero and mentor. We are told that Darwin’s wife Emma wished he were buried closer to Lyell.)

I am not overly sentimental but this visit has a powerful effect. I think Charles Darwin is the greatest scientist who ever lived—yes, even greater than Sir Isaac Newton whose huge statue overshadows Darwin’s humble marker in the floor. Natural selection is one of the greatest scientific ideas of all time. Darwin discovered it and he deserves enormous praise for his achievement. But Charles Darwin died on April 19, 1882 and that was a long time ago.

Temple Universit Students Respond

 
Last week I posted an article about Intelligent Design Creationists giving a talk at Temple University. I wondered what the students must have been thinking to invite two Young Earth Creationists (Marcus Ross, Paul Nelson) to come and talk to them as part of a series entitled: DECIDE FOR YOURSELF: Evolution and Intelligent Design.

Two Temple University students who attended the event have now responded. Both choose to remain anonymous. You can read their comments at [Marcus Ross, Michael Behe, and Paul Nelson at Temple University].

Here are some teasers ..

To start, your judgmental and self-righteous words have proven to me that your whole position is without credibility as you refused to attend Temple's event.

and .....

Because you chose not to participate, is it really right to take potshots at students from the web? Are you in a superior position professionally, educationally, or morally to condemn the students and therefore the other professional educators under whose approval and encouragement the students are working? Your intolerant conceit is more disagreeable than the students' supposed ignorance.

Gee, I wonder what they "decided for themselves" at the lectures? Anyone wanna take a wild guess?

Nobel Laureate: Aaron Klug

 

The Nobel Prize in Chemistry 1982.



Aaron Klug (1926- ): "for his development of crystallographic electron microscopy and his structural elucidation of biologically important nucleic acid-protein complexes"

Aaron Klug won the Nobel Prize in 1982 for his work on a special technique for solving the structures of large molecules that can't be crystallized. He used it to determine the structure of the nucleosome. Here's how Klug's contribution is described in the presentation speech.

Large molecular aggregates can seldom be obtained in a form which allows structural determination by X-ray diffraction. The investigator who has been awarded with this year's Nobel Prize in chemistry, Aaron Klug, has developed a method to study the structure of molecular aggregates from biological systems. His technique is based on an ingenious combination of electron microscopy with principles taken from diffraction methods. Electron microscopy has long been used to depict the structural components of the cell, but its power of resolution is after, limited by a lack of contrast in the picture. Klug has shown that even picture; seemingly lacking in contrast may contain a large amount of structural information, which can be made available by a mathematical manipulation of the picture.

With this technique, in combination with other methods of structural chemistry, Klug has inter alia investigated viruses and chromatin of the cell nucleus. His virus studies have illuminated an important biochemical principle, according to which the complicated molecular aggregates in the cell are formed spontaneously from their components. The chromatin investigations have provided clues to the structural control of the reading of the genetic message in DNA. In a long-term perspective they will undoubtedly be of crucial importance for our understanding of the nature of cancer, in which the control of the growth and division of cells by the genetic material no longer functions.

Klug started working on tobacco mosaic virus in 1954 when be began a collaboration with Rosalind Franklin who had just abandoned DNA. Klug and Franklin remained close associates until she died a few years later.

Klug solved the structure of TMV using X-ray diffraction but this proved inadequate for other large structures. In order to solve the structures of more complex viruses (e.g., bacteriophage T4) and chromatin, Klug turned to high resolution electron microscopy. He developed techniques for assembling and refining multiple images with the aid of complex computer programs. Basically, he was able to solve the three dimensional shape using multiple two dimensional images as shown in the diagram (right) from his Nobel Lecture.

Klug's work has been modified an improved over the years. Today the electron microscopic images are much better and special low temperature electron microscopes (cryo-EM) can be used to obain images from material that would be destroyed at normal temperature. The enormous increase in computing power and modern software have led to the solution of many complex structures such as the pyruvate dehydrogenase complex.

The Structure of the Pyruvate Dehydrogenase Complex

 
The pyruvate dehydrogenase complex catalyzes the reaction that converts pyruvate to acetyl-CoA with the release of CO₂. The reaction is coupled to the reduction of NAD⁺ to NADH₂ [Pyruvate Dehydrogenase Reaction]. The three components of the complex, E₁, E₂, and E₃ catalyze different steps.

The size of the pyruvate dehydrogenase complex is enormous. It is several times bigger than a ribosome. In bacteria these complexes are located in the cytosol and in eukaryotic cells they are found in the mitochondrial matrix. Pyruvate dehydrogenase complexes are also present in chloroplasts.

The eukaryotic pyruvate dehydrogenase complex is the largest multienzyme complex known. The core of the complex is formed from 60 E2 subunits arranged in the shape of a pentagonal dodecahedron (12 pentagons joined at their edges to form a ball). This shape has 20 vertices and each vertex is occupied by an E2 trimer. Each of the E2 subunits has a linker region projecting upward from the surface. This linker contacts an outer ring of E1 subunits that surround the inner core. The linker region contains the lipoamide swinging arm.

The outer shell has 60 E1 subunits. Each E1 enzyme contacts one of the underlying E2 enzymes and makes additional contacts with its neighbors. The E1 enzyme consists of two α subunits and two β subunits (α2β2) so it is considerably larger than the E2 enzyme of the core. The E3 enzyme (an α2 dimer) lies in the center of the pentagon formed by the core E2 enzymes. There are 12 E3 enzymes in the complete complex corresponding to the 12 pentagons in the pentagonal dodecahedron shape. In eukaryotes, the E3 enzymes are associated with a small binding protein (BP) that’s part of the complex.

The model shown above has been constructed from high resolution electron microscopy images of pyruvate dehydrogenase complexes at low temperature (cryo-EM) (below). In this technique, a large number of individual images are combined and a three-dimensional image is built with the help of a computer. The model is then matched with the structures of any of the individual subunits that have been solved by X-ray crystallography or NMR.

A similar pyruvate dehydrogenase complex is present in many species of bacteria although some, such as gram negative bacteria, have a smaller version where there are only 24 E2 enzymes in the core. In these bacteria, the core enzymes are arranged as a cube with one trimer at each of the 8 vertices. The E2 subunits of the two different bacterial enzymes and the eukaryotic mitochondrial and chloroplast versions are all closely related. However, the gram negative bacterial enzymes contain E1 enzymes that are unrelated to the eukaryotic versions.

So far, it has not been possible to grow large crystals of the entire pyruvate dehydrogenase complex on Earth. Experiments were undertaken to grow crystals on the International Space Station where the absence of gravity might have led to better results. Unfortunately, none of the esperiments were successful so, for the time being, the best model of the pyruvate dehydrogenase complex is the one constructed from the cryo-EM images.


[This is a slightly modified version of material in Horton et al. (2006) Principles of Biochemistry 4th ed.©L.A. Moran and Pearson/Prentice Hall]

Pyruvate Dehydrogenase Reaction

 
Pyruvate dehydrogenase catalyzes the conversion of pyruvate to acetyl-Coenzyme A (acetyl-CoA). The reaction is coupled to the reduction of NAD⁺ to NADH. The reaction is an example of an oxidative decarboxylation since the other product is carbon dioxide (CO₂). [Pyruvate] [Fritz Lipmann and Coenzyme A]

Acetyl-CoA is subsequently used up in the citric acid cycle and in fatty acid synthesis.

This is a very complicated reaction. It turns out that the enzyme pyruvate dehydrogenase is actually a complex of several different activities. From now on I'll refer to it as the pyruvate dehydrogenase complex (PDC).

The first step in the reaction is the decarboxylation step and it requires a special cofactor called thiamine pyrophosphate (TPP). This is vitamin B₁ and it explains why that vitamin is essential. Carbon dioxide is released in this step and the remaining 2-carbon fragment of pyruvate is attached to TPP. This part of the reaction is catalyzed by a part of PDC composed of E₁ subunits.

In the next step, the 2-carbon fragment is transferred to a "swinging arm" composed of a lipid arm (blue zigzag) and a head containing two sulfur (S) atoms. The swinging arm actually swings to carry the red acetyl group from one active site in the complex to another. The second site is where the acetyl group is attached to CoA. This part of the reaction is carried out by the E₂ subunits in the complex.

The swinging arm carries excess electrons from the previous reaction in the form of two -SH groups. The next site visited by the swinging arm is the site where electrons are passed to another cofactor called FAD. This is the dihydrolipoamide dehydrogenase activity and it's the E₃ subunits that do the job. Electrons are then passed from FADH₂ to NAD⁺ to produce NADH₂.

The complete reaction is a classic example of an electron transport chain involving three groups: the lipoamide head of the swinging arm, FAD, and NAD⁺. In the next article we'll look at the structure of the pyruvate dehydrogenase complex. It's one of the largest multienzyme complexes found in living cells.

Recourse to the Miraculous is Always a Regressive, Obfuscating Move

 
The same issue of Skeptical Inquirer that contained the Michael Ruse article [Appeasers and Other Atheists] also has an article by Frederick Crews.

Crews, F. (2007) Follies of the Wise. Skeptical Inquirer March/April 2007 pp.27-31.

Crews addresses the same issue as Ruse; namely whether it's a good idea to distinguish between Intelligent Design Creationists and Theistic Evolutionists. However, he delves deeper into the issue that Ruse does. I'm tempted to say that Crews is being more scholarly than Ruse.

Whenever we (e.g., PZ, Dawkins etc.) try to make the case that Theistic Evolution is just as fuzzy-headed as Intelligent Design Creationism we are accused of over-stepping the limits of science. While everyone recognizes that scientists must practice methodological naturalism, there seem to be lots of people who don't know what that is. They seem to think that it's okay to believe in miracles and still brag about being scientific. I've tried to point out the inconsistencies in such a position in my essay [Theistic Evolution: The Fallacy of the Middle Ground]. As Crews says below, "recourse to the miraculous is always a regressive, obfuscating move." This applies to Intelligent Design Creationism of course, but it also applies to the Theistic Evolution of Ken Miller, Francis Collins, and Simon Conway-Morris. I just don't see how atheists can dismiss the miracles of Dembski, Denton, and Behe while accommodating the miracles of Miller, Collins, and Conway-Morris. That makes no sense to me.

Crews takes a different approach. He argues that metaphysical naturalism is a valid and rational extension of methodological naturalism. This is contrary to Ruse and to the people at NCSE (e.g., Eugenie Scott). I present the Crews argument below. Let me know what you think. Personally I agree with him, even though I'm prepared to argue that most of the so-called "science" in books by Theistic Evolutionists is in violation of methodological naturalism not just metaphysical naturalism.

... some scientists and philosophers who are privately indifferent or hostile to transcendent claims nevertheless seek an accommodation with them. They do so from the best of motives, in order to stem the infiltration of bumpkin "creation science" or its slick city cousin "intelligent design," into biology curricula. Their hope is to show that scientific research and education have no bearing on issues of ultimate meaning and hence needn't be feared by the pious. To that end, they emphasize that science exemplifies only methodological naturalism, whereby technical reasons alone are cited for excluding nonmaterial factors from reasoning about causes and effects. Hence, they insist, the practice of science doesn't entail metaphysical naturalism, or the atheist's claim that spiritual causation is not only inadmissible but altogether unreal.

In one sense this is an impregnable argument. Even when science is conducted by ardent believers, it has to disregard theological claims because those claims typically entail no unambiguous, real-world implications, much less quantitative ones, that might be tested for their supportive or falsifying weight. The allegation that God was responsible for a given natural fact can't be either established or refuted by any finding; it is simply devoid of scientific interest. And thus it is true enough that scientists stand under no logical compulsion to profess metaphysical naturalism.

Any God worthy of the name has to be capable of miracles, and each of the great Western religions attributes a number of very special miracles to their conception of God. What can science say about a miracle? Nothing. By definition, the miraculous is beyond explanation, beyond our understanding, beyond science.

Ken Miller in "Finding Darwin's God" p. 239Quite obviously, however, trust in the supernatural does get shaken by the overall advancement of science. This is an effect not of strict logic, but of an irreversible shrinkage in mystery's terrain. Ever since Darwin forged an exit from the previously airtight argument of design, the accumulation of corroborated materialist explanations has left the theologian's "God of the gaps" with less and less to do. An acquaintance with scientific laws and their uniform application is hardly compatible with faith-based tales about walking on water, a casting out of devils,and resurrection of the dead.

Metaphysical naturalism may be undiplomatic, but it is favored by the totality of evidence at hand. Only a secular Darwinian perspective, I believe, can make general sense of humankind and its works. Our species appears to have constituted an adaptive experiment in the partial and imperfect substitution of culture for instinct, with all the liability to self-deception and fanaticism that such an experiment involves. We chronically strain against our animality by inhabiting self-fashioned webs of significance—myths, theologies, theories—that are more likely than not to generate illusory and often murderous "wisdom." That is the price we pay for the same faculty of abstraction and pattern drawing that enables us to be not mere occupiers of an ecological niche but planners, explorers, and, yes, scientists, who can piece together facts about our world and our own emergence and makeup.

Here it may be objected that myths, theologies, and theories themselves, as nonmaterial things that can nevertheless set in motion great social movements and collisions of armies, confound a materialist or metaphysically naturalist perspective. Not at all. We materialists don't deny the force of ideas; we merely say that the minds precipitating them are wholly situated within brains that, like everything else about which we possess some fairly dependable information, seem to have emerged without any need for miracles. Although it is not a provable point, it is a necessary aid to clear thought, because now that scientific rationality has conclusively shown its formidable explanatory power, recourse to the miraculous is always a regressive, obfuscating move.

Pyruvate

Pyruvate is a very important molecule in living cells. It was Monday's Molecule #22. It's important because it's one of the essential 3-carbon (3C) intermediates in biochemical pathways. It's required in both biosynthesis and degradation pathways. If you only have to know a few molecules in the cell then this one should be near the top of your list.

Let's start by learning a little bit of organic chemistry. The simplest organic molecule with only two carbon atoms is called ethane (CH₃CH₃). The one with three carbons is propane and the one with four is butane. The alcohols are called ethanol, propanol, and butanol respectively. The acids are ethanoic acid (CH₃COOH), propanoic acid (CH₃CH₂COOH), and butanoic acid (CH₃CH₂CH₂COOH). Ethanoic acid is more commonly known as acetic acid because it's found in vinegar and the Latin word for vinegar is acetum.

Pyruvate can also be called pyruvic acid. In a minute I'll explain why we call it pyruvate instead. It's related to propanoic acid (CH₃CH₂COOH) except that it has an extra oxygen on the middle carbon. The -C=O group is called a keto group and molecules that have it are ketones.

The proper chemical name for pyruvate is 2-oxopropanoic acid because it's propanoic acid with an extra oxygen (oxo-) on carbon atom #2. Keto acids usually have trivial names that aren't related to the names of the simple organic molecules and that's why pyruvate is the name everyone uses. The reason why keto acids are common in biochemistry is because they are quite reactive.

Acids are compounds that can easily give up a proton (H⁺). Protons are what makes an acid an acid. The more protons you have in solution the more acidic the solution will be. The term pH refers to the inverse of the concentration of H⁺. The lower the pH the higher the concentration of protons.

Pyruvic acid is an acid because when it's dissolved in water the proton from the carboxylate (-COOH) group dissociates leaving the negatively charged pyruvate and a free proton. The three components exist in equilibrium—that's what the double arrows mean. At any given time there will always be some pyruvic acid, some pyruvate, and some protons. The concentrations won't change because the dissociation/association reactions are at equilibrium.

The relative concentrations of pyruvic acid and pyruvate at equilibrium depend on the properties of the molecule and the conditions inside the cell. In this particular case we know for certain that there's very little pyruvic acid inside the cell. Almost all of the molecules are in the form of pyruvate. Pyruvate is the biologically significant molecule and that's why biochemists always refer to these molecules as acetate, pyruvate, butyrate, etc.

In all species, pyruvate is required for the synthesis of the amino acid alanine. It's also required for the synthesis of 6-carbon (6C) sugars such as glucose. In fact, you can think of gluconeogenesis (glucose synthesis) as just a series of steps where two pyruvate molecules are joined to make one glucose molecule (3C + 3C → 6C).

Pyruvate itself is synthesized from simpler molecules in bacteria. One common pathway is to add a carbon from carbon dioxide to a 2-carbon acetate molecule (1C + 2C → 3C). In plants and bacteria the primary product of the carbon fixing cycle (Calvin cycle) is a 3-carbon compound called glyceraldehyde 3-phosphate. It is rapidly converted to pyruvate.

Pyruvate can be activated to a phosphorylated derivative called phosphoenolpyruvate (PEP). PEP is a high energy compound used as an energy source in many reactions. (It has considerably more energy than ATP.) The direct conversion shown in the diagram below is confined to bacteria. In eukaryotes the conversion follows an indirect pathway.

There are many other fates of pyruvate as shown above. Pathways leading to synthesis of additional amino acids go through oxaloacetate. Oxaloacetate is also one of the components of the citric acid cycle. The synthesis of lipids and fatty acids requires acetyl-coenzyme A or acetyl-CoA, which is made from pyruvate in a reaction catalyzed by pyruvate dehydrogenase (more about this later—it's the main theme of this series).

Most students first encounter pyruvate as the end product of glycolysis. Glycolysis is the pathway for breaking down glucose. It is the opposite of gluconeogenesis because a 6-carbon compound is degraded to two 2-carbon compounds (6C → 3C + 3C). Even though this is a minor pathway in most kingdoms, it is important in animals and, most especially, in mammals like us. This is why glycolysis has traditionally received so much attention in schools. That's about to change.

In some species the accumulation of pyruvate leads to other pathways. One of them is synthesis of lactate, which often serves as a temporary storage depot for 3-carbon compounds. Another is synthesis of ethanol, which involves chopping off one of the carbons in the form of carbon dioxide (CO₂) and excretion of ethanol. Humans love those species that excrete ethanol. Over several millennia humans have selected strains of yeast that do a very efficient job.

Nobody needs to memorize all the pathways involving pyruvate. Not even my biochemistry students. The point here is simply to illustrate the central importance of pyruvate so you'll see why it's something you should know about.

Of all the fates of pyruvate, the one that is most interesting is the conversion to acetyl-CoA. As mentioned above, acetyl-CoA is required for fatty acid synthesis but it also serves as the substrate for the citric acid cycle where the acetate part of pyruvate is fully oxidized to CO₂. Note that the reaction catalyzed by pyruvate dehydrogenase not only makes acetyl-CoA but also accomplishes the first step in the complete oxidation of pyruvate to 3 molecules of CO₂. The energy released in this breakdown is captured by NADH.

Dicumarol and Warfarin Inhibit Blood Clotting

 
Many of the blood coagulation factors are post-translationally modified in various ways. One of these modifications is unusual and it is only seen in these factors and a few other specialized proteins. This modification converts a specific glutamyl side chain to a γ-carboxyglutamyl derivative [Vitamin K].

The carboxylation reaction is catalyzed by vitamin K-dependent carboxylase and it is coupled to the conversion of vitamin K to its oxidized form. In order for the enzyme to modify additional factors, the oxidized form of vitamin K has to be converted back to the reduced form. Recall that vitamin K is an essential vitamin in animals. It can be obtained from plants or from intestinal bacteria.

Recycling of vitamin K is catalyzed by K reductase. The mechanism involves oxidation of two sulfhydryl (-SH groups) to form a disulphide bridge [Disulfide Bridges]. The carboxlase and reductase reactions are required for synthesis of prothrombin, protein C, Protein S, and Factors VII, IX, and X because these proteins must bind C²⁺ and the γ-carboxyglutamyl group is an excellent cheator of Ca²⁺.

A vitamin K deficiency means that the carboxylation reaction cannot proceed and this leads to accumulation of inactive clotting factors in the liver. Blood clots cannot form and severe hemorraging can lead to death.

The carboxylation of clotting factors can also be prevented by inhibiting the K reductase reaction. There are many drugs that inhibit this reaction. They are related to warfarin (left) [Monday's Molecule #19]. The best known ones are dicoumarol and coumarin. These drigs, especially warfarin, are used frequently to prevent clotting in patients who have suffered a stroke or otherwise have tendencies to exhibit thrombosis.

Since the drugs prevent synthesis of clotting fators, they take a few days to have an effect. They are usually administerd with heparin, which has an immediate effect on blood clot formation.

Wafarin was a common rat poison in the past since rats are very sensitive to inhibition of K reductase. After eating food laced with wafarin for several days, the rats would die of internal bleeding.