Sunday, March 19, 2017

Why ATP?

Inside cells, the major energy carrier is ATP. Why, and what might have come before?

Two weeks ago, we learned about the intricate mechanism by which organisms transduce energy from a proton gradient into ATP, using a rotating, motor-like enzyme: the ATP synthase. Proton gradients are one important way for organisms to store and distribute their energy, just as we do macroscopically with fossil fuels, and the body does with glucose, distributed through the blood. Inside cells, ATP is the primary short-term form of energy distribution, with longer term stores taking the form of various carbohydrates, lipids, and, in general, all the components of the cell, which can be phagocytosed in times of need (or even entire cells, which can commit suicide and be consumed by others when needed).

But what is so great about ATP? It comes up constantly as the extra ingredient that makes difficult reactions go forward. Drug efflux pumps use ATP. Actin uses ATP to create physical force during its filament assembly and projection, as does myosin, which is the motor that pulls on actin to create muscle action. Likewise, dynein, which moves things along microtubules- another form of locomotion used in cell division and nerve cell organization, among much else. Translation uses a net of four ATP per amino acid incorporated, in the form of one ATP=>AMP (worth two ATP=>ADP), and two GTP, which are close cousins of ATP. ATP fuels our chemical purification factories, such as the kidneys, whose various chemical pumps all depend on a master gradient driven by the ATP-using sodium / potassium antiporter. Countless regulatory pathways use ATP to attach a phosphate to a protein, thus changing its activity, often dramatically. Whenever our enzymes are doing something that is energetically impossible on its own, it is a good bet that ATP is supplying the extra oomph. And life naturally depends on many such impossible steps.

ATP, the molecule. The orange groups are called phosphoanhydride groups, or phosphate groups for short.  Each of those groups carries a negative charge.

The last bond in ATP, which is broken (via hydrolysis with water) to make ADP + phosphate, provides about 30 kJ/mol. In comparison, a hydrogen bond, such as those that hold DNA strands together, are about 8 kJ/mol, and a normal carbon-carbon bond has about 360 kJ/mol. It takes a lot of energy to build up organic molecules, which is why such extraordinary measures like photosynthesis were developed during evolution, (taking about 3,100 kJ/mol worth of ATP and NADPH to produce one sugar molecule). But most other processes that just need a chemical nudge are well within the range of that ADP-P phosphate bond. Breaking the second-to-last instead of the last bond, to release diphosphate and AMP, yields about 41 kJ/mol, for a little extra energy when needed.

Sometimes the phosphate bond is referred to as "high-energy", but that doesn't mean it is a strong bond. One of the strongest bonds is the triple bond between the two atoms of atmospheric nitrogen, which takes 945 kJ/mol to break. Quite the opposite- instead of requiring lots of energy to break, the phosphate bond gives up those 30 kJ/mol energy when hydrolyzed (that is to say, the reaction is exothermic). Those negative charges really want to get away from each other! That makes it an energy store instead of an energy sink. But due to the its high negative charge, it is not easily attacked by water, which means it is kinetically stable and does not spontaneously degrade- again, an important property for an energy store. It takes an small enzymatic nudge to do the job- something our proteins are very good at.

 Indeed, the weakness of the phosphate bond is its strong point, as it makes an excellent "leaving group". That means that it reacts enthusiastically, taking away two electrons when a nucleophilic attack is directed against its neighbor. Hydrolysis by water is the most common mode of attack, or by a target compound or protein that ends up with the phosphate group attached. The phosphate's negative charge is also beneficial, so that chemical intermediates to which it is attached are instantly given a charge, which confines them within the membrane. This can be a significant issue for the many small molecules of metabolic reaction chains.

Table from Westheimer, discussing the utility of phosphate for organic chemistry. Human chemists do not have enzymes at their disposal, so have to use much more caustic and active leaving groups than the chemists of molecular biology can.

But there is a problem from an evolutionary perspective, which is that phosphorous is hard to come by in geological terms. This is a debatable point, but many researchers contend that mineral phosphates are all highly insoluble, and have been since the Earth was formed. Phosphorous could thus be likened to gold- a precious and rare element that changes hands frequently as a medium of exchange, but whose actual abundance is exceedingly low. For example, our bodies are thought to break and reform over 400 pounds of ATP per day, but have only 0.1 pound on hand at any moment.

Whatever the geological case, one research group was inspired to look for possible pre-phosphorous chemistries in the early history of life. They propose that ATP and its phosphate-carrying colleagues may have been a later development in early biotic chemistry, though it was well-entrenched by the time of the last common ancestor of all life, abbreviated LUCA. They took a somewhat round-about route in their analysis, trolling through all the known reactions among existing organisms (KEGG) to find those which do not rely on phosphate. Then they tried to assemble from those as much of a basic metabolism as possible. What they came up with (a phosphate-free metabolism) is interesting.

This metabolism is highly dependent on thioesters, which place sulfur in the place of oxygen in bonds with carbon. The thioester bond is worth, in free energy of hydrolysis terms, about as much as the phosphate bond, but it is not as good a leaving group as phosphate, and does not reliably confer charge on its targets. This chemistry is still used in many biological reactions, however, by way of coenzyme A. With this chemistry, and with numerous metal-containing enzymes, they can access about 315 reactions and 260 metabolites of the roughly 700 metabolites of core metabolism- an impressive achievement, really. The set of reactions they come up with are biased towards those of core metabolism, those using iron and other metal cofactors, and those known to have been key to early life, and to those accessible to shorter genes and proteins with a minimal complement of amino acids.

Naturally, to pull in reactions from all sorts of odd and currently-existing bacteria to stand in for the  possibilities of early evolution is hazardous, but it does indicate that phosphate-free biology is conceivable, if inefficient and incomplete. But it is the geological debate about phosphorous / phosphate availability that needs to be resolved first before this issue becomes pertinant and interesting at all.

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