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A machine to teach with: ATPase as a core cellular mechanism

Angry cartoon firecrackerBoth studies and common sense indicate that common threads running through our teaching provide students with reinforcement of both thread components and the things they connect. This approach also highlights conveying principles rather than fact collecting as our learning objective. The details of a basic ATP hydrolysis reaction illustrate both key principles (how enzymes actually implement their abstract aspects [speed up reaction; lower reaction barrier], roles for specific amino acids and protein folding), value of understanding chemistry in thinking about biology) as well as providing students with a tool that they’ll see over and over… and over again: the core mechanism is found in nucleotide addition, kinase and phosphatase reactions, pre-mRNA splicing, the timing mechanisms of GTPases [tubulin, EF-Tu, GPCRs], and… oh yeah: virtually every ATP-driven or -coupled reaction in the cell!

Building on concrete: the sorcery of enzymes

One of my great frustrations with the college biology textbooks I have seen is the fact that they take beginning learners and demand that they learn difficult new concepts in the abstract often with no concrete example provided. Nowhere is this more true than discussions of enzymes, where discussions of reaction intermediates, barriers, the lowering thereof, etc. are glibly tossed about with nary an enzyme or reaction in sight. When this is the case, we have only ourselves to blame when students ‘learn’ by associating the terms used to describe these critical ideas with other terms and fail to anchor them to any meaning.

Happily, at the heart of life’s energy transactions lies an answer to this and many other challenges. No textbook or intro Bio course escapes without the introduction of ATP as ‘the energy currency of the cell’. Alas, many DO sail on without identifying where that energy is and how itgets out to be spent, harnessed, etc. By going in depth into the (readily understood) mechanism of an ATPase, we can provide students with a touchstone for their thinking about enzymes, an understanding of (90%?) of the energy-driven reactions in the cell, and a theme that will be returned to over and over again during the course of the course.

ATP: Who’s got the energy?

I’ve covered the basics of energy in ATP and how I choose to teach it in a previous post. The central idea is that there is no mystical ‘high energy bond’; there are several bonds that are FORCING positively charged phosphates to remain in close proximity to one another–a state passionately opposed by the negative charges thereon. Tho confusion arises because it is indeed the case that hydrolyzing (not ‘breaking’) those bonds (the distinction is that a chemist’s broken bond is a real mess; a biologist’s usually means ‘thing A and thing B are no longer satisfying their outer shells with each other, but rather by new relationships with other players; often ‘other’ means the pieces of water) allows the phosphates to move away from one another, converting the potential energy of closely placed like charges to the kinetic energy of the phosphates flying apart (including pushing whatever ‘levers’ are present in the protein that is ‘holding’ them).

Let me out! Setting a phosphate free

The key question then becomes “What is it going to take to ‘break’ a phosphate bond off”? Per above, we’re going to keep everyone’s outer shells satisfied at all times, so for the phosphate to leave, we must find something for it to (covalently) partner with OTHER THAN the oxygen attached to the rest of the ADP that will be left behind. Earth being the watery planet, it is unsurprising that the simplest ‘tool’ for this job is a water molecule; with the oxygen as attacking group, a free electron pair of the water is converted to the pair of electrons in a covalent bond to the outermost (gamma) phosphate, ‘satisfying’ it and allowing it to give up the electron pair constituting the covalent bond between it and the beta phosphate (thus leaving the beta phosphorus ‘satisfied’ with what it has).

A wonderful visual demonstration of this reaction is this University of Surrey (Great Britain) video here:

Sn2 reaction video (for ATP, think of ‘activated’ water instead of hydroxide and gamma phosphate instead of ethyl group, with ADP instead of bromine atom)

Please note that there are some vital things to see here, illustrating a lot of Intro Bio content but in a much more concrete and comprehensible way:

  • importance of orientation and activation of the water (closer resemblance to OH- is better)
  • high energy state (transition where there is a LINEAR relationship between attacker/leaver, and PLANAR arrangement of the attacked group (midway point of the ‘umbrella’ blowing inside out)
  • claimed greater stability of the product

There are a fair number of ‘correspondences’ to understand; while I love the video, in our case, it’s the oxygens of the phosphate that go planar, and ADP that ‘leaves’. A little trick I use in lecture to make a point about the ‘discomfort’ of the planar intermediate is to tell students to pretend their hand is a phosphorous and their index, middle, and ring fingers are the unbound oxygens. Their wrist is the oxygen leading to the other phosphates. I tell them spread their fingers out as widely as possible to create a tetrahedron with their wrist, and to then use their other palm to smush the fingers further and further apart until planar. If done correctly, this HURTS. Trust me. I then clarify that they should not finish inverting the umbrella by breaking their fingers, because the analogy breaks down at this point :-).

Being an enzyme: an interactive exercise

Having worked to explain why ATP thinks the reaction is difficult, I then walk them through some other challenges. What is the charge on the oxygens at your ‘fingertips’ in the tetrahedron? (full or partial negative). How does the water need to orient to form the bond with the phosphorous (lead with oxygen–I point out here that waters NOT oriented correctly are a waste of our time). How easy is it going to be to get the water oxygen past the ‘guard’ oxygens of the tetrahedron? (not!). How eager are the phosphate oxygens to go planar (not, as we have discussed above).

It’s likely useful to point out at this point that these reasons are why the dynamite of ATP is nonetheless stable in the cell–a lot of energy is bound up, but it takes external assistance to release it.

Now comes the fun part. I ask them to spout the rote definition of an enzyme (you can always count on a chorus of “something that speeds up a reaction, but that is not used up and does not change the equilibrium”, a litany that too often means nothing to them. “OK,” I say “Knowing as you now do, what is HARD about using a water to hydrolyze off the gamma phosphate, what can you do to make it easier?” Usually, this doesn’t quite work; rather than thinking about the facts in front of them, I believe they are struggling to do something with deltaH or wrestle with the facts that all we’ve really taught them is that enzymes are magic.

So I offer a hint: is there anything in that list of hard/unlikely things that you can make more likely? Usually the first thing I get is “position the water?”. BANG!! I say with great enthusiasm and authority. You’re an enzyme! Generally, there are a fair number of confused faces. “Did you speed up the reaction?” I ask. Slowly, uncertainly, I get nods. “Isn’t that what enzymes do?”. Ditto. “So the difference between doing that and being an enzyme would be?”. It still usually falls on me to reassure them that yes, yes they are an enzyme. “Are you a very GOOD enzyme?”. Maybe not. “OK, what OTHER things could you do to make unlikely/energetically unfavorable things more likely/favorable? The ice broken, and sometimes with some prodding, I can usually elicit

  • Activate the water by ‘pulling’ on one of the hydrogens
  • strain the leaving group::gamma phosphorous bond to make it more ready to break
  • (this one usually takes the most work or must be pointed out) make the ‘fingers’ more comfortable in their planar state. How to do this?
    • ask them about those oxygens… they’re negatively charge
    • ask them how you make a negative ion happy: generally by providing them a positive charge to snuggle up to
    • ask them where they would put these to give aid and comfort to the PLANAR oxygens of the transition state “Wherever the oxygens would be then.” Yup. Again, you’re an enzyme

Sciencing it up

All of this constitutes a hypothesis or prediction about what an ATPase will look like and how it will work. It also exposes one of the most important values I try to convey in my teaching: You can think your way to solutions if you have some pieces to think with and think carefully within the laws of chemistry/physics. What makes all of this heavenly is that all of this is EXACTLY how real-life ATPases in their bodies work. A wonderful example is this movie:

Start here and select the movie “F1-ATPase catalytic site binding and hydrolysis of…” (though they’re all massively cool). Note that this movie explicitly shows the water, surrounding protein structure, and some of the specific sidechains and H-bonds involved

Tying it in

While looking at the movie (I recommend playing it through a couple of times, calling attention to the water, the H-bonding, the planar intermediate… do be aware that the movie reverses from time to time to allow the watcher to get full value out). In many textbooks, the amino acid family is introduced in the abstract–“they do stuff in proteins and are the building blocks of the machines of life.” True, but wouldn’t it be lovely to show them at work… in a real protein… that was actually folded… in such a way that their precise positioning was clearly consequential?

All wishes granted! Several of the sidechains are labeled, and their role in positioning, comforting, or pulling can be readily perceived. Tie these actions directly to the ‘how to be an enzyme’ list above. You may want to ask of a lysine “Could a glutamate or leucine do that? Why/Why not?”. This also highlights the importance of folding and the “other 99%” of the protein–how did those amino acids come to be EXACTLY where they were needed, oriented as they were? Would they achieve this if free in solution?

Looking forward

One of the great values of teaching this chemistry is that it can be made to appear many, MANY times during the semester, providing not only an opportunity to review this content, but also to point out that there are a smaller number of THEMES and principles that unify biology, often allowing avoidance of massive amounts of minutia. Here are some places where you’ll find this exact chemistry:

  • 3′ OH attach of 5′ triphosphate in adding DNA, RNA
  • self-destruction of RNA chain via 2’OH attack of 5′ – 3′ phosphodiester bond (partly accounting for the superiority of DNA over RNA)
  • lariat formation in pre-mRNA splicing (2′ OH attach of branch point on 5′ splice site)
  • phosphorylation of serine, threonine, tyrosine in protein (and removal of these phosphates)
  • ATP-coupled machines (motors, pretty much everything else as well)
  • The GTPase timers (tubulin, EF-Tu, G-protein coupled receptors)
  • pretty sure I’m missing many others!

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