Corante

About this Author
DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

Chemistry and Drug Data: Drugbank
Emolecules
ChemSpider
Chempedia Lab
Synthetic Pages
Organic Chemistry Portal
PubChem
Not Voodoo
DailyMed
Druglib
Clinicaltrials.gov

Chemistry and Pharma Blogs:
Org Prep Daily
The Haystack
Kilomentor
A New Merck, Reviewed
Liberal Arts Chemistry
Electron Pusher
All Things Metathesis
C&E News Blogs
Chemiotics II
Chemical Space
Noel O'Blog
In Vivo Blog
Terra Sigilatta
BBSRC/Douglas Kell
ChemBark
Realizations in Biostatistics
Chemjobber
Pharmalot
ChemSpider Blog
Pharmagossip
Med-Chemist
Organic Chem - Education & Industry
Pharma Strategy Blog
No Name No Slogan
Practical Fragments
SimBioSys
The Curious Wavefunction
Natural Product Man
Fragment Literature
Chemistry World Blog
Synthetic Nature
Chemistry Blog
Synthesizing Ideas
Business|Bytes|Genes|Molecules
Eye on FDA
Chemical Forums
Depth-First
Symyx Blog
Sceptical Chymist
Lamentations on Chemistry
Computational Organic Chemistry
Mining Drugs
Henry Rzepa


Science Blogs and News:
Bad Science
The Loom
Uncertain Principles
Fierce Biotech
Blogs for Industry
Omics! Omics!
Young Female Scientist
Notional Slurry
Nobel Intent
SciTech Daily
Science Blog
FuturePundit
Aetiology
Gene Expression (I)
Gene Expression (II)
Sciencebase
Pharyngula
Adventures in Ethics and Science
Transterrestrial Musings
Slashdot Science
Cosmic Variance
Biology News Net


Medical Blogs
DB's Medical Rants
Science-Based Medicine
GruntDoc
Respectful Insolence
Diabetes Mine


Economics and Business
Marginal Revolution
The Volokh Conspiracy
Knowledge Problem


Politics / Current Events
Virginia Postrel
Instapundit
Belmont Club
Mickey Kaus


Belles Lettres
Uncouth Reflections
Arts and Letters Daily
In the Pipeline: Don't miss Derek Lowe's excellent commentary on drug discovery and the pharma industry in general at In the Pipeline

In the Pipeline

« Shootmenowicene | Main | Good Forum for a Response on Drug Innovation? »

August 16, 2012

Just How Do Enzymes Work?

Email This Entry

Posted by Derek

How do enzymes work? People have been trying to answer that, in detail, for decades. There's no point in trying to do it without running down all those details, either, because we already know the broad picture: enzymes work by bringing reactive groups together under extremely favorable conditions so that reaction rates speed up tremendously. Great! But how do they bring those things together, how does their reactivity change, and what kinds of favorable conditions are we talking about here?

And some of this we know, too. You can see, in many enzyme active sites, that the protein is stabilizing the transition state of the reaction, lowering its energy so it's easier to jump over the hump to product. It wouldn't surprise me to see the energies of some starting materials being raised to effect that same barrier-lowering, although I don't know of any examples of that off the top of my head. But even this level of detail raises still more questions: what interactions are these that lower and raise these energies? How much of a price is paid, thermodynamically, to do these things, and how does that break out into entropic and enthalpic terms?

Some of those answers are known, to some degree, in some systems. But still more questions remain. One of the big ones has been the degree to which protein motion contributes to enzyme action. Now, we can see some big conformational changes taking place with some proteins, but what about the normal background motions? Intellectually, it makes sense that enzymes would have learned, over the millennia, to take advantage of this, since it's for sure that their structures are always vibrating. But proving that is another thing entirely.

Modern spectroscopy may have done the trick. This new paper from groups at Manchester and Oxford reports painstaking studies on B-12 dependent ethanolamine ammonia lyase. Not an enzyme I'd ever heard of, that one, but "enzymes I've never heard of" is a rather roomy category. It's an interesting one, though, partly because it goes through a free radical mechanism, and partly because it manages to speed things up by about a trillion-fold over the plain solution rate.

Just how it does that has been a mystery. There's no sign of any major enzyme conformational change as the substrate binds, for one thing. But using stopped-flow techniques with IR spectroscopy, as well as ultrafast time-resolved IR, there seem to be structural changes going on at the time scale of the actual reaction. It's hard to see this stuff, but it appears to be there - so what is it? Isotopic labeling experiments seem to say that these IR peaks represent a change in the protein, not the B12 cofactor. (There are plenty of cofactor changes going on, too, and teasing these new peaks out of all that signal was no small feat).

So this could be evidence for protein motion being important right at the enzymatic reaction itself. But I should point out that not everyone's buying that. Nature Chemistry had two back-to-back articles earlier this year, the first advocating this idea, and the second shooting it down. The case against this proposal - which would modify transition-state theory as it's usually understood - is that there can be a number of conformations with different reactivities, some of which take advantage of quantum-mechanical tunneling effects, but all of which perform "traditional" transition-state chemistry, each in their own way. Invoking fast motions (on the femtosecond time scale) to explain things is, in this view, a layer of complexity too far.

I realize that all this can sound pretty esoteric - it does even to full-time chemists, and if you're not a chemist, you probably stopped reading quite a while ago. But we really do need to figure out exactly how enzymes do their jobs, because we'd like to be able to do the same thing. Enzymatic reactions are, in most cases, so vastly superior to our own ways of doing chemistry that learning to make them to order would revolutionize things in several fields at once. We know this chemistry can be done - we see it happen, and the fact that we're alive and walking around depends on it - but we can't do it ourselves. Yet.

Comments (23) + TrackBacks (0) | Category: Biological News | Chemical News


COMMENTS

1. Angry Med Chem Guy on August 16, 2012 11:23 AM writes...

One of the most intriguing theory's that I have read, regarding the mechanism of enzymes and how they accomplish those mind blowing rates, is described by chreodes.

http://tinyurl.com/9cg95h5

Permalink to Comment

2. Angry Med Chem Guy on August 16, 2012 11:32 AM writes...

I'm sure that was a new concept for you Derek.

Permalink to Comment

3. Paul on August 16, 2012 11:35 AM writes...

Enzymatic reactions are, in most cases, so vastly superior to our own ways of doing chemistry that learning to make them to order would revolutionize things in several fields at once.

You don't even have to be as good as natural enzymes to have significant economic implications. If you can make an analogue of carbonic anhydrase with even a fraction of its activity, for example, it could be useful for reducing the cost of separating CO2 from gas streams.

Permalink to Comment

4. Anon on August 16, 2012 11:45 AM writes...

Question:
If you could make a blockbuster custom enzyme to solve a problem, what would it be?

Permalink to Comment

5. Brian on August 16, 2012 12:01 PM writes...

Dorothee Kern (HHMI at Brandeis) wrote a few papers in Nature a few years ago about the hierarchy of structural dynamics of proteins at different time-scales (i.e. from individual bond vibrations to global conformational changes). The review/perspective-type piece was a very interesting read. Sounds like she drinks the koolaid, but the jury is still out as to the extent these motions participate in catalysis.

Permalink to Comment

6. Watson on August 16, 2012 12:37 PM writes...

Our lab is currently investigating the dynamics of shootmenowicene gluronidation by PacManase. We have a proposal under consideration to do 5-second MD simulations on Anton, which we feel will significantly contribute to the field.

Permalink to Comment

7. luysii on August 16, 2012 12:48 PM writes...

You are certainly correct that people have been thinking about enzymes and how they work for a long time. I remember hearing Henry Eyring (who developed a good deal of transition state theory) in the late 50s, describing his idea of an elastomeric rack, how motions of lots of parts of the protein while each individually small, could pull on the bonds of the substrates lowering the activation energy. This would be an example of increasing reaction rates by increasing the energy of the participants, rather than lowering that of the transition state. The actual paper came out later [ Proc. Natl. Acad. Sci. vol. 68 pp. 2241 --> '71 ]

It isn't surprising that the B12 doesn't move much during catalysis It has a 'near porphyrin' ring holding the catalytic Co atom. All the porphyrin atoms are there, but it isn't fully desaturated in two of the 4 rings.

Permalink to Comment

8. Pharmacologyrules on August 16, 2012 3:05 PM writes...

I thought the enzyme that chemists sre most informed aboute is alcohol dehydrogenase.

Permalink to Comment

9. Rob on August 16, 2012 3:09 PM writes...

(something I actually work on).
There are a couple of problems with over-generalizing one mechanistic study - there are lot's of enzymes - and it is unlikely that they all use the same features to enhance rates. In the retroviral protease world, for example, the rate of the protease reaction is sometimes coupled to the maturation of the virus (Out of the Weber lab - engineering RSV protease to be as fast as HIV protease kills the virus because it upsets maturation kinetics). Similarly different substrates might have different slow steps - so that simple transition state theory isn't always portable across all substrates all the time. (Can cite references here - but the margin of the post is too small).

That said, it is not inconceivable that an enzyme could enhance reactivity by being extra stabilizing of the transition state in one micro-conformation and then a few picoseconds later being less stabilizing. This gets a little closer to a Maxwell's demon than is ideal - but as long as the average behavior doesn't have the enzyme doing an energy-less measurement of the substrate it should be physically OK. Basically the reaction rate could be different for different micro-conformations and the best rate micro-conformation might not be the equilibrium conformation.

Since Latex probably won't work here I'll try to describe it in words rather than equations.

Suppose we have 2 micro-states (for simplicity), then the total rate would be P(state 1)*rate(state 1) + P(state 2)*rate(state 2). If the rates are very different, but the two P(state)'s are similar then the micro-state with the fastest reaction will dominate. If the dominating state is only accessible by protein motion, then thermal and isotope effects from protein should be seen. Of course if the dominating state doesn't require motion, then only limited thermal and isotope effects will be seen.

Permalink to Comment

10. Computationally Entertained on August 16, 2012 3:45 PM writes...

@6,

pacmanase actually exists! LOL

http://www.alibaba.com/product-free/103713692/PACMANASE_WASTE_DIGESTING_CONCENTRATED_LIQUID.html

Permalink to Comment

11. dearieme on August 17, 2012 6:19 AM writes...

Delighted to see such questions asked. When I asked a special case of Derek's question years ago, the answers I got lay somewhere on the tautology - brush off spectrum.

Permalink to Comment

12. Rick Wobbe on August 17, 2012 7:54 AM writes...

Don't forget that enzymes do all this in aqueous solution! Adapting what's learned from these studies to organic catalysts in organic solutions has been and will be very challenging.

Permalink to Comment

13. Rick Wobbe on August 17, 2012 8:02 AM writes...

This seems like one more reason to look more closely at non-competitive/uncompetitive (in biochemical, not marketing, terms) inhibitors, aka non-active site inhibitors.

Permalink to Comment

14. Claire on August 17, 2012 8:31 AM writes...

13 'This seems like one more reason to look more closely at non-competitive/uncompetitive (in biochemical, not marketing, terms) inhibitors, aka non-active site inhibitors.'

I'd be really keen if we did more work on compounds with alternative mechanisms.

I've never fully understood why anyone would want to make an ATP competitive kinase inhibitor when the cellular concentration of ATP is ~2mM.

Permalink to Comment

15. Chmrat on August 17, 2012 8:44 AM writes...

@9, Rob, I take your point, but I feel we fail as chemists if we cannot come up with generalized mechanisms. That would mean everything has to be memorized, which seems to differ from a typical chemistry approach. I am sure that details will change with each case but I hope we can still lump mechanisms together into small numbers of parent classes once these highly sophisticated studies gain even more momentum. This desire for conceptual simplicity may be in vain, of course.

Permalink to Comment

16. Angry Med Chem Guy on August 17, 2012 9:57 AM writes...

@14 Because ATP typically has a Km of 10-150 uM for kinases.

That's why.

Permalink to Comment

17. Claire on August 17, 2012 10:36 AM writes...

16. Angry Med Chem Guy on August 17, 2012 9:57 AM writes...

@14 Because ATP typically has a Km of 10-150 uM for kinases.

That's my point. If you have a 1 nM inhibitor and run the assay at Km ATP (which is pretty standard for HTS)...let's say 10 uM [ATP], you get a 100 x drop off when you go into the cell. It's obviously less of a problem with enzymes that have a higher Km.

I've seen quite a few projects wondering why their cpds aren't as potent as they were hoping and when you crunch the numbers you can explain almost all of the drop off through increased competition with ATP in the cell vs the enzyme assay.

Permalink to Comment

18. Ian Greig on August 17, 2012 1:03 PM writes...

This paper cannot make any claims as to the origin of enzyme catalysis as it does not compare a reaction occurring in solution to one occurring in the enzyme.

Rather it provides an interesting characterization of an enzyme's mechanism.

What remains unmentioned is that coupling between 'environment' (protein, water...) motions and chemical bonding changes will also occur in uncatalyzed (water) reaction - as charge pattern change during a typical reaction the extents to which solvent molecules are ordered around different atomic centres will change.

The origins of numerous enzymes' catalytic power have been shown to arise from their ability to minimise the energy associated with solvent reorganization. This point has long been made particularly by Warshel (e.g. Chem. Rev. 2006, 3210 or J. Biol. Chem. 1998, 27035) yet the catalysis - protein dynamics line of thought seems to be a unjustifiably persistent meme .

Permalink to Comment

19. darwin on August 17, 2012 6:00 PM writes...

Enzymes work by the same mechanism as the refrigerator light

Permalink to Comment

20. anonymous on August 17, 2012 7:49 PM writes...

"16. Angry Med Chem Guy on August 17, 2012 9:57 AM writes...

@14 Because ATP typically has a Km of 10-150 uM for kinases.

That's my point. If you have a 1 nM inhibitor and run the assay at Km ATP (which is pretty standard for HTS)...let's say 10 uM [ATP], you get a 100 x drop off when you go into the cell. It's obviously less of a problem with enzymes that have a higher Km.

I've seen quite a few projects wondering why their cpds aren't as potent as they were hoping and when you crunch the numbers you can explain almost all of the drop off through increased competition with ATP in the cell vs the enzyme assay."

Ah, but if your excellent Med Chem team makes a low pM ATP competitive inhibitor for you, you're still in FINE shape (assuming the ADME Gods are shining on you). In other words, it's all about [I]/Ki vs. [S]/Km

Permalink to Comment

21. Claire on August 20, 2012 3:18 AM writes...

@20 "Ah, but if your excellent Med Chem team makes a low pM ATP competitive inhibitor for you, you're still in FINE shape (assuming the ADME Gods are shining on you). In other words, it's all about [I]/Ki vs. [S]/Km"

Yes, but that's the point, if you're working on an ATP competitive series then you often have to be low pM (and then you're usually in all sorts of problems with tight binding limits in your enzyme assay...) but if you work with alternative MoA then you don't need such low affinity. That's the bit I don't get - why work on a series where you know you're aiming for low pM when there are alternatives, which in my experience are usually never even considered by the med chem team. (Maybe other pahrma companies do things differently?)

Permalink to Comment

22. anonymous on August 20, 2012 7:30 PM writes...

@21 Claire: Come on! A clever (woman?) like yourself can solve the tight binding issue...that's the LEAST of your problems!!! BTW, one cannot always find non-ATP competitive kinase inhibitors and there often isn't an "alternative MOA" to target besides a kinase. Finally, an intelligent Med Chemist will listen to a "good" alternative and if yours' won't, find one who will !!!

Permalink to Comment

23. Rick Wobbe on August 21, 2012 8:49 AM writes...

If I understand Claire's point correctly, it's that we could do more to exploit non-competitive, uncompetitive, allosteric and/or non-active site inhibitors than is generally done. In my experience, substrate-, cofactor- or agonist-competitive inhibitors are often prioritized above these other types of inhibitors and it's easy to see why: their binding and the consequences thereof are relatively easier to envision and model. In contrast, although we've had nice examples of small molecule allosteric inhibitors for well over half a century, progress understanding their mechanism at the molecular-atomic level has been relatively slower. (Do they still teach the R/T conformational change model of hemoglobin in undergrad Biochemistry?) To the extent that these inhibitors operate by altering the distribution of conformational states (e.g. stabilizing an inactive conformation), studies like the Russell et al paper Derek cited help us better understand and (hopefully) exploit this mechanism more effectively than we have thus far. But I don't want to put words in Claire's mouth...

Permalink to Comment

POST A COMMENT




Remember Me?



EMAIL THIS ENTRY TO A FRIEND

Email this entry to:

Your email address:

Message (optional):




RELATED ENTRIES
The Worst Seminar
Conference in Basel
Messed-Up Clinical Studies: A First-Hand Report
Pharma and Ebola
Lilly Steps In for AstraZeneca's Secretase Inhibitor
Update on Alnylam (And the Direction of Things to Come)
There Must Have Been Multiple Chances to Catch This
Weirdly, Tramadol Is Not a Natural Product After All