Why HisH doesn't fire until it sees the whites of PRFAR's eyes

The enzyme imidazole glycerophosphate synthase (IGPS) can be a bit of a lump. If you bind just one substrate it doesn't do anything, even though its two active sites are separated by more than 30 Å. Only if the second substrate also binds does catalysis actually go at anything like a respectable rate. In a recent paper in Structure researchers from Yale report evidence that this change of pace results from a change in dynamics.

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The role of dynamics in catalysis

For some enzymes, dynamics on the millisecond timescale play a critical role in catalysis. I don't think this is a particularly controversial or unclear statement, but then, I know what I mean by it. In the process of communication, however, the intended meaning sometimes gets lost or transformed. A statement that addresses an entire catalytic cycle, for instance, might be interpreted as addressing only the chemical step. This seems to have happened in a pair of papers that concern the transfer of energy from conformational rearrangements to a chemical reaction.

Consider a reaction scheme in which an enzyme loosely associates with substrates (E.S), then "closes" to form a tight, catalytically-competent complex that then undergoes a reaction with the rate k_chem:

Pisliakov et al. (1) ask whether the closing process can accelerate k_chem. They ask this question primarily because a group from Harvard University proposed that this was possible in a paper printed last year in J. Phys. Chem. B (2). In that paper, Min et al. performed some simulations suggesting that such an acceleration was at least possible, and consistent with some enzymatic data. Pisliakov et al. approach the question with simulations of the reaction of the phosphotransfer enzyme Adk with 2 ADP molecules to form ATP and AMP. As part of the catalytic cycle, the enzyme goes from an open state (PDB: 4AKE) where the ATP and AMP binding sites are exposed to solvent, to a closed state (PDB: 1ANK) where the substrates are shielded from the surrounding solution by ATP and AMP "lids" that close down over the active site.


One can, perhaps, imagine that when the enzyme closes around the substrates, some motion will occur that promotes the transfer of a phosphate group from one molecule to another. Pisliakov et al. use a three-tiered system of simulations to address the question, as a way of trying to get around the difficulty of dealing with the long timescales required. Their simulations allow them to adjust the energy barrier to match the experimental rates or accelerate the reaction so that the whole pathway can be simulated. In general, they find that conformational fluctuations do not enhance the chemical reaction rate in this system.

I have two main concerns about the science that was performed here. The first is that the energy barriers in the long-timescale experiment appear to be improperly paramaterized. In estimating these barriers for the phosphotransfer reaction in Adk, Pisliakov et al. used 260 /s as k_chem. However, although the actual reaction carried out by Adk follows an extremely complex scheme, the analysis performed by Wolf-Watz et al. utilized a simplified scheme that combined all post-association steps into a single k_cat. This is why the concordance between k_cat and k_open justifies the conclusion that lid-opening is rate-limiting. In principle, the experiments used for that paper are incapable of separating the opening and closing steps from the chemical step. Therefore we have no experimental knowledge of the phosphotransfer rate, except that it is greater than 260 /s. This perplexing error appears to have originated with Min et al., but I am surprised Warshel's group did not catch it.

This is not a major problem because the bulk of the conclusions of the experiment were drawn from a different simulation in which the energy barriers were lower, but this leads to my second concern. If the structural transition involves a very smooth and coherent rearrangement of the protein, then simply manipulating energy barriers should not result in a serious error of analysis. In reality, however, ensemble motions of protein elements are not going to be so directed or uniform. Structural rearrangements are not highly singular steps, but involve a large number of intermediates and transition states. Motions in the late stages of the structural transition that promote catalysis may well be missed by simplified models, or accelerated beyond productivity by lowering the energy barrier.

That said, I'm not particularly surprised that Pisliakov et al. find that energy from the conformational coordinate does not transfer to the chemical coordinate, nor do I disagree with the finding. Despite what Pisliakov et al. appear to believe, the papers that have come out of Dorothee's group don't argue that the millisecond motions contribute directly to the chemistry. Doro doesn't believe that for a second. Neither do I. The importance of dynamics has little to do with shoving the reaction along the chemistry coordinate, but everything to do with getting substrates bound and into a state where chemistry is possible.

Dynamics allow an enzyme to reconcile incompatible functional requirements. To efficiently function as a phosphotransfer enzyme (as opposed to a hydrolytic phosphatase), Adk must expel water from the active site during catalysis. If the active site is inaccessible to solution, however, there is no way for the substrates to diffuse into it. It is difficult to create a single, rigid fold that can accommodate both these demands, but by fluctuating between two states the problem is resolved quite easily. So yes, the dynamics are essential to catalysis, but that does not imply that the conformational and chemical energy coordinates are coupled.

More perplexing is the discussion of the hierarchy of motion, which Pisliakov et al. take to mean that nanosecond motions somehow contribute to the chemical coordinate. As I discussed when that paper was initially published, the question being addressed was whether and how motions on the fast timescale (ps-ns) in Adk were related to the slower (ms) motions of the lids. In a hierarchy of motion, fast timescale fluctuations enable or promote slow timescale dynamics. In the case of Adk, this means that nanosecond flexibility at structural hinges allow the millisecond motions of the ATP and AMP lids. It was not implied, then or since, that the nanosecond motions in question make a direct contribution to movement along the chemical coordinate. This is not to say that there are no researchers who believe that ns motions contribute to catalysis — I've previously mentioned some work on hydrogen tunneling that makes precisely this argument. In the specific case of Adk, however, the contribution of ns motions to catalysis consists entirely in their enabling of the slower ensemble motions of the nucleotide binding domains, and nobody but the Warshel group has suggested otherwise.

There is an ongoing disconnect in the literature concerning the role of dynamics in catalysis. While it is true that in many cases rates of structural transitions correlate with rates of catalysis, this does not imply that the conformational transition coordinate is linked to the chemical reaction coordinate by direct transfer of energy. It is more likely that the dynamics of the enzyme contribute to catalysis by generating reaction-competent states from reaction-incompetent states. This is not to say that dynamics cannot possibly make a contribution to phenomena such as hydrogen tunneling, but it strikes me as unlikely that motions on the millisecond timescale will contribute to a chemical coordinate. Experiments, rather than simulations, will be the ultimate test of the idea. However, in principle, this hypothesis can only be tested experimentally on enzymes where the conformational changes do not limit the chemical reaction rate. Because the rate of the chemical step is unknown in Adk, it may not be an appropriate model system for addressing this question.

1. Pisliakov, A., Cao, J., Kamerlin, S., & Warshel, A. (2009). Enzyme millisecond conformational dynamics do not catalyze the chemical step Proceedings of the National Academy of Sciences, 106 (41), 17359-17364 DOI: 10.1073/pnas.0909150106

2. Min, W., Xie, X., & Bagchi, B. (2008). Two-Dimensional Reaction Free Energy Surfaces of Catalytic Reaction: Effects of Protein Conformational Dynamics on Enzyme Catalysis The Journal of Physical Chemistry B, 112 (2), 454-466 DOI: 10.1021/jp076533c

3. Wolf-Watz, M., Thai, V., Henzler-Wildman, K., Hadjipavlou, G., Eisenmesser, E., & Kern, D. (2004). Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair Nature Structural & Molecular Biology, 11 (10), 945-949 DOI: 10.1038/nsmb821

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Microwave Pfu CelB 5 minutes for highest activity

Like many bachelors, I regularly eat meals heated up in a microwave oven. I'd like to think I eat a somewhat lower percentage of frozen dinners than others in my situation, but even when it comes to food I cooked myself I usually don't have the time or patience to cook a recipe for one every night. That means I'm often eating reheated leftovers from the trusty Radar Range. The microwave oven works using dielectric heating, a process in which the movements of dipolar bonds are coupled to an oscillating external field. Water, for instance, is a molecule with a large dipole moment, and when you cook something in a microwave a lot of the heating occurs by the accelerated movements of water molecules. In principle this kind of excitation should also occur for other kinds of polar molecules, and thus we come to an interesting study from the lab of Alex Dieters, in which microwave radiation was used to activate a cellulase from a hyperthermophile.

Protein backbones consist of series of peptide bonds which include a carbonyl group, a classic example of a polar bond. Naturally, one might expect that the motion of these groups would be excited by microwave radiation. However, it does not directly follow that additional motion of the peptide backbone will actually accelerate chemical reactions, because this motion may be chaotic or unproductive. Moreover, from the fact that microwaves cook things (like eggs), we know that microwave radiation does a good job of denaturing proteins, sometimes at lower temperatures than we expect. Both of these problems can conceivably be avoided by studying a hyperthermophilic protein.

Proteins from hyperthermophiles such as Pyrococcus furiosus tend to be stable and optimally active at very high temperatures, at or even exceeding the boiling point of water. At lower temperatures, they retain their stability, but tend to become inactive. In many cases this reduction in activity appears to result from squelching internal motions that may be necessary to bind or properly orient substrates. Young et al. decided to study the β-glucosidase CelB from P. furiosus as a way of understanding whether microwaves might enhance enzymatic catalysis. Because CelB has optimal activity at 110° C it should be possible to see a significant difference in activity if microwave activation works. The stability of this protein at high temperatures also suggests that you will not accidentally cook it.

Sharp readers will have noticed an obvious problem with this idea—because heat activates this protein, and microwaves heat aqueous solutions, we must incorporate some kind of control in order to determine the pure effect of the radiation as opposed to the temperature. Young et al. resolve this problem by monitoring the heating of the sample during microwave irradiation, and then using a normal thermal apparatus to match this temperature profile (Figure 2). When a reaction reached 40° C using either heating method, it was quenched by the addition of a basic solution and the concentration of products was measured. Simply heating the Pfu CelB reaction to 40° C produced negligible activity, but microwaving it increased the activity by 4 orders of magnitude (i.e. a factor of 10,000). Less dramatic, but still significant, effects were observed for two other hyperthermophilic enzymes, but an enzyme from a mesophilic organism (the almond) was not activated by microwave irradiation.

That the microwaves caused increased backbone motion was supported by the finding that irradiating Pfu CelB at 75° C caused it to denature; this temperature is well below the normal melting temperature of this enzyme (115° C). The authors attribute the differences in activation between CelB and the other hyperthermophiles to their lower optimal activity temperatures, but it is also possible that the particular motions enhanced by microwaves are simply not as productive in those molecules. Although all the dipoles should be affected in similar ways by microwaves, they are all oriented differently with respect to each other in the protein molecule. As a result, the induced motion may be chaotic, perhaps specifically so, and therefore the activation of a particular thermophile may depend on the nature of the motions needed for its catalytic cycle. Enzymes that require large ensemble motions of subdomains, such as adenylate kinase, might not be activated as much as a protein that simply needs to be melted a little. Examining the differences in structural dynamics of enzymes differentially activated by microwaves may be an interesting area of future study.

While microwave activation is unlikely to revolutionize some of the more common uses of hyperthermophilic proteins (i.e. PCR), it does have promise. In ligations, for instance, hyperthermophilic enzymes can not be used at present because many DNA inserts denature at the optimal active temperature. With microwave activation, it may be possible to employ extremophilic ligases in these reactions, gaining the benefits of their speed and durability without having to worry about accidentally melting your DNA. Depending on the enzymes available, this technique may also prove valuable in improving mobile medical laboratories and developing novel diagnostic tools for field work.

1. Young, D.D., Nichols, J., Kelly, R.M., Deiters, A. (2008). Microwave Activation of Enzymatic Catalysis. Journal of the American Chemical Society DOI: 10.1021/ja802404g

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Enzymes almost as good as Ma Nature used to make

Biological systems have the interesting property that most of the reactions enabling life processes are, when left to their own devices, exceedingly slow. To reach the timescales that we associate with "living", these reactions must be sped up, which requires the presence of enzymes. Because they significantly enhance reaction rates under conditions that can be encountered almost anywhere, the design of artificial enzymes is an active area of research. In two papers this month, David Baker's lab describes notable success in designing enzymes in silico to have specific activities with significant (10⁶-fold) rate enhancement.

As a graduate student at UNC, I was fortunate to interact frequently with Richard Wolfenden, who did a great deal of work to find out just how good enzymes are at what they do (1). The fact is that there is a wide range of activities and rate enhancements. The proline isomerase cyclophilin, for instance, achieves a modest 10⁶-fold rate increase, depending on the substrate. In contrast, arginine decarboxylase achieves an amazing rate enhancement of about 10¹⁹. Many reactions we think nothing of, such as hydrolysis of a phosphodiester bond (found in nucleic acids) would take millions of years in pure neutral water at 25° C. Of course, deviations from neutral pH and the presence of other molecules greatly enhance these rates, and obviously the same is true of changes in temperature, but this is a useful starting point for comparing enzymes to basal rates.

In the works at hand, collaborative teams involving several labs coordinated by David Baker designed enzymes to perform a novel retro-aldol reaction (2) and the Kemp elimination from 5-nitro-benzisoxazole (3) (a proton abstraction causing a ring to open). The retro-aldol paper is fascinating, particularly because of the multi-step nature of the reaction, but I'm going to focus on the Nature paper because its results are more complete, in that they implemented an appropriate wet-lab extension to the computational procedure.

The fundamental strategy of both papers is the same. For most enzymes it is believed that catalysis occurs because the transition state, the moment when the chemical reaction has the highest energy, is stabilized by the functional groups of the enzyme (see (1), among others). Using their knowledge of chemistry, the researchers of these groups predicted a transition state, and then positioned functional groups of side chains in such a way that they would stabilize this predicted state. They also placed potential bases in an appropriate geometry to attack protons as necessary. This done, they used a program based on Baker's ROSETTA to predict sequences that would fold to produce this geometry. This required a somewhat more complicated process in the case of the retro-aldol reaction due to its multiple steps.

One interesting outcome was that TIM barrels were a popular choice of this algorithm in both papers. The final results in the Röthlisberger paper are all based on backbones identified by CATH as TIM-barrel folds (explore these scaffolds at the PDB: 1thf, 1a53, 1h61, 1jcl). As the authors note, the TIM barrel is a very common catalytic scaffold in nature, in part because the central β-strands provide a convenient way to orient side-chains towards the catalytic pocket. In both papers, the structures predicted using the ROSETTA algorithm were shown to be very close to the actual result, although they only checked successful catalysts. A comparison of the failed designs to their predicted structures may be of great use in refining the computational approach.

As the above paragraph implies, the groups did in fact succeed in designing enzymes that achieved significant rate enhancements. In the case of the Kemp elimination, the eight enzymes reported had ~5x10³ - 2x10⁵ -fold increases in rate over the spontaneous reaction in a very slightly basic solution. This amounted to actual k_cat (reaction rate) values of 0.006 - 0.29 s^-1, which is significantly slower than is common for enzymes.

In order to improve these results, Röthlisberger et al. turned to the process that produced our own prodigious enzymes in the first place, i.e. evolution. Using a relatively standard in vitro evolution approach, they altered one of the early successes, KE07, which had a k_cat of 0.018 s^-1. Keep in mind, this was not the best computational design result, just one of the first that worked. This in vitro evolution procedure, in just a few rounds, produced an enzyme with a k_cat of 1.37 s^-1. While this is still slow for an enzyme, it represents a rate enhancement of ~1x10⁶ over the spontaneous reaction in solution, an acceleration comparable to that of a modest enzyme like cyclophilin.

This is nowhere near a complete journey. I've already mentioned that the enzymes produced in these experiments are still quite slow in comparison to the genuine article, and the rate enhancements are still modest. The specificity of the enzymes also has yet to be proven—can these proteins distinguish their targets from a sea of similar molecules, or are they promiscuous catalysts? A further dissection of the failed designs is essential to refining the computational approach employed. More careful consideration of effects beyond the secondary shell, and (as the authors note) backbone dynamics and loop positioning may prove particularly helpful in future iterations.

So, we are not all the way to the creation of a truly proficient man-made enzyme, but this is a tremendous step in that direction. The combination of wet lab and computational approaches proved to be very successful in this case. In principle, it should be possible to incorporate all that was learned in the in vitro evolution experiments into the design algorithm from the start. We will not be designing custom catalysts for biofuel production and bioremediation tomorrow or next week. These results, however, demonstrate substantial promise for the future.

In particular, the retro-aldol paper suggests that this approach will work for multi-step reactions. However, provided that the intermediates are stable and soluble this will not be strictly necessary. So long as efficient catalysts can be designed for each step, the ability of ROSETTA to design protein-protein interfaces will make it possible to assemble functional synthetic or catabolic enzyme cassettes to achieve very complex chemistry with tremendous accelerations over basal rates.

1. Wolfenden, R., Snider, M. (2001). The Depth of Chemical Time and the Power of Enzymes as Catalysts. Accounts of Chemical Research, 34 (12), 938-945. DOI: 10.1021/ar000058i

2. Jiang, L., Althoff, E.A., Clemente, F.R., Doyle, L., Rothlisberger, D., Zanghellini, A., Gallaher, J.L., Betker, J.L., Tanaka, F., Barbas, C.F., Hilvert, D., Houk, K.N., Stoddard, B.L., Baker, D. (2008). De Novo Computational Design of Retro-Aldol Enzymes. Science, 319(5868), 1387-1391. DOI: 10.1126/science.1152692

3. Röthlisberger, D., Khersonsky, O., Wollacott, A.M., Jiang, L., DeChancie, J., Betker, J., Gallaher, J.L., Althoff, E.A., Zanghellini, A., Dym, O., Albeck, S., Houk, K.N., Tawfik, D.S., Baker, D. (2008). Kemp elimination catalysts by computational enzyme design. Nature DOI: 10.1038/nature06879

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Dynamics and tunneling in soybean lipoxygenase

Because even the simplest case of enzymatic catalysis involves multiple steps (i.e. association, chemistry, and dissociation), semantic issues have obscured the question of what role protein structural dynamics play. For instance, the opening of the lids in Adenylate kinase appears to be rate-limiting, but there is as yet no evidence that dynamics play any role in the phosphotranfer. Thus, one can argue that dynamics are critical (because they are rate-limiting) or irrelevant (because they contribute nothing to the chemical step). Some authors, Arieh Warshel for example, have argued that the latter finding is general—that the highly successful transition-state stabilization model leaves no place for dynamics in chemical catalysis. Some researchers, however, believe that dynamics may play a significant role in chemistry when catalysis proceeds by other routes, especially hydrogen tunneling. In this vein, Matthew Meyer, Diana Tomchick, and Judith Klinman presented evidence in last week's Proceedings of the National Academy of Sciences that protein dynamics play a significant role in the chemical step of catalysis for the protein soybean lipoxygenase (SLO-1).

Hydrogen tunneling refers to the idea that a hydrogen may in some instances "tunnel through" an energy barrier, going directly from substrate to product without wasting any time or kT in actually surmounting that barrier. A reaction that primarily reflects tunneling should have three properties. The reaction rate should vary only weakly with temperature, because tunneling mostly divorces the rate from kT. Using an Arrhenius plot, this translates to a low calculated energy of activation. Replacing the reactive hydrogen with deuterium should enormously inhibit the reaction—a large kinetic isotope effect (KIE)—because the doubling of mass makes tunneling less likely. Nonetheless, the difference in the energies of activation (ΔE_a) for hydrogen and deuterium calculated from an Arrhenius plot should also be small, if tunneling is the main mechanism. Conceivably an enzyme could enhance the rate of such a reaction by using dynamics to promote favorable vibrational modes for the tunneling to occur, or to temporarily adopt a disfavored structure that puts reactive groups at a better distance for tunneling.

SLO-1 exhibits all three features, and so Klinman's group has used it as model system to try and understand whether and how enzymes promote tunneling reactions. In this case they have performed a series of mutations of isoleucine 553. In addition to existing data on WT and I553A, they analyzed the KIEs, crystal structures, and activation energies of I553L, I553V, and I553G mutants. They find that the protein as a whole is not much distorted by any of these mutations, nor do any of them appear to have significant effects on the binding pocket or substrate dissociation constant—the K_d for each mutant is around 3 μM, while WT is around 10 μM. Yet as the bulk of the mutated residue decreases, the ΔE_a increases significantly. In addition, the kinetic isotope effects decrease much more sharply with temperature than for WT. Again, the magnitude of this increased temperature dependence appears to vary inversely with the bulk of the side chain at I553. From these features, and a decline in the magnitude of the pre-exponential factor, the authors argue that dynamics play an important role in encouraging the tunneling reaction.

Well, you know how I love long-range dynamic effects in proteins. But I don't quite buy it here. An argument based on negatives is never very satisfying in any case, and here we have a lot of questions. For one thing, to say that the structure hasn't changed significantly seems overly simplistic to me. The lowest-energy structure may not have been seriously deformed, but absent crystal packing and with a little extra kT around, the average structure might be different. Additionally, these structures were obtained in the absence of ligand. Even though the energetics of binding do not appear to differ significantly for these mutants, the structure of the enzyme or ligand may have changed in the bound state. Even if I accept that the existing structures argue for a completely identical binding site in the free state, and this could realistically be debated, that's no guarantee that the same is true of the bound state.

I'm not denying that the evidence is strongly suggestive, and getting direct data may be difficult given the size of the protein. However, almost all of these side chains are methylated. That means that perdeuteration of the protein in concert with specific side-chain labeling could be especially fruitful in directly observing the dynamics of the pocket during catalysis. It should also in principle be possible to label other side chains in the region to determine how mutations affect the whole area. It is likely that the substrate can also be labeled, possibly with a nucleus or tag that is poorly relaxed. T₂ is likely to be a major challenge here, but not necessarily an insurmountable one, especially since the enzyme appears to be folded and active up to around 50 °C. The presence of the iron is certainly a complicating factor; however, the dynamics should be the same whether catalysis is occurring or not, so possibly it could be substituted with a non-paramagnetic metal. It wouldn't be the easiest project in the world, but it should be doable.

I think these results are intriguing, and I'd love to hear Warshel's take on them. Nonetheless, I feel I have to reserve judgment at least until dynamic changes in the pocket that seem to correlate with the kinetic observations are directly demonstrated by NMR or some other method.

Meyer, M.P., Tomchick, D.R., Klinman, J.P. (2008). Enzyme structure and dynamics affect hydrogen tunneling: The impact of a remote side chain (I553) in soybean lipoxygenase-1. Proceedings of the National Academy of Sciences, 105(4), 1146-1151. DOI: 10.1073/pnas.0710643105 - OPEN ACCESS ARTICLE

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Small-molecule allosteric catalyst

An interesting pair of articles showed up this week, both from Chad Mirkin's lab at the Nanotechnology Center of Northwestern University. You can find them both in the August 22 edition of JACS. The essence of these articles is that Mirkin's group has created a group of macrocyclic catalysts that can be allosterically regulated by small ligands. The basic principle is shown below (figure taken from the second paper):

The catalytic molecule has a small intrinsic ability to catalyze the chemical conversion of the small molecules, as shown on the left. The addition of the "Analytes" in this case Cl- and CO, displaces the sulfur coordinating groups that are weakly bound to the rhodium or copper metal centers. Removing the sulfur coordination allows the molecule to undergo a conformational change, opening up to adopt a new structure that is vastly more effective as a catalyst. This mimics the allosteric regulation of protein catalysts and binding proteins by ligands.

The approach is a pretty clever bit of molecular engineering, and an important step forward for nanotechnology. Though it remains to be seen how well these molecules survive under field conditions (reducing/oxidizing environments, complex solutions), they have a feature that will be absolutely essential for functional nanomachines, namely that they can be regulated. The ability to turn our microscopic tools on and off will be an important part of producing useful nanomachines, to say nothing of avoiding the "gray goo" of sci-fi nightmares.

If you're interested, check out these articles:
Kuwabara, J. Stern, C.L., and Mirkin, C.A. "A Coordination Chemistry Approach to a Multieffector Enzyme Mimic", J. Am. Chem. Soc. 129 (2007) p. 10074-10075.

Masar III, M.S., Gianneschi, N.C., Oliveri, C.G., Stern, C.L., Nguyen, S.T., and Mirkin, C.A. "Allosterically Regulated Supramolecular Catalysis of Acyl Transfer Reactions for Signal Amplification and Detection of Small Molecules" J. Am. Chem. Soc. 129 (2007) p. 10149-10158.

I recommend the first article for lay readers, and the second for the hardcore.

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