Enzymes are nature’s best catalysts. The how and why of it are the central questions of enzymology. Over the decades, enzymologists have come a long way in understanding and even designing enzyme function. But three new results are highlighting previously unappreciated complexities of enzyme function, complexities that are often neglected by enzyme designers.
First, the Bustamente lab debuts a provocative explanation of recently-observed a curiosity of enzyme function: enzyme diffusivity increases during reaction with substrates, at least for some enzymes. That fact itself was initially a surprise, and would likely never have been appreciated but for nifty new fluorescence-based single-molecule tracking techniques.
But the new paper offers the first cogent, general explanation of the effect: the energy released during substrate turnover causes an asymmetric expansion of the enzyme, pushing against the surrounding solvent and accelerating the enzyme molecule. The prevailing theory until now had been that the energy of reaction was quickly dissipated into the surrounding solvent, to be lost as heat. Previous groups who had noted the anomalous diffusion were focused on only a few types of enzyme (primarily catalase or urease). Their hypotheses centered on microbubbles or micro-gradients in pH, effects specific to their particular enzyme of interest. The new paper’s more general results dispel those hypotheses too. A. Joshua Ward also adds a lot of valuable commentary.
A second recent paper comes from Martina Havenith’s group, along with collaborators and co-workers. They report using terahertz absorption spectroscopy to study the dynamics of the water involved in the enzymatic reaction of a matrix metalloproteinase. Terahertz spectroscopy, for those of you that don’t know (a set that until a few minutes ago included me!), is like the UV or infrared spectroscopy most of us know and love, except it uses longer wavelengths (from around 10,000 to 100,000 nm). Water absorption at these wavelengths is controlled by inter-molecular interactions such as H-bond exchange or libration. So changes in the THz spectrum indicate changes in the structure of liquid water: longer-lasting or fewer H-bonds, for example, or fewer librations. Terahertz spectroscopy is for biomolecular analysis is fairly new, but the first wave of practitioners are discovering very interesting things about how solutes change the structure of the water they’re dissolved in; Havenith herself has written a very nice review.
Back to enzymes. The textbook view is that once a substrate reacts in the enzyme’s active site and is released, that the enzyme is back in its initial state—ready to react with another substrate and repeat the process over again. The second reaction proceeds the same as the first, and so on. An always made (but rarely stated) assumption of this textbook view is that the water surrounding the enzyme stays the same from cycle to cycle. THz spectroscopy permits a test of that assumption, and the authors find that it doesn’t hold, at least in their particular system of interest. Enzymes can modulate the structure of the water they’re dissolved in, and these modulations can (a) be dependent on catalytic activity, and (b) take place over longer time scales than the enzyme reaction itself. The clear implication is that the change in water structure could then affect enzyme reactivity or other interesting biological properties. (Turning that could into a does, however, awaits further research.)
Third, a new vibrational spectroscopy technique for measuring local electric fields has found that highly localized, very large electric fields can directly contribute to an enzyme’s catalytic effect. Folks have appreciated the contribution of electric fields to substrate binding for a while, but once the substrate arrives at the active site, just how much they affected catalysis was a matter of debate. Electric fields are very difficult to measure at the tiny length scales of enzyme active sites. That’s where Stark spectroscopy comes in. Stark spectroscopy, based on the electrochromic effect, is perhaps nature’s smallest electrometer: it can measure electric fields over length scales as small as chemical bonds (i.e. a few angstroms). The method examines shifts in molecular vibration frequency in response to an electric field. And an obvious extension, used by the Stanford authors, is to calibrate these shifts against known electric fields, and then to repeat the spectroscopy when the molecule moves into an environment of interest.
Working with ketosteroid isomerase, chemists in Steven Boxer’s group at Stanford were able to measure the electric field along a key C-O double bond in a substrate analog as it sat in the enzyme’s active site. And they did this not only for the wild-type enzyme, but for a series of mutants. Those mutants were predicted to change the electric field at the key C-O double bond. The new Stark spectroscopy technique showed that the mutants (with one exception) did just that, but also that these changes in the local C=O electric field correlated strongly with catalytic proficiency (ΔG† varied linearly with electric field for all you transition state theorists out there). The strength of this trend implied that the electric field effect was responsible for 70% of ketosteroid isomerase’s catalytic activity.The findings aren’t just relevant for ketosteroid isomerase. An accompanying commentary by Peter Hildebrandt puts it well:
It is very likely that the electric field–dependent acceleration of elementary reactions is a general concept in biological catalysis and perhaps also in chemical catalysis, as suggested, for instance, for zeolite-based catalytic reactions…
The exquisite measurements in these three studies have revealed new layers to the onion of complexity that is enzyme function. It remains to be seen whether these traits are selected for by evolution, or merely quirky side-products of selection for other properties. More research, as they say, is required.