Here I am, back again after a long hiatus talking about the effects of perchlorate salts on chymotrypsin, but this time it’s not bad news! The literature on the effects of chaotropic molecules (perchlorates included) on enzyme activity and stability overwhelmingly shows that they are deleterious to enzymes, something I have been able to show myself through my own research. But this time, I get to write about how perchlorate salts can actually increase enzyme activity at low temperatures.
So how did this research idea even come about? It all started when two of my research interests, extremophilic enzymes and perchlorates, basically collided into each other. All of my previous research has shown that perchlorate salts reduced the enzymatic activity and structural stability of my model enzyme, chymotrypsin. On the face of it, these effects are bad news for biochemistry and life in perchlorate rich environments. But is structural instability always a bad thing?
When might an enzyme being less stable, actually be beneficial for its activity? This brings us to the psychrophilic enzymes, which come from organisms that are uniquely adapted to life in cold environments. Psychrophilic enzymes are generally characterised by a few notable features; they exhibit greater enzymatic activity at low temperatures compared to their mesophilic and thermophilic counterparts, which is obviously beneficial for the organism expressing them. These psychrophilic enzymes also tend to lose their activity at lower temperatures and are more susceptible to unfolding by chaotropic molecules. An important point here is that the structural instability and low temperature activity of psychrophilic enzymes are not separate phenomena, they are linked, and wonderfully so. Because psychrophilic enzymes are less stable, they are routinely reported to have increased flexibility, and it is this flexibility which actually allows them to be active at low temperatures! You can even increase the low temperature activity of an enzyme by simply making it more flexible by introducing a single glycine substitution. Quick caveat: this doesn’t always work, but it works often enough that we can view it as a general rule. We can also look at the flexibility-activity relationship from a thermodynamic viewpoint, which really brings the whole picture together. The key to increasing low temperature enzyme activity is to make the reaction require less activation energy (make ΔH‡ smaller). As psychrophilic enzymes have less stabilising interactions, fewer of these interactions need to be broken in order to facilitate catalysis and as a result you need less energy input to start the reaction. This isn’t a free meal, the reduced activation energy comes with a price. As there are less stabilising interactions, and the enzyme is more flexible as a result, it means that to form the activated enzyme-substrate complex, you must pay a larger entropic cost (ΔS‡ becomes more negative). These are the thermodynamic hallmarks of psychrophilic enzymes, a lower energy of activation and a more negative entropy of activation.
After researching all this, I realised that I might be able to link the psychrophilic activity-flexibility relationship to my own work with perchlorates and chymotrypsin. My thought was, if perchlorate salts partially unfold my enzyme, thus reducing the number of stabilising interactions, would this make my enzyme more flexible and then potentially more active at low temperatures? I became convinced that this should be the case when I found a couple of rare examples of chaotropic molecules increasing enzyme activity at room temperature. All I had to do now was test the hypothesis.
The resulting data did indeed show that perchlorate salts (0.25M) could increase the activity of chymotrypsin at 5°C by ~10% despite perchlorates lowering chymotrypsin activity at ambient temperatures. The thermodynamic analysis suggested that this stemmed from a reduced enthalpy of activation, as had been predicted, but that the effect was diminished by an increasingly negative entropy of activation. These two facets suggested to me that perchlorate induced flexibility was probably the cause of this increased enzyme activity. However the picture wasn’t that simple. Higher concentrations of perchlorate salts (0.5M) did not increase the enzyme activity over the assayed temperature range, however the results suggested that by ~0°C these higher perchlorate concentrations should confer increased activity. So what emerged was this complex interaction between enzyme activity, temperature, and perchlorate concentrations. It’s worth noting in general that enzymes don’t work frozen in ice, and perchlorate salts depress the freezing point of water, so provided an enzyme can remain sufficiently folded to be active, perchlorate salts effectively offer an avenue for activity where there’d be no activity without them at subzero temperatures.
So perchlorate salts aren’t necessarily as bad for biology as we think, at least in colder environments. But that then raises the question of how cells behave at low temperatures in the presence of perchlorates. A question which will hopefully be answered and published soon.
