Speak of the devil....

...and the devil, he shall appear.

A recent discussion at the Curious Wavefunction briefly touched upon the role of (macro)molecular crowding in biochemical studies.  I am presently preparing to see whether a certain set of experiments are feasible, and in some of the potential tests, I am adding a fair amount of crowding agents to my samples. 

The samples that have those crowding agents are presently the only ones that exhibit any enzymatic activity.  The other samples are dead, biochemically speaking.  One of the "dead" samples does exhibit complex formation in one of my alternate tests, but that's it.  Further tests are needed, of course, and it's certainly possible that I can retain enzymatic activity in the other samples upon logically adjusting my current protocols.  But perhaps there's a lesson here to be learned.




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SAXS And Promiscuity - Or, What Your Biochemistry Text Doesn't Cover.

Glycolysis is one of those things you learn about as an undergraduate (in high school as well, to be fair, but in a good bit more detail in an undergraduate biochemistry course) and – at least in my experience – it was presented as a topic that had already been well-explored and thoroughly annotated. After all, if it hadn’t been, would they have put it down for posterity in a textbook?

Snickering aside, I was pretty intrigued to see the following paper the other day for more than just being another entry in my “clearly, plenty of mechanistic detail was glossed over in my biochem text” list. Basically, the research team utilized a combination of crystallography, small-angle x-ray scattering (SAXS), and computational model to develop a scheme for the mechanism of phosphoglycerate kinase. They propose that the enzyme has a preferred “open” conformation where substrates (1,3-bisphosphoglycerate and ADP) can bind (separated by over 15 angstroms), and then a “closed” conformation, where the domains “fold in” on one another, bringing the substrates together for chemistry and which exposes a hydrophobic patch, which they suggest drives the preference for the “open” conformation. In the supplemental info, they do have some movies for download which make for fun viewing.

It is a nice example of what some in the structural biology field have been pulling for, an integration of high-resolution methods with lower-resolution methods that can provide additional insight into dynamics at the domain scale and above. Just as a representative example of this thinking is the SIBYLS beamline at Lawrence Berkeley Lab (SIBYLS – Structurally Integrated Biology for Life Sciences, where they possess the ability to do both crystallography and SAXS at the same station). They’ve also got a fairly lengthy review linked to on that page that describes the interplay between crystallography, SAXS, and computational methods.

In the spirit of Wavefunction’s link post the other week, at least, I stumbled across this recent paper on stochastic ensembles, conformationally adaptive teamwork, and enzymatic detoxification today. I am still working through the paper, but – given that one of the authors has written rather extensively on atypical (non-Michaelis-Menten) kinetics in enzymes – he is putting forth a new set of organizing thoughts for understanding the unusual substrate binding and catalytic properties of detoxification enzymes (which frequently have multiple isoforms differentially expressed in tissues). These enzymes are not only promiscuous in terms of the substrates they’ll work with, but are also involved in multiple metabolic processes. So it's hardly as straightforward as biochem texts are fond of portraying with those nice, neat flow charts. I have occasionally considered this as a possible reason for all those secondary metabolites in plants that no one can figure out why they're present in the first place - you have a bunch of enzymes floating around in the cells and given enough time, stuff happens. But that is perhaps another post for another day. Read more!