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Mistreating Enzymes For a Good Cause

What happens when you chuck an active enzyme into the wrong solvent? Well, it stops working (or at the very least, it stops working as well as it did). And how do you know which one is the wrong solvent? Why, those are the ones that make it stop working.

That round trip is to illustrate that there’s often really no way to predict this sort of thing. The only way to know if a particular protein will still be functional in Buffer X or with a few per cent of Solvent Y is to check it and see. Better-known proteins have already had such work done on them, so the pH tolerance of widely used enzymes and their ability to work in (say) 2% DMSO is already established. But this is a pile of empirical data, every time, because we don’t have a very detailed picture of the ways that protein activity can go haywire as the solvent environment changes.

This paper (among others) is an attempt to bring some light into this area. It may sound odd or tedious if you’re outside the field, but this is yet another way to get more of a grip on the mighty Protein Folding Problem: given a particular sequence of amino acids, what three-dimensional protein structure can you expect to form? Living systems are exquisitely arranged around just that behavior; protein structure is a fundamental attribute of life. Our existence depends on these folding events being controllable and on repetitive, reproducible changes in protein structures. And in the other direction, there’s a lot of interest in getting enzymes and other proteins to perform under nonphysiological conditions and in nonaqueous solvents, for industrial uses and the like. So understanding what happens when a working protein starts to become deranged by solvent effects is a worthwhile approach.

In this work (from a multi-center team in Germany), the test enzyme is HMG CoA reductase, the famous target of the statin drugs and a very well-studied system. To give you an idea of how complex the situation is, adding any simple alcohol co-solvents decreased enzyme activity as the concentration went up, as you’d expect. Small amounts of DMSO, on the other hand, actually increased activity, although higher concentrations brought it back down. But DMF, also in the polar aprotic solvent category, hurt activity without ever helping it. Dioxane was the most destructive solvent for enzyme activity, but THF had hardly any effect (and in fact, up at 20% by volume, actually helped activity a bit). Acetonitrile was similar to THF; at 10% by volume it also increased activity. Acetone, though, was nearly as bad as dioxane.

Through small-angle X-ray studies, calorimetry, melting point shifts, and other techniques, the team was able to unravel some of the various mechanisms that lead to this suite of outcomes. The alcohol solvents, particularly ethanol, actually act as inhibitors in the active site as well as affecting the structure of the protein as a whole. On the other hand, the increased rate with a solvent like acetonitrile seems to be due to increased flexibility of the protein structure. You could certainly imagine that going either way, though – too much flexibility allows the protein to flop around to inactive conformations, whereas making a bit more flexible can be sort of “greasing” it for faster turnover. DMSO seems especially powerful for causing structural changes as you move to higher concentrations. These can occur as structurally important water molecules are stripped off the protein, or as solvent molecules themselves start making new (and destructive) interactions themselves.

On the other hand, dioxane’s nasty effects are apparently still a mystery, as are the differences between it and THF. In general, the paper concludes with one of those “Sure is a lot more work to do” situations. “The interactions investigated are often peculiar and, at the present level of knowledge, a holistic theory (based on first principles or empirics) cannot be inferred“, the paper says, and I can well believe it. This is a hard problem that gets into some fundamental principles, and it’s going to be a while before we really have a grip on it.

22 comments on “Mistreating Enzymes For a Good Cause”

  1. John Wayne says:

    I’m expecting to read articles about how Big Pharma is keeping the fact that vodka is just as useful for lowering cholesterol levels as the poisons they overcharge for.

    1. Billy says:

      You sound like you have an idea for a grad school project. Time to write a grant proposal and get to work. Who knows, you might win the Nobel.

    2. GladToMoveToProcess says:

      The cholesterol-lowering effect of ethanol is well known. Way back when, we had a lipid lowering project, with a doctor from NYU consultant. He also worked at Bellevue, and had done a lot of autopsies on homeless people who died on the streets. He remarked, “their livers were shot to hell, but their arteries were clean as a whistle.”

      1. Vader says:

        *blink blink*

        Wait, seriously? Confounders?

        I would not be surprised if ethanol lowers plasma cholesterol. I would be surprised if this improves longevity. Niacin?

      2. electrochemist says:

        This is a difference between lowering cholesterol concentration in serum and removing deposits from artery walls (or preventing deposition in the first place). It was the latter that was the observable from autopsies, right?

      3. DrOcto says:

        Can’t block your arteries if you can’t afford a hamburger.

        1. AR says:

          Legit. I think the thinking is now that the deposits are caused and exacerbated by inflammation as opposed to eating too much fat, and a big cause of that could be excess sugar in the diet.

      4. Me says:

        I once read that most alcoholics’s health issues are related to malnutrition rather than to effects of alcohol….

    3. Lab insect(the bug) says:

      Sweet analysis brother!!

  2. Mol Biologist says:

    Hmm interesting fact that vodka was invented by D. Mendeleev.

      1. Mol Biologist says:

        Correction: His proposal identified the potential for new elements such as germanium. https://en.wikipedia.org/wiki/Dmitri_Mendeleev

  3. Rhodium says:

    Except for immunology, I don’t know of anything in chemical biology more confusing than the origin of hydrophobicity. Keeping track of how it changes with temperature, and why proteins can denature in the cold is like trying to juggle five or six balls.

    1. Trans Cript says:

      I was recently surprised to learn that the hydrophobic effect at high temperatures is actually enthalpically driven, i.e. the interaction between water and hydrophobic molecules is enthalpically unfavorable. So at low temperatures it’s entropically driven and at high temperatures it’s enthalpically driven,

    2. chiz says:

      Ric Pashley has been arguing for some time that hydrophobicity is mainly driven by gas nanobubbles and that solubility is different in degassed water. If true then dissolved gases may be having an effect in other solvents too. There doesn’t appear to be have been any research, that I’ve heard of, into whether the amount of dissolved gas, and the manner in which it is dissolved, might explain some of the ‘modified water’ claims that some people make. From memory there have been claims for altered ion solubility in one of those other waters.

  4. a. nonymaus says:

    It’s not surprising that adding co-solvents to water can increase activity of enzymes given that most were evolved to operate in cells. There is a medium where between all of the sugars, ions, lipids, and other proteins, what water there is has a lot to interact with. Not that this gives me the ability to predict which co-solvent to try.

  5. PI says:

    I prefer mistreating grad students ( lab insects) for a good cause……curing cancer!!!, er and padding my portfolio to give to pharma as an offering.

  6. John Campbell says:

    I remember a talk 35 years ago on superoxide dismutase and one thing stuck in my mind was the lecturer said SOD was soluble in chloroform, in fact it could be extracted from aqueous solution with chloroform. Can anyone confirm this?

  7. Vaudaux says:

    One of the ways to release periplasmic proteins from E coli is to treat the bacteria with chloroform, which somehow disrupts the outer membrane without having much effect on the overall structure of the bacterial cell. This is not the same as extracting the proteins into chloroform, but might be what the lecturer was referring to.

    1. John Campbell says:

      Thanks, that may be the case, it was a long time ago.

  8. Molecular Pharmacology Guy says:

    The influence of solvents on protein function for chemical synthesis brings a tangentially related point to mind: the influence of DMSO on cellular phenotypes; i.e. the potential impact of this solvent on a large number of proteins.

    DMSO concentration is usually expressed as a very low percentage of cell medium (e.g. 1%). However, just 1% DMSO translates to a molarity of 141 mM DMSO! In other words, DMSO is present at a level only observed with sodium chloride in physiology… Though it is a very small molecule, can we really expect this high concentration to be innocuous in terms of biology?

    Unsurprisingly, DMSO impact on protein function (e.g. BET domains – PMID 21804994) and overall cellular phenotypes (e.g. gene expression – PMID 22105179) have been documented previously.

  9. gippgig says:

    Off topic but may be of interest:
    Partnering with Industry for Better Education in Drug Discovery
    http://www.hopkinsmedicine.org/research/advancements-in-research/fundamentals/in-depth/thinking-outside-the-box-the-johns-hopkins-medimmune-scholars-program-is-widening-students-career-opportunities

    Also, I’ve been regularly e-mailing lists of problems with blog entries but many definite errors have not been corrected.

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