How do enzymes survive the snow? Adapting adenylate kinase to the cold.

Regardless of the weather, our bodies have evolved to function optimally at 37 ℃. However, we are an insignificant speck of the vast array of species out there, where many, if not most, species are operating at temperatures that could either freeze or frazzle us! Enzymes are the key players in catalysing the many biochemical reactions that go on within cells, however, like with non-enzymatic reactions, the rate of the reaction drops in conjunction with the temperature. Slow rates of reaction are bad for an organism maximising its fitness to survive. How then do organisms cope at low temperatures? In a recent Nature paper, Saavedra et al.1 use adenylate kinase to demonstrate how enzymes can cope with the cold by increasing their disorder with flexible amino acids like glycine far from their active site.


cold enzyme
Figure 1: An enzyme looking cool when it’s cool



So, what are enzymes? Affinity and turnover

The textbook definition of an enzyme are macromolecular biological catalysts… i.e they are proteins (mostly) that speed up the rate of a reaction by providing a lower energy pathway for the reaction to occur.

There is a whole suite of enzymes that come in different sizes and shapes, that catalyse different reactions and have differing efficacy to increase the rate of a reaction. Two key enzymatic properties that are key to the rest of this discussion are affinity and turnover.

AFFINITY: simply meaning a liking for something, affinity in the context of enzymes refers to the strength of an interaction with its reactants (substrates in biological lingo). Affinity can be measured as a constant, the Michaelis constant (K), with a lower Km indicative of a higher enzyme-substrate affinity.

TURNOVER: enzyme turnover refers to the number of conversions of substrate –> product per second for a given concentration of an enzyme. This enzyme property is symbolised by Kcat (a constant for catalytic turnover), with a higher Kcat supporting a higher turnover of substrate to product.

In this manner, an enzyme that has a low Km and a high Kcat can increase the rate of a reaction greater than an enzyme with a high Km and a low Kcat.

Comparing related enzymes from organisms living in cold temperatures (cryophiles) and physiological temperatures (mesophilic), have shown that they can catalyse reactions at similar rates. Given that reactions are slower at low temperatures, including motion of the enzymes themselves, how have the enzymes been adapted?

Glycine = gloves

Many previous studies have analysed various enzymes to answer this question2. A reoccurring theme is the presence of the small amino acid, glycine, in external positions of proteins, that replace bulkier amino acids present in the mesophilic equivalent enzymes. Being smaller, glycine causes less interference with the enzymes protein structure, but that besides, how do these changes, far from the active centre of the enzyme, alter the enzyme’s catalytic rate at low temperatures?

Giving gloves to adenylate kinase

To uncover the mechanisms that enable enzymes to operate at low temperatures, Saavedra’s team used an enzyme essential to the bacterium, Escherichia coli (E. coli), adenylate kinase. This enzyme catalyses the conversion of two substrates, ATP and AMP, to two molecules of ADP.

The structure of adenylate kinase has been well characterised, with the enzyme showing three domains; a lid (LID), a core (CORE) and an AMP binding site (AMPBD) domain. The LID undergoes a conformational change when the enzyme transitions from an “open” to “closed” state (Figure 2). The CORE domain is the main site for the catalytic activity.


adenyltte kinase
Figure 2: Structural changes in adenylate kinase


To understand the functional effects of glycine residues, targeted mutations of residues to glycine were made in surface-exposed regions in the LID and AMPBD of adenylate kinase. Using differential scanning calorimetry (DSC), to study the effect of the mutations on the proteins dynamics and isothermal titration calorimetry (ITC), to look at substrate affinity, it was found that mutations in the LID, but not the AMPBD, increased the percentage of proteins in the “open” state and showed reduced affinity to substrate.

This supports a role of the LID domain in substrate affinity.

What about turnover?

To examine the effect of mutations on turnover, the authors measured and compared the turnover rates of the wild type and glycine-mutated enzymes at a range of temperatures. In contrast to affinity, LID mutations showed no effect on Kcat, whilst Kcat significantly increased in the AMPBD­ mutants at each temperature tested.

Conformational entropy = measuring the number of conformations

Whilst the activation enthalpy (energy needed for the reaction to occur) was similar between the normal and mutated proteins, the AMPBD­ mutants displayed a lower activation entropy at each temperature. Since entropy is a measure of the number of conformations, the results (with some further analysis) strongly suggested that conformational changes in the AMPBD, not the LID, were rate-limiting for product-release, supporting the changes seen in enzyme turnover.


So, how does this help with the cold?

Well, the results from the LID mutations, stabilised the “open”, unfolded state of adenylate kinase. This unfolding is disfavoured at lower temperatures as it requires high energy to perform the transition – thus, the glycine mutation enables the unfolded state at low temperatures to match the proportion of normal enzymes in this state at physiological temperatures.

Similarly, the AMPBD glycine mutations enable product release at low temperatures to match that of normal enzymes at physiological temperatures.

Thus, these entropy-enhancing mutations seem to be key for the success of enzymes at low temperatures.

However, the really fascinating finding by Saavedra’s team is the division of affinity and turnover to the LID and AMPBD domains, respectively. This work will be useful for protein engineering and directed evolution approaches, but how these dynamics could be designed or selected for on a large scale will be the next challenges to tackle.

Further reading

  1. Saavedra, H. G., Wrabl, J. O., Anderson, J. A., Li, J. & Hilser, V. J. Dynamic allostery can drive cold adaptation in enzymes. Nature 1 (2018). doi:10.1038/s41586-018-0183-2
  2. Fields, P. A. & Somero, G. N. Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes. Proc. Natl. Acad. Sci. U. S. A. 95, 11476–81 (1998).

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