Remember entropy – take III

Pop quiz.  How would you make an enzyme in a cold dwelling organism (0 Centrigrade) as catalytically competent as its brothers living in us at 37 C?

We know that reactions go faster the hotter it is, because there is more kinetic energy of the reactants to play with.  So how do you make an enzyme move more when it’s cold and there is less kinetic energy to play with.

Well for most cold tolerance enzymes (psychrophilic enzymes — a great scrabble word), evolution mutates surface amino acids to glycine.  Why glycine?  Well it’s lighter, and there is no side chain to get in the way  when the backbone moves.  The mutations aren’t in the active site but far away.   This means more wiggling of the backbone — which means more entropy of the backbone.

The following papers [ Nature vol. 558 pp. 195 – 196, 324 – 218 ’81 ] studied adenylate kinase, an enzyme found in most eukaryotes  which catalyzes

ATP + AMP < — > 2 ADP.

They studied the enzyme from E. Coli which happily lives within us at 37 C, and mutated a few surface valines and isoleucines to glycine, lowered the temperature and found the enzyme works as well (the catalytic rate of the mutated enzyme at 0 C is the same as the rate of the unmutated enzyme at 37).

Chemists have been studying transition state theory since the days of Eyring, and reaction rates are inversely proportional the the amount of free energy (not enthalpy) to raise the enzyme to the transition state.

F = H – TS (Free energy = enthalpy – Temperature * Entropy).

So to increase speed decrease the enthalpy of activation (deltaH) or increase the amount of entropy.

It is possible to separately measure enthalpy and entropies of activation, and the authors did just that (figure 4 p. 326) and showed that the enthalpy of activation of the mutated enzyme (glycine added) was the same as the unmutated enzyme, but that the free energy of activation of the mutated enzyme was less because of an increase in entropy (due to unfolding of different parts of the enzyme).

Determining these two parameters takes an enormous amount of work (see the story from grad school at the end). You have to determine rate constants at various temperatures, plot the rate constant divided by temperature and then measure the slope of the line you get to obtain the enthalpy of activation.   Activation entropy is determined by the intercepts of the straight line (which hopefully IS straight) with the X axis.  Determining the various data points is incredibly tedious and uninteresting.

So enzymes  of cold tolerant organisms are using entropy to make their enzymes work.

Grad school story — back in the day, studies of organic reaction mechanisms were very involved with kinetic measurements (that’s where Sn1 and Sn2 actually come from).  I saw the following happen several times, and resolved never get sucked in to having to actually do kinetic measurements.  Some hapless wretch would present his kinetic data to a seminar, only to have Frank Westheimer think of something else and suggest another 6 months of kinetic measurements, so back he went to the lab for yet more drudgery.

 

 

Advertisements
Post a comment or leave a trackback: Trackback URL.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s

%d bloggers like this: