In the previous guide, I showed how you could obtain information about the enzyme by changing the substrate,
pH, and the enzyme. Why not change the solvent? Attempts have been made to do this for the last 100 years.
- First, water miscible solvents like ethanol and acetone were added. If the water concentration
was high enough, activity remained.
- Biphasic mixtures were made in which an aqueous solution of an enzyme was emulsified in a water immiscible
solvent like chloroform or ethylacetate. The substrate would partition into both phases, while the product hopefully would
end up into the organic phase.
- Nearly nonaqueous solvents were used, with a few % water at less than the solubility limits of water.
- Finally, anhydrous organic solvents (0.01% water) were used. It is this later case that is most astonishing,
since at first glance it is hard to believe that enzymatic activity was retained.
It is important to realize that in this last case, the enzyme is not in solution. It is rather in suspension and acts as a heterogeneous
catalyst, much like palladium acts as a heterogeneous catalyst in the hydrogenation of alkenes. The suspension must be mixed
vigorously and then sonicated to produce small suspended particles, so diffusion of reactants into the enzyme and out is not
rate limiting. Let's explore the activity of chymotrypsin in a nonpolar solvent. Consider the following questions.
- Why aren't the enzymes inactive? Surely it must seem ridiculous that they aren't,
since as we learned earlier, proteins are not that stable. A 100 amino acid protein on average is stabilized only about 10
kcal/mol over the denatured state, or the equivalent of a few H bond. Surely the hydrophobic effect, one of the dominant contributors
to protein folding and stability, would not stabilize the native structure of enzymes in nonpolar organic solvents, and the
protein would denature. It doesn't however! Maybe the real question should
be not whether water is necessary, but rather how much water is necessary. The enzyme can't "see" more than a monolayer or
so of water around it. The data suggests that the nature of the organic solvent is very important. The
most hydrophobic solvents are best in terms of their ability to maintain active enzymes! Chymotrypsin
retains 104 more activity in octane than pyridine (see kcat/Km below), which is more
hydrophilic than octane. The more polar the solvent, the more it can strip bound water away from the protein. If you add 1.5%
water to acetone, the bound water increases from 1.2 to 2.4%, and the activity of chymotrypsin increases 1000 fold.
Chymotrypsin Activity in Organic Solvents
|H2O bound to enzyme (%, w/w)|
- How active are enzymes in nonpolar solvents? Enzymes are often studied in model
transesterification reactions. Typical reaction conditions are enzyme at 1 mg/ml, with one substrate, an ester such as N-acteyl-L-Phe-ethyl
ester,at 2-12 mM, and the other substrate an alcohol, such as n-propanol (instead of being water as in a typical hydrolysis
reaction) at 0.25-1.5 M. The more concentrated alcohol replaces the alcohol (ethanol) esterified in the ester. Michaelis-Menten
kinetics are followed, with biomolecular rate constants of 1010 > than without the enzyme.
- How much water do the enzymes need? 1 molecule of chymotrypsin in octane has
< 50 molecules of water associated and can demonstrate activity. To form a monolayer requires about 500 water molecules.
Water can be added which presumably leads to more bound water and higher activity.
- How stable are the enzymes? Denaturation requires conformational flexibility,
which apparently requires water. The half-life of chymotrypsin in water at 60oC is minutes, but in octane at 100oC
it is hours. At 20oC, the half-life in water is a few days, but in octane it is > 6 months. Remember two things
contribute to stability. The protein can denature at high temperatures. Also since chymotrypsin is a protease, it can cleave
itself in a autoproteolytic reaction.
Half-Life of Chymotrypsin Activity in Water and Octane
||> 6 months|
Now consider competitive inhibitors. Napthalene binds 18 times more tightly than 1-napthoic acid, but
in octane, the chymotrypsin binds napthoic acid 310 times as tightly. Likewise the ratio of [kcat/Km (L isomer)]/[kcat/Km
(D isomer)] of N-acetyl-D- or N-acetyl-L-Ala-chloroethyl esters is 1000-10,000 in water, but less than 10 in octane.
Chymotrypsin Inhibition Constants in Water and Octane
Inhibition Constant Ki (nM)
- Can new reactions be carried out in nonpolar solvents? The quick answers is yes,
since reactions in aqueous solutions can be unfavorable due to low Keq's, side reactions, or insolubility of reactants. Consider
lipases which cleave fatty acid esters by hydrolysis in aqueous solutions. In nonaqueous solutions, reactions such as transesterification
or ammonolysis can be performed.
Enzymes are clearly active in organic solvents which appears to contradict our central concepts of protein
stability. Two reasons could could explain this stability.
- It is possible that from a thermodynamic view, the enzyme is stable in organic solvents. However, as
was discussed above, this is inconceivable given the delicate balance of noncovalent
and hydrophobic interactions required for protein stability.
- The second reason must win the day: the protein is unable to unfold from a kinetic point of view. Conformational
flexibility is required for denaturation. This must require water as the solvent.
A specific example helps illustrate the effects of different solvents on chymotrypsin activity. Dry chymotrypsin
can be dissolved in DMSO, a water miscible solvent. In this solvent it is completely and irreversibly denatured. If it is
now diluted 50X with acetone with 3% water, no activity is observed. (In the final dilution, the concentrations of solvents
are 98% acetone, 2.9% water, and 2% DMSO.) However, if dry chymotrypsin was added to a mixture of 98% acetone, 2.9% water,
and 2% DMSO, the enzyme is very active. We end up with the same final solvent state, but in the first case the enzyme
has no activity while in the second case it retains activity.
Dry enzymes added to a concentrated water-miscible organic solvent (like DMSO) will dissolve and
surely denature, but will retain activity when added to a concentrated water-immiscible solvent (like octane), in which the
enzyme will not dissolve but stay in suspension.
It appears the enzymes have very restricted conformational mobility in nonpolar solvents. By lyophilizing
(freeze-drying) the enzyme against a specific ligand, a given conformation of a protein can be trapped or literally imprinted
onto the enzyme. For example, if the enzyme is dialyzed against a competitive inhibitor (which can be extracted by the organic
solvent), freeze-dried to remove water, and then added to a nonpolar solvent, the enzyme activity of the "imprinted" enzyme
in nonpolar solvents is as much as 100x as great as when no inhibitor was present during the dialysis. If chymotrypsin
is lyophilized from solutions of different pHs, the resulting curve of V/Km for ester hydrolysis in octane is bell-shaped
with the initial rise in activity reaching half-maximum activity at a pH of around 6.0 and a fall in activity reaching half-maximum
at pH of approximately 9.
Enzymes in organic solvent allow new routes to organic solvents. Enzymes, which are so useful in
synthetic reactions, are:
- stereoselective - can differentiate between enantiomers and between prochiral substrates
- regioselective - can differentiate between identical functional groups in a single substrate
- chemoselective - can differentiate between different functional groups in a substrate (such as between
a hydroxyl group and an amine for an acylation reaction)
Enzyme in anhydrous organic solvents are so useful (from a synthetic point) not only since new types of
reactions can be catalyzed (such as transesterification, ammonolysis, thiolysis) but also because the stereoselectivity, regioselectivity,
and chemoselectivity of the enzyme often changes from activities of the enzyme in water.