BIOCHEMISTRY - DR. JAKUBOWSKI
Learning Goals/Objectives for Chapter 7D: After class and this reading, students will be able to
In the previous chapter, 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.
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.
Chymotrypsin Activity in Organic Solvents
|H2O bound to enzyme (%, w/w)
Half-Life of Chymotrypsin Activity in Water and Octane
|> 6 months
Chymotrypsin Specificity Changes in
Water and Octane
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)
Enzymes are clearly active in organic solvents which appears to contradict our central concepts of protein stability. Two reasons could could explain this stability.
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.
Use of enzymes in organic solvent allows new routes to organic synthesis. Enzymes, which are so useful in synthetic reactions, are:
Enzyme in anhydrous organic solvents are 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.
Organic Reactions in Water?
Organic reactions are usually conducted in organic solvents, since many organic molecules react with water, and the reagents and products are usually not soluble in water. In a manner analogous to using an enzyme as a heterogeneous catalyst in nonpolar solvent, Sharpless is pioneering a technique to conduct organic reactions in water. They (Narayan et al.) have shown that many unimolecular and bimolecular reactions occur faster in water than in organic solvents. As in enzyme catalysis in nonpolar solvent, the reactions must be mixed vigorously to disperse reactants in micro-drops (a suspension) in water, greatly increasing the surface area that might allow water to act on transition states or intermediates to stabilize them through hydrogen bonding. They called these reactions "on water" reactions since reactants usually float on water. They have performed cycloadditions, alkene reactions, Claisen rearrangments, and nucleophilic substitution reactions using this process. One cycloaddition reaction went to completion in ten minutes at room temperature, compared to 18 hours in methanol and 120 in toluene. Adding nonpolar solvent at certain times greatly increased the rate of the reaction.
Klijn, J and Engberts, J. Organic chemistry: Fast reactions 'on water'. Nature 435, 746-747 (9 June 2005) | doi:10.1038/435746a
Narayan, S. et al. Angew. Chem. Intl. Edn 44, 3275 (2005)