Structure and activity of trypsin in reverse micelles

The kinetic properties of trypsin have been studied in reverse micelles formed by two surfactant systems, namely bis(2‐ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, and hexadecyltrimethyl ammonium bromide (CTAB) in chloroform/isooctane (1:1, by vol.). Three substrates have been used, namely N α ‐benzoyl‐ l ‐Arg ethyl ester, N α ‐benzoyl‐ l ‐Phe‐ l ‐Val‐ l ‐Arg p ‐nitroanilide (BzPheValArg‐NH‐Np) in AOT and N α ‐benzyloxycarbonyl‐ l ‐Lys p ‐nitrophenyl ester (ZLysO‐Np) in CTAB. One of the main aims of the work was to compare the behaviour of trypsin in reverse micelles with that of α‐chymotrypsin, for which an enhancement of k cat had been observed with respect to aqueous solutions. The pH profile is not significantly altered in reverse micelles with respect to water, however the kinetic parameters ( k cat and K m ) differ widely from one another, and are markedly affected by the micellar conditions, in particular by the water content w o , ( w o = [H 2 O]/[AOT]). Whereas in the case of BzPheValArg‐NH‐Np k cat is much smaller than in water, in the case of ZLysO‐Np at pH 3.2 (but not at pH 6.0) a slight enhancement with respect to water is observed. On the basis of rapid kinetic spectrophotometry (stopped‐flow) and solvent isotope effect studies, this enhancement is ascribed to a change in the rate‐limiting step (acylation rather than hydrolysis). As in the case of α‐chymotrypsin, the maximal found for all substrates at rather small w o values (below 12), which is taken to suggest that the enzyme works better when is surrounded by only a few layers of tightly bound water. Spectroscopic studies [ultraviolet absorption, circular dichroism (CD) and fluorescence] have been carried out as a function of w o . Whereas the absorption properties are practically unchanged, the CD spectrum in AOT micelles has a lower intensity than in water, which is interpreted as a partial unfolding. The intensity is partly restored when Ca 2+ ions are added, indicating that the micellar environment may cause a partial denaturation by depleting it of calcium ions. Fluorescence data show that the emission properties of the protein in reverse micelles match those in aqueous solution at around w o = 13 approx., whereas λ max shifts towards the red by increasing w o , indicating an exposure of the tryptophan residues and probably an unfolding of the whole protein, at w o values above 15. Finally the reaction between trypsin and its specific macromolecular Kunitz inhibitor from soybeans is studied. The stoichiometry of inhibition and the general inhibition behaviour is as in water, suggesting that exchange and recognition of macromolecules in reverse micelles are as fast as in the case of low‐molecular‐mass inserted molecules.