the development of experimental techniques that allow the Catalysis, stability, and thermostability of yeast hexokinase were study of water-protein interactions (4-6). An experimental determined in the microenvironments of two organic solvent/Triapproach that allows the study of water-protein interactions ton X-100/phospholipids systems. In the abscence of enzyme, with different, but low, amounts of water, is to entrap prophase diagrams showed two transparent/turbid transitions, and teins in the interior of reverse micelles (7-10). In these reverse micelles were only observed in the second region of transsystems it is possible to vary the size of the micelle and, parency (T2), where particle size as a function of water content hence, the amount of water in contact with the protein. The shows a minima (see previous paper in this issue). In the present catalytic activity of enzymes entrapped in reverse micelles work, enzyme activity was detected throughout the four regions generally increases as the amount of water is increased (7, of the phase diagrams of these systems. Catalysis increased with water content; nevertheless, the maximum activities that were 8, 11). The increase in activity, observed with increasing reached in the toluene and propylbenzene systems were 30 and amounts of water has been ascribed to a higher flexibility 1.6%, respectively, of the activity in all aqueous media. Because of the protein molecule, and/or shifts in the properties of in the T2 region in the propylbenzene system, micelles are much intramicellar water toward those of bulk water (8).
smaller than in toluene (see preceding paper), it would appear
It has also been reported that at low levels of water, enthat expression of catalysis depends on the size of the micelles. zymes dispersed in organic solvents (12), or entrapped in However, a comparison of the dimensions of hexokinase and those reverse micelles formed with phospholipids (with or without of reverse micelles in the T2 region, suggests that in this region, Triton X-100) exhibit a high thermostability (13, 14). In hexokinase entrapment increases the inner volume of the micelle. the latter conditions enzymes may carry out catalysis at tem-High enzyme thermostability was only observed in the first transperatures that in all water media cause rapid denaturation parent region (T1) of the system that contained phospholipids. In this region, hexokinase induced the formation of reverse micelles . Thus it has been proposed that in this low water envifrom dispersed surfactant monomers. There is a striking similarity ronment, enzymes are relatively rigid and that this accounts in the dimensions of hexokinase entrapped in reverse micelles as for their low catalytic rates and high thermostability (8). To determined by dynamic light scattering measurements in the T1 understand the systems in which these observations have region with those of hexokinase as obtained from X ray diffraction been made, in the preceding paper, we characterized by studies of the enzyme in a crystalline environment. This suggest phase boundary titrations and dynamic light scattering, the that high thermostability, and low catalytic rates result from resystems formed by an aqueous solution, Triton X-100, and strictions in mobility imposed by a low water environment. ᭧ 1998 phospholipids in toluene or propylbenzene. Four regions