Advances in Enzymology and Related Areas of Molecular Biology: Meister/Advances

## PIERRE DOUZOU 1. ## Study of Enzyme-Substrate Intermediates Hydrolytic Enzymes: Tryptic Hydrolysis of Benzoyl Arginine Ethyl Ester (BAEE) a. Horse Radish Peroxidase b. Bacterial Luciferase c. Bacterial Cytochrorne Pa, G. IV. Conclusion Acknowledgments References Rapid Mixing of Micromicellar Solutions and Related Problems 58 61 61 63 64 65 70 72 72 I. Aqueous Media Withstanding Subzero Temperatures: Unfreezable Water While fast kinetic techniques are an essential and definitely established part of enzyme studies, allowing experiments to be performed in increasingly shorter time periods, lack of information about what they mean on a molecular level is responsible for the current interest in attempting to carry out a number of reactions in more accessible time periods at lower temperatures. Enzyme studies at subzero temperatures have been reviewed in this series and elsewhere (1-4). Up to now, they have consisted mostly of the investigation of enzyme reactions that normally occur in seconds and milliseconds. These are slowed to the time scales of hours, minutes, or seconds with the stabilization and determination of many intermediates and occasionally with kinetic studies of elementary steps. In spite of an increasing number of successful cryoenzymologic studies, two main problems characterize and rule this new area, namely, (1) the presence of high concentrations of organic solvents used as "antifreeze" that might influence reaction pathways and (2) the absolute necessity of finding a way of circumventing the technical barrier caused by the difficulty of mixing two solutions of high viscosity in a time period in the millisecond range, a difficulty encountered with most mixed solvents at subzero and sometimes at normal temperatures. A series of suitable tests are used to check whether detected intermediates in aqueous-organic solvents are on the actual catalytic pathway observed under normal conditions with fast kinetic techniques, but in spite of very encouraging results, it appears that cosolvents can reversibly, but significantly, change the respective values of substrate affinity, heat of formation, and activation energies through changes in substrate partitioning between the active site and the bulk solvent, in protein conformation, PIERRE DOUZOU TABLE 11 Dielectric Constant of Water as a Function of Temperature Measured values Estimated values Temperature, 'C +20 +10 . O -10 -20 -30 -40 D 80.4 84.2 88.1 92 96 100 104 We have seen that the properties of the droplets in the metastable supercooled states indicated a tendency toward a more open packing of water molecules, possibly an increased similarity to ice. Thus the micropolarity of such droplets could be markedly different from that of bulk water in the test tube and further ANS fluorescence analysis is needed to decide whether such a difference actually exists. A last, but interesting, property of water-in-oil emulsions is that molecular oxygen can undergo unrestricted diffusion. C. SOLUTIONS OF PROTEINS ## PIERRE DOUZOU After slight photodissociation ( I lo%), the rate of formation of HbCO could be described by a second-order rate constant for the whole reaction. The value of this constant was found to be 7 X 10' M-' sec-' at -55°C. This is not far from what would be expected from Gibson's value of 6.9 x lo8 at 19°C and activation energy (10 kcal) determined in aqueous solution. We were aware that factors other than temperature (that is chemical composition, organization, and viscosity of the solvent) may play a part in determining the rates, but the agreement seemed to show that, at least for this reaction, the secondary effects were not very involved. Many of the uncertainties about the chemical and physical properties of the solvent should disappear when one considers the kinetics of the reaction at a given temperature (say -55°C) and compares the time course of formation of HbCO after slight photodissociation with that after high initial photodissociation. The first reaction is strictly monophasic. After high initial photodissociation, the kinetics are heterogeneous and can be fitted to Gibson's scheme in terms of the constants I\*, I', and k,; a good fit was obtained following Gibson's procedure with I\* = 9.75 x lo9 PIERRE DOUZOU mixed solvents. However, the water-in-oil emulsion procedure is very far from being problem-free and lacks the facility of the mixed-solvent technique. The turbidity of the emulsions precludes analytical techniques that rely on optical transparency. However, recent exploratory studies using EPR on supercooled emulsions of hemoproteins show promise (F. Franks, unpublished data), and, in principle, the same should apply to NMR-based techniques. The emulsion method cannot at present be applied to reactions that proceed rapidly at 0°C or in conjunction with proteins of membrane origin that are likely to be adsorbed at the oil-water interface. Finally light-induced processes and flash photolysis studies might be suitable in water-in-oil emulsions, given the diffusibility of gaseous ligands in silicone oils and the fact that the high turbidity is not a major obstacle when dual-wavelength spectrophotometry is available. However, we turned to new procedures to try to find better conditions for investigating enzyme-catalyzed reactions in fluid aqueous environments at subzero temperatures. Water-in-Oil Microemulsions ## A. MICELLE FORMATION IN NONAQUEOUS MEDIA: REVERSE MICELLES A microemulsion is a clear, transparent, and stable system consisting of essentially monodisperse oil-in-water or water-in-oil droplets with diameters generally in the range of 10-200 nm. Microemulsions are transparent because of their small particle size; they are spherical aggregates of oil or water dispersed in the other liquid and stabilized by an interfacial film of one or more surfactants. Micelle formation in nonaqueous solution (polar solvents), although recognized for some time, has been investigated systematically only in recent years both experimentally and theoretically. Amphiphilic surfactants, characterized by molecules that possess at the same time a hydrophilic group, which tends to be water soluble and hydrocarbon insoluble, and a lipophilic group which tends to be hydrocarbon soluble and water insoluble, tend to be miscible with both water and hydrocarbon. Suitable amphiphilic surfactants dissolved in a hydrocarbon are able to solubilize water, which, in the absence of these compounds, is Advances in Enzymology and Related Areas of Molecular Biology is a seminal series in the field of biochemistry, offering researchers access to authoritative reviews of the latest discoveries in all areas of enzymology and molecular biology. These landmark volumes date back to 1941, providing an unrivaled view of the historical development of enzymology. The series offers researchers the latest understanding of enzymes, their mechanisms, reactions and evolution, roles in complex biological process, and their application in both the laboratory and industry. Each volume in the series features contributions by leading pioneers and investigators in the field from around the world. All articles are carefully edited to ensure thoroughness, quality, and readability. With its wide range of topics and long historical pedigree, Advances in Enzymology and Related Areas of Molecular Biology can be used not only by students and researchers in molecular biology, biochemistry, and enzymology, but also by any scientist interested in the discovery of an enzyme, its properties, and its applications.