Enzymes conformational studies

In one case, a small peptide with enzyme-like capability has been claimed. On the basis of model building and conformation studies, the peptide Glu-Phe-Ala-Ala-Glu-Glu-Phe-Ala-Ser-Phe was synthesized in the hope that the carboxyl groups in the center of the model would act like the carboxyl groups in lysozyme 17). The kinetic data in this article come from assays of cell wall lysis of M. lysodeikticus, chitin hydrolysis, and dextran hydrolysis. All of these assays are turbidimetric. Although details of the assay procedures were not given, the final equilibrium positions are apparently different for the reaction catalyzed by lysozyme and the reaction catalyzed by the decapeptide. Similar peptide models for proteases were made on the basis of empirical rules for predicting polypeptide conformations. These materials had no amidase activity and esterase activity only slightly better than that of histidine 59, 60). 

In order to be exploitable for extraction and purification of proteins/enzymes, RMs should exhibit two characteristic features. First, they should be capable of solubilizing proteins selectively. This protein uptake is referred to as forward extraction. Second, they should be able to release these proteins into aqueous phase so that a quantitative recovery of the purified protein can be obtained, which is referred to as back extraction. A schematic representation of protein solubilization in RMs from aqueous phase is shown in Fig. 2. In a number of recent publications, extraction and purification of proteins (both forward and back extraction) has been demonstrated using various reverse micellar systems [44,46-48]. In Table 2, exclusively various enzymes/proteins that are extracted using RMs as well as the stability and conformational studies of various enzymes in RMs are summarized. The studies revealed that the extraction process is generally controlled by various factors such as concentration and type of surfactant, pH and ionic strength of the aqueous phase, concentration and type of CO-surfactants, salts, charge of the protein, temperature, water content, size and shape of reverse micelles, etc. By manipulating these parameters selective sepa-... 

Only the quantities of water introduce in both solvents are not similar, because the water is necessary for the active enzyme conformation and not for the reaction. The water effect was studied elsewhere [13,14],... 

Traditional steady-state kinetic studies rely on indirect observation of catalysis by monitoring the accumulation of product or consumption of substrate as a consequence of many reaction cycles with a trace of catalyst. Conclusions are limited to inference of the possible pathways for the order of addition of multiple substrates and release of products and quantification of two bulk kinetic parameters, kcat and kcaJKm- The parameter kcat defines the maximum rate of conversion of enzyme-bound substrate to product released into solution, but it cannot be used to establish whether the maximum rate of reaction is limited by enzyme conformational changes, rates of chemical reaction, or rates of product release per se it does, however, set a lower... 

Our understanding of the principles underlying the catalytic activity of enzymes has increased greatly in recent years. Enzymes are proteins and we know now that the polypeptide chain of a globular protein molecule will assume spontaneously a well-defined conformation (3). This native conformation of the macromolecule is essential to the function of the enzyme. Recent studies show that enzyme crystals, into which substrate molecules may penetrate by diffusion, have similar catalytic characteristics as enzyme solutions (4) and this result tends to strengthen our belief that the conformation of the enzyme molecule, as deduced from X-ray diffraction studies of enzyme crystals, is identical, or at least very similar, to the conformation responsible for the catalytic activity under physiological conditions. The structures of some enzymes, e.g., lysozyme (5), ribonuclease (6), and carboxypeptidase A (7), have been determined they are all consistent with the general belief that... 

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