Enzymes substitution

It is quite true that we have no specificity as yet. However, I refuse to become a prisoner of enzymologists. We are not trying to make enzymes we are making catalysts. We are not aiming toward enzyme substitutes for insertion in cellular systems. There specificity is crucial, for cells would not have evolved if enzymes were nonspecific (and hence lethal in a cell). With our broad catalysts, we have choice of the process to be accelerated by selection of the substrate added. 

Shovers, J. and Bavisotto, V. S. 1967. Fermentation derived enzyme substitute for animal rennet. J. Dairy Sci. 50, 942-942. 

Harms, H. K., Bertele-Harms, R. M., and Bruer-Kleis, D. (1987), Enzyme-substitution therapy with the yeast Saccharomyces cerevisiae in congenital sucrase-isomaltase deficiency, N. Engl. J. Med., 316,1306-1309. 

A number of derivatives of this complex have been prepared (see Multi-Heme Cytochromes Enzymes). Substitution of carbonyl ligands by other donors, such as that shown in equation (15), gives rise to a wide range of complexes. 

Antigens, haptens, and antibodies radiolabeled with or are commonly used as tracers in immunoassay. These nuclides can be introduced directly into functional groups normally present in proteins and other macromolecules or into suitable derivatives that can be synthesized by a variety of chemical procedures. The most widely used iodination methods have been direct chemical or enzymic substitution of hydrogen in tyrosine or related groups using chloramine-T or lactoperoxidase, respectively. These methods are described in separate chapters in this volume. 

The chemical modeling of active sites of enzymes can help to gain deeper insight into their catalytic mechanisms and might also lead to enzyme substitutes which are reliable and easy-to-handle tools for organic synthesis. Recently, it... 

C. Erlanson-Albertsson and O. Wisln. Enzyme substitution in pancreatic disease is colipase activity sufficient Scand. J. Gastroenterol. 27 108 (1992). 

Stereochemical information is important in the analysis of most reaction mechanisms. This is true for all substitution reactions at atoms with substituents in tetrahedral array, such as saturated carbon and tetravalent phosphorus. Enzymic substitution at phosphorus in phosphoric esters and phosphoanhydrides is not an exception to the rule. There are experimental complications, however, in that all naturally occurring biological phosphates have two or more chemically equivalent oxygens, so that none has chirally substituted phosphorus. Inasmuch as an asymmetric arrangement of substituents is required for stereochemical analysis, P-chiral substrates for stereochemical studies of phosphotransferases and nucleotidyltransferases must be synthesized with sulfur or heavy isotopes of oxygen as substituents in an asymmetric array. 

Much more research will be required to address the question of the true nature of the transition states for enzymic substitution at phosphorus. The catalytic reaction pathways are now well understood with respect to the question of double-displacement and single-displacement mechanisms. Information about the nature of the transition states will require much more information about the structures of active sites, as well as many more structure-function studies on the enzymes for comparison with mechanistic information about comparable nonenzymic reactions. 

The author s research In the held of enzymic substitution at phosphorus is supported by Grant No. GM 30480 from the National Institute of General Medical Sciences. 

An interesting and potentially useful observation in the mutational work is that the substitution of leucine for Hisl43o decreased the activity to 0.2% of wild type. This significant activity allowed the deuterium kinetic isotope effect for the reaction of [ 1- H2] propane-1,2-diol to be measured. The value obtained turned out to be 2 for ° cat, about one-fifth to one-sixth of that for wild-type enzyme. Substitution of alanine gave about 1.5% activity and a deuterium kinetic isotope effect of 5-6. It appears that the hydrogen transfer is not solely rate limiting in these variants. 

For glyceraldehyde-3-phosphate dehydrogenase, glutamate dehydrogenase, and the NADP-linked oxidative decarboxylases, which have three substrates in one direction, initial rate measurements with a fixed concentration of any one of the three substrates also conform to Eq. (1). This again rules out any form of enzyme-substitution mechanism in which free product is formed before all the substrates have combined with the enzyme and indicates the involvement of a quaternary enzyme complex. The appropriate generalized form of Eq. (1) is... 

The rate equations for the several possible enzyme-substitution mechanisms all lack the last term of this equation. The kinetic coefficients in Eq. (2) can be estimated by primary, secondary, and tertiary plots 21). 

Ynenol lactones are also proposed to inactivate serine proteases irreversibly by alkylation of the active site histidine. Acylation of elastase by ynenol lactones produces an electrophilic allenone intermediate (Fig. 51) which covalently modifies and inactivates the enzyme with a partition ratio of 1.7 (Copp et al., 1987). Direct addition of the allenone carboxylic acid is without effect, demonstrating that the inactivator must be tethered in the active site to allow reaction with the enzyme. Substitution a to the lactone carbonyl is required for loss of activity, whereas the rate of inactivation is decreased by substitution at the acetylene terminus, suggesting that allene formation is slowed or that nucleophilic attack on the allene is hindered. 

Other double-stranded molecules have been examined and found to show inducing and toxic effects [119], The polyadenylic-polyuridylic acid complex (poly A U) is a much weaker inducer, and less toxic, than poly I C but it is much less thermally stable and more readily degraded by enzymes. Substitution of the phosphate groups by thiophosphate gave a more stable complex and increased its ability to stimulate interferon production [120]. 

All enzymes are proteins, which are linear sequences of amino acids linked by peptide bonds. The folding of these sequences determined the secondary structure (such as a-helix, p-sheet or p-turn) and tertiary structure. Therefore, the properties of an en me are actually presumed from its sequence of amino acids. Some amino acids, dubbed hot spots , especially the ones in the active site where substrate binds, are sensitive to catalytic properties of an enzyme. Substitution of these important amino acids can significantly improve the activity or enantioselectivity toward a certain reaction. Protein stability is also maintained by the intramolecular and intermolecular interactions between residues of amino acids, including van der Waals forces, hydrophobic forces, electrostatic forces, hydrogen bonds and disulfide bonds. Detailed analysis of these amino acids, usually located in the protein surface, sheds light on the protein design for better thermostability. 

In another recycling system we co-immobolized cytochrome bz and laccase in a gelatin layer in front of an oxygen electrode. In the presence of the cytochrome bz substrate, lactate, BQ is reduced to H2Q. Therefore, this enzyme substitutes the cathode of the previous cycling system (Figure 6). However, the substrate recyling is not restricted to the phase boundary (as with electrochemical recyling) but it proceeds in the total volume of the enzyme layer. At the optimum pH of cytochrome b2 (pH 6.5) and lactate saturation a maximum amplification of 500 has been obtained. 

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