Enzyme testing considerations

So we had these latter enzymes under consideration as well. Rune Stjernholm and I made crude extracts of the propionic acid bacteria and using the proper substrates we looked for C02 conversion to the expected compounds. As a test of COa fixation we simply bubbled C02 through the incubation mixture after making it acid and then determined if there was radioactivity remaining in the mixture. We soon found with P-enolpyruvate... 

Aleshin and coworkers (49) have reported the X-ray crystal structure at 2.2-A resolution of a G2-type variant produced by Aspergillus awamori. Meanwhile, an attempt was made to determine the amino acid residues that participate in the substrate binding and catalysis provided by G2 of A. niger (52). The results of the chemical approach indicated that the Asp-176, Glu-179, and Glu-180 form an acidic cluster crucial to the functioning of the enzyme. This conclusion was then tested by site-specific mutagenesis of these amino acid residues, which were replaced, one at a time, with Asn, Gin, and Gin, respectively (53). The substitution at Glu-179 provided an inactive protein. The other two substitutions affected the kinetic parameters but were not of crucial importance to the maintenance of activity. The crystal structure (49) supports the conclusion that Glu-179 functions as the catalytic acid but Asp-17 6 does not appear to be a good candidate for provision of catalytic base. Thus, there still exists considerable uncertainty as to how the disaccharide is accepted into the combining site for hydrolysis. Nevertheless, the kind of scheme presented by Svensson and coworkers (52) almost surely prevails. 

As mentioned above, to apply to insects a conclusion drawn directly from tests on mammals may sometimes be misleading.3 For instance, American cockroaches have a remarkably high tolerance for acetylcholine,4 but, on the other hand, a substance showing some of the pharmacological properties of acetylcholine does accumulate in flies and cockroaches poisoned with D.D.T. Similarly, Hopf, working with locusts, was unable to demonstrate any increase in toxicity of eserine or T.E.P.P. resulting from the subsequent injection of acetylcholine. From this, Lord and Potter infer that acetylcholine may not be directly involved in the insecticidal action of organo-phosphorus compounds, either because the enzymes which hydrolyse acetylcholine are not inhibited to any considerable extent in vivo or because the functions performed by acetylcholine in mammals are performed by another substance in insects. 

With isotopes it has been possible to show that all enzyme-catalyzed reactions are stereospecific. Before the availability of isotopes, there was no way of testing this generalization. Of course there are some apparent exceptions to prove the rule. Bently has listed a considerable number (2>, Table XIII, Chapter 6). The most interesting one to me seems to be luciferase, but that is an exception that isn t an exception. Thus, the enzyme luciferase acts on its substrate luciferin (2), in the presence of ATP and O2, to oxidize the luciferin to oxyluciferin (3). The reaction consists of an initial activation of the substrate by ATP to give luciferyl adenylate, after which the oxidation takes place. When the natural enantiomer (synthesized from D-cysteine) is activated and oxidized, light is emitted. The other enantiomer is also acted on by the enzyme, and is converted to the adenylate, but oxyluciferin is not formed, and there is no bioluminescence 37,38,38a)... 

Equations 2.26 and 2.27 carmot be solved analytically except for a series of limiting cases considered by Bartlett and Pratt [147,192]. Since fine control of film thickness and organization can be achieved with LbL self-assembled enzyme polyelectrolyte multilayers, these different cases of the kinetic case-diagram for amperometric enzyme electrodes could be tested [147]. For the enzyme multilayer with entrapped mediator in the mediator-limited kinetics (enzyme-mediator reaction rate-determining step), two kinetic cases deserve consideration in this system in both cases I and II, there is no substrate dependence since the kinetics are mediator limited and the current is potential dependent, since the mediator concentration is potential dependent. Since diffusion is fast as compared to enzyme kinetics, mediator and substrate are both approximately at their bulk concentrations throughout the film in case I. The current is first order in both mediator and enzyme concentration and k, the enzyme reoxidation rate. It increases linearly with film thickness since there is no... 

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