Advances in Enzymology and Related Areas of Molecular by Eric J. Toone

By Eric J. Toone

This publication covers important advances in enzymology, explaining the habit of enzymes and the way they are often applied to advance novel medicinal drugs, synthesize recognized and novel compounds, and comprehend evolutionary methods.

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Extra resources for Advances in Enzymology and Related Areas of Molecular Biology (Volume 77)

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The gray object denotes AcrD, with its periplasmic domain shown as ellipses. The black rectangle denotes AcrA, and the substrate (streptomycin, 70 mM) is shown as “S”. Curve 1, AcrD proteoliposomes containing Mg2þ and AcrA inside, but with no streptomycin. Curve 2, the same proteoliposome as in curve 1, to which streptomycin was added to the external medium. Curve 3, AcrD-proteoliposomes made on the same day that contained Mg2þ , AcrA, and streptomycin in the intravesicular space. ] was added to the more acidic intravesicular space, corresponding to the periplasm (curve 3).

Point mutations can also be introduced into the transmembrane domain by site-directed mutagenesis. In 1999, inspection of amino acid sequence of an RND-family toxic cation efflux pump CzcA of Ralstonia sp. and other RND pumps showed that there are several conserved charged residues in STRUCTURE AND MECHANISM OF RND-TYPE MULTIDRUG EFFLUX PUMPS 23 the transmembrane domain, including Asp402, Asp408, and Glu415 (84). Changing these residues into nonacidic residues abolished the cation pumping activity.

5 mM for rhodamine 6G to 74 mM for ciprofloxacin. However, the method does not give reliable information on binding stoichiometry, and because the authors did not use, as controls, mutant AcrB proteins that are altered in substrate binding, it is unclear whether the binding occurred to the true binding sites or to some extraneous pockets that bind lipophilic molecules in a nonspecific manner. The efflux of fluorescent dyes from cells after their energization was followed in several studies. Typically, cells are preloaded with dyes that become fluorescent only within the membrane or the cytosol.

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