Eric J. Toone's Advances in Enzymology and Related Areas of Molecular PDF

By Eric J. Toone

ISBN-10: 0470638354

ISBN-13: 9780470638354

This publication covers important advances in enzymology, explaining the habit of enzymes and the way they are often applied to increase novel medicines, synthesize recognized and novel compounds, and comprehend evolutionary techniques.

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

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However, there were several mutants in the transmembrane domain, and their phenotypes remain to be explained. 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).

Hints on the binding affinity of various substrates were obtained by using them as competitive inhibitors in the reconstitution assay of AcrB (67) (see below). In this assay, which depended on the export of fluorescent phospholipids by the purified AcrB protein, taurocholate, a conjugated bile salt, inhibited the reaction most strongly (presumably by competing as a substrate), the 50% inhibition occurring at 15 mM. In contrast, antibiotics were less efficient inhibitors, and cloxacillin and erythromycin caused 50% inhibition only around 100 mM.

The low turnover number was as predicted in early studies, on the ground that the tripartite pumps may have to deal only with the small number of drug molecules that have trickled through the effective outer membrane barrier (7). Interestingly, when the assay was extended to cephalosporins that were clinically useful, such as cephalothin, cefamandole, and cephaloridine, kinetics with strong positive cooperativity was observed (Figure 5B). Although it is not clear why such kinetics were not observed with nitrocefin, we note that nitrocefin has a much lower Km value than that of these compounds.

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Advances in Enzymology and Related Areas of Molecular Biology (Volume 77) by Eric J. Toone

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