By Eric J. Toone
This booklet covers important advances in enzymology, explaining the habit of enzymes and the way they are often applied to increase novel medications, synthesize recognized and novel compounds, and comprehend evolutionary methods.
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Additional resources for Advances in Enzymology and Related Areas of Molecular Biology (Volume 77)
Since b-lactams are hydrolyzed by the periplasmic b-lactamase, and since we know the Km and Vmax values of the enzyme, we can calculate the periplasmic concentration of b-lactams, Cp, from the Michaelis–Menten equation if we determine spectrophotometrically the b-lactam hydrolysis rate by intact cells. Once the periplasmic concentration Cp is known, the rate at which the b-lactams STRUCTURE AND MECHANISM OF RND-TYPE MULTIDRUG EFFLUX PUMPS 17 cross the outer membrane by simple diffusion through porin channels (Vin) can be calculated from the permeability coefficient of the OM, because it is proportional to the concentration difference of the b-lactam between the external medium and the periplasm.
Indeed, Phe386Ala mutation hardly affected susceptibility of E. coli to most drugs when tested with expression by the vector pSPORT1 (88). Nevertheless, Hearn and coworkers (89), using a Pseudomonas fluorescens homolog of AcrB, EmhB, expressed from a medium copy number plasmid, found that decreased efflux of dequalinium was produced by the same change (Phe386Ala) as well as the change of Asn99 to alanine. Furthermore, changing Asp101, which is located at the “ceiling” of the central cavity, into alanine decreased the efflux of most drugs as well as of polycyclic hydrocarbons.
A giant stride in this area was recently made by the Koronakis group (53). They have refined the data obtained previously for the MexA crystal (47) and succeeded in obtaining the structure of the hitherto missing domain containing the N-terminus and the large C-terminal segment. This is now known to form the fourth domain or membrane-proximal domain of the adaptor, containing an a/b structure with a characteristic concave surface (Figure 3). By using this MexA structure as a template, they built the complete structure of AcrA.