No intermediate channelling in stepwise hydrolysis of fluorescein di‐β‐D‐galactoside by β‐galactosidase

Abstract

In the enzymic hydrolysis of biopolymers such as polypeptides, polysaccharides or nucleic acids, two extreme patterns of action are conceivable [l]. The enzyme, after forming an enzyme-substrate complex, may begin to catalyze hydrolysis of mult.iple bonds in what has been described as zipper fashion until it comes to the end of the chain. Alternatively, the hydrolysis of only one bond per effective encounter is catalyzed, the classical random action. Between these extremes is the case of 'multiple attack', where several bonds are hydrolyzed before the enzyme-substrate complex dissociates. Examples for the hydrolysis of more than one bond per enzyme-substrate encounter seem to be relatively rare. Multiple attack has been reported in the hydrolysis of starch by a-and P-amylases [l, 21. 'Processivity' (as it is called there) is observed in enzymes processing the highly charged DNAs or RNAs and might be a feature of their polymerase function, retained e.g. in the partially processive hydrolysis by the RNase H activity of retroviral reverse transcriptase [3]. Processive hydrolysis is also found with ribonuclease I1 [4] and exonuclease I from Escherichiu coli and some further exonucleases As to cases where only a few specific bonds are to be hydrolyzed in a substrate molecule, partial processive cleavage was described in the hydrolysis of polynucleotides with two EcoRI sites by EcoRI endonuclease [6, 71. We have obtained evidence that in the release of the kallidin decapeptide from bovine kininogen by tissue kallikrein the two peptide bonds hydrolyzed may be cleaved at a single enzyme-substrate encounter [8, 91. Therefore, we were highly interested in a recent report [lo] stating that in the hydrolysis of fluorescein di-P-galactoside (FDG) by P-galactosidase intermediate channelling does occur.

Evaluation of Multidrug Efflux Pump Inhibitors by a New Method Using Microfluidic Channels

“…Fluorescein-di-β- d -galactopyranoside (FDG, Figure 1) is a fluorogenic compound that is non-fluorescent until it is hydrolyzed by β-galactosidase in the cytoplasm of Escherichia coli to produce a highly fluorescent dye, fluorescein [19], [20], [21]. We first confirmed that both FDG and fluorescein are substrates of RND pumps in E. coli .…”

Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa

“…Fluorescein-di-β- D -galactopyranoside (FDG) is hydrolyzed in the cytoplasm of E. coli by β-galacotsidase to produce fluorescein ( Russo-Marie et al, 1993 ; Fieldler and Hinz, 1994 ; Yang and Hu, 2004 ). Both FDG and fluorescein were shown to be efflux-pump substrates and could be used to develop transport assays with E. coli cells expressing the pseudomonal MexAB-OprM and MexXY-OprM efflux systems ( Matsumoto et al, 2011 ).…”

A Microfluidic Device for Simple and Rapid Evaluation of Multidrug Efflux Pump Inhibitors

“…We utilized an appropriate substrate, fluorescein-di-β- d -galactopyranoside (FDG), for a visual assay. FDG is non-fluorescent until it is hydrolysed by β-galactosidase in the cytoplasm of Escherichia coli to produce a highly fluorescent dye, fluorescein (Russo-Marie et al, 1993; Fieldler and Hinz, 1994; Yang and Hu, 2004). We confirmed that both FDG and fluorescein are substrates of RND pumps in E. coli .…”