Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase

Abstract

Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.

Global Kinetic Mechanism of Microsomal Glutathione Transferase 1 and Insights into Dynamic Enzyme Activation

“…MGST1 is an integral membrane enzyme facing the challenge of conjugating a hydrophilic substrate (GSH) with a hydrophobic electrophile entering the enzyme from the membrane phase (Figure 8) 15 .…”

“…MGST1 is an integral membrane enzyme facing the challenge of conjugating a hydrophilic substrate (GSH) with a hydrophobic electrophile entering the enzyme from the membrane phase (Figure 8). 15 A cleft between subunits opens to the membrane phase, allowing hydrophobic substrate access. 11 The cleft is covered by a loop that must open and close over GSH entering from the cytosol.…”

“…The MAPEG superfamily plays an important role in cellular protection and eicosanoid signaling . Members of this family use glutathione to catalyze transformations of lipophilic substrates harvested from the lipid bilayer, − with the exception of FLAP that is thought to present arachidonic acid to 5-lipoxygenase …”

Kinetic Behavior of Glutathione Transferases: Understanding Cellular Protection from Reactive Intermediates

“…Based on the stereochemistry of product formation in cells it could be demonstrated that conjugation is predominantly catalyzed by MGST1, even though the GST catalytic capacity of isolated cytosolic and membrane fractions were equal in vitro. In general, cytosolic GSTs cannot access membrane-embedded substrates [ 89 ], and act upon the fraction that equilibrates out from the membrane phase (governed by logP [ 71 ]). Membrane-bound MGST1, conversely, cannot access the cytosolic substrate fraction [ 89 ].…”

Fluorescence-based monitoring of electronic state and ion exchange kinetics with FCS and related techniques: from T-jump measurements to fluorescence fluctuations

“…Since TRAST does not rely on fluctuation measurements of single molecules, molecular brightness is not a strongly limiting issue, and low fluorescence signals can be compensated by higher concentrations of fluorescent molecules in the sample, as an alternative to increased excitation irradiances. Thereby, also relatively low brightness compounds, such as autofluorescent species (Hevekerl et al 2016 ), can be studied.…”