A simple alternate substrate test can help determine the aqueous or bilayer location of binding sites for hydrophobic ligands/substrates on membrane proteins.

Abstract

It is of general interest to determine the location of binding sites in membrane protein receptors and enzymes. A simple method is described that relies on the use of alternate substrates with defined hydrophobicity and decipherable trends in relative kcat/Km values. The rates at first-order conditions (low substrate concentrations) for at least two substrates are determined at a defined lipid/detergent volume fraction. Ratios of the rates (which at first-order conditions are linearly related to substrate concentration) are compared to the predicted partitioning of the substrates. Since the relative rates depend also on the kcat/Km ratios, the contribution of this parameter and its limits is discussed. When alternate substrates that fulfill reasonable criteria regarding kcat/Km values are used, the location of binding sites can be tentatively predicted. Examples are given describing evaluation of the location of the xenobiotic binding site in detoxication enzymes (microsomal glutathione transferase and cytochrome P450). Furthermore, it is argued that unspecific binding sites for hydrophobic molecules, such as those that are important in many detoxication enzymes, can only benefit from hydrophobic interactions facing the aqueous compartment of the cell. The membrane location of many drug-metabolizing enzymes thus implies that an aqueous active site located close to the membrane is advantageous, an advantage that could be realized if a concentration gradient of hydrophobic molecules extends into the aqueous phase at lipid interfaces.