EVIDENCE FOR AN ESSENTIAL HISTIDINE RESIDUE LOCATED IN THE BINDING-SITE OF THE CYSTEINE-SPECIFIC LYSOSOMAL TRANSPORT PROTEIN

Previously, we observed that the activity of the cysteine-specific lys osomal transport system increases 7-10-fold between pH 6 and 7.3 to be maximally active in the neutral pH range. To understand what factors contribute to this pH dependence, different chemical modifying agents were used to probe the nature of amino acid residues residing in the t ransport protein binding site. Diethyl pyrocarbonate (1 mM) and N-ethy lmaleimide (5 mM) each strongly inactivated lysosomal cysteine uptake greater than or equal to 88%, whereas dicyclohexylcarbodiimide (2.5 mM ), phenylisothiocyanate (2 mM), N-acetylimidazole (33 mM), and phenylg lyoxal (2 mM) had a moderate to small effect. Maximal inactivation by DEPC occurs within 12-15 min upon exposure to DEPC concentrations 2 gr eater than or equal to mM. DEPC inactivation is consistent with modifi cation of a histidine residue, displaying no inactivation at pH < 6, h alf-maximal inactivation at pH 6.6, and maximal inactivation at pH gre ater than or equal to 7.3. The close correspondence of DEPC inactivati on to the pH activity curve of cysteine uptake suggests the large incr ease in lysosomal cysteine transport activity between pH 6 and 7.3 ref lects deprotonation of an essential histidine residue. The substrate, L-cysteine (4 mM), fully protects the transport protein from DEPC inac tivation suggesting that this histidine residue is located in the carr ier's substrate binding site. Finally, part of the pH dependence of th e lysosomal cysteine carrier appears to be due to responsiveness to th e lysosomal transmembrane proton gradient as indicated by lysosomal me mbrane vesicles which display a 1.5-fold greater rate of cysteine upta ke when pH 7.4(out) > pH 5.3(in) than when pH 7.4(out) = pH 7.4(in).