Histidine-193 of rat glucosylceramide synthase resides in a UDP-glucose- and inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol)-binding region: a biochemical and mutational study

Glucosylceramide synthase (GCS) catalyses the transfer of glucose from UDP- glucose (UDP-Glc) to ceramide to form grucosylceramide, the common precurso r of most higher-order glycosphingolipids. Inhibition of GCS activity has b een proposed as a possible target of chemotherapeutic agents for a number o f diseases, including cancer. Design of new GCS inhibitors with desirable p harmaceutical properties is hampered by lack of knowledge of the secondary structure or catalytic mechanism of the GCS protein. Thus we cloned the rat homologue of GCS to begin studies to identify its catalytic regions. The h istidine-modifying agent diethyl pyrocarbonate (DEPC) inhibited recombinant rat GCS expressed in bacteria; this inhibition was rapidly reversible by h ydroxylamine and could be diminished by preincubation of GCS with UDP-Glc. These data suggest that DEPC acts on histidine residues within or near the UDP-Glc-binding site of GCS. Mutant proteins were expressed in which the ei ght histidine residues in GCS were individually replaced by other amino aci ds. H193A (His(193) --> Ala) and H193N (His(193) --> Asn) mutants were unaf fected by 0.1 mM DEPC, a concentration that inhibited other histidine mutan ts and the wild-type enzyme by at least 60%. These results indicate that Hi s(193) is the primary target of DEPC and is at, or near, the UDP-Glc-bindin g site of GCS. His(193) mutants were also insensitive to the GCS inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol, at concentrations which inhibited the wild-type enzyme by, 80%. These results have significa nce for both an understanding of the GCS active site and also for the possi ble design of new and specific inhibitors of GCS.