PMID- 10393098
OWN - NLM
STAT- MEDLINE
DCOM- 19990908
LR  - 20181113
IS  - 0264-6021 (Print)
IS  - 0264-6021 (Linking)
VI  - 341 ( Pt 2)
DP  - 1999 Jul 15
TI  - 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.
PG  - 395-400
AB  - Glucosylceramide synthase (GCS) catalyses the transfer of glucose from
      UDP-glucose (UDP-Glc) to ceramide to form glucosylceramide, the common precursor 
      of most higher-order glycosphingolipids. Inhibition of GCS activity has been
      proposed as a possible target of chemotherapeutic agents for a number of
      diseases, including cancer. Design of new GCS inhibitors with desirable
      pharmaceutical 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
      histidine-modifying agent diethyl pyrocarbonate (DEPC) inhibited recombinant rat 
      GCS expressed in bacteria; this inhibition was rapidly reversible by
      hydroxylamine 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 eight
      histidine residues in GCS were individually replaced by other amino acids. H193A 
      (His193-->Ala) and H193N (His193-->Asn) mutants were unaffected by 0.1 mM DEPC, a
      concentration that inhibited other histidine mutants and the wild-type enzyme by 
      at least 60%. These results indicate that His193 is the primary target of DEPC
      and is at, or near, the UDP-Glc-binding site of GCS. His193 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 significance for both an
      understanding of the GCS active site and also for the possible design of new and 
      specific inhibitors of GCS.
FAU - Wu, K
AU  - Wu K
AD  - Mayo Clinic and Foundation, Thoracic Diseases Research Unit, Department of
      Biochemistry and Molecular Biology, 200 First Street, S.W., Rochester, MN 55905, 
      USA.
FAU - Marks, D L
AU  - Marks DL
FAU - Watanabe, R
AU  - Watanabe R
FAU - Paul, P
AU  - Paul P
FAU - Rajan, N
AU  - Rajan N
FAU - Pagano, R E
AU  - Pagano RE
LA  - eng
SI  - GENBANK/AF047707
GR  - GM-22942/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - Biochem J
JT  - The Biochemical journal
JID - 2984726R
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Morpholines)
RN  - 4QD397987E (Histidine)
RN  - 73257-80-4 (RV 538)
RN  - EC 2.4.1.- (Glucosyltransferases)
RN  - EC 2.4.1.80 (ceramide glucosyltransferase)
RN  - V50K1D7P4Y (Uridine Diphosphate Glucose)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Binding Sites
MH  - Enzyme Inhibitors/chemistry/metabolism
MH  - Glucosyltransferases/*chemistry/*genetics/metabolism
MH  - Histidine
MH  - Humans
MH  - Molecular Sequence Data
MH  - Morpholines/*chemistry/metabolism
MH  - Mutagenesis, Site-Directed
MH  - Point Mutation
MH  - Rats
MH  - Sequence Alignment
MH  - Uridine Diphosphate Glucose/*chemistry/*genetics/metabolism
PMC - PMC1220372
EDAT- 1999/07/07 00:00
MHDA- 1999/07/07 00:01
CRDT- 1999/07/07 00:00
PHST- 1999/07/07 00:00 [pubmed]
PHST- 1999/07/07 00:01 [medline]
PHST- 1999/07/07 00:00 [entrez]
PST - ppublish
SO  - Biochem J. 1999 Jul 15;341 ( Pt 2):395-400.