PMID- 10037775
OWN - NLM
STAT- MEDLINE
DCOM- 19990330
LR  - 20190508
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 274
IP  - 10
DP  - 1999 Mar 5
TI  - Molecular enzymology of mammalian Delta1-pyrroline-5-carboxylate synthase.
      Alternative splice donor utilization generates isoforms with different
      sensitivity to ornithine inhibition.
PG  - 6754-62
AB  - Delta1-Pyrroline-5-carboxylate synthase (P5CS; EC not assigned), a mitochondrial 
      inner membrane, ATP- and NADPH-dependent, bifunctional enzyme, catalyzes the
      reduction of glutamate to Delta1-pyrroline-5-carboxylate, a critical step in the 
      de novo biosynthesis of proline and ornithine. We utilized published plant P5CS
      sequence to search the expressed sequence tag data base and cloned two
      full-length human P5CS cDNAs differing in length by 6 base pairs (bp) in the open
      reading frame. The short cDNA has a 2379-bp open reading frame encoding a protein
      of 793 residues; the long cDNA, generated by "exon sliding," a form of
      alternative splicing, contains an additional 6-bp insert following bp +711 of the
      short form resulting in inclusion of two additional amino acids in the region
      predicted to be the gamma-glutamyl kinase active site of P5CS. The long form
      predominates in all tissues examined except gut. We also isolated the
      corresponding long and short murine P5CS transcripts. To confirm the identity of 
      the putative P5CS cDNAs, we expressed both human forms in gamma-glutamyl kinase- 
      and gamma-glutamyl phosphate reductase-deficient strains of Saccharomyces
      cerevisiae and showed that they conferred the proline prototrophy. Additionally, 
      we found expression of the murine putative P5CS cDNAs conferred proline
      prototrophy to P5CS-deficient Chinese hamster ovary cells (CHO-K1). We utilized
      stable CHO-K1 cell transformants to compare the biochemical characteristics of
      the long and short murine P5CS isoforms. We found that both confer P5CS activity 
      and that the short isoform is inhibited by L-ornithine with a Ki of approximately
      0.25 mM. Surprisingly, the long isoform is insensitive to ornithine inhibition.
      Thus, the two amino acid insert in the long isoform abolishes feedback inhibition
      of P5CS activity by L-ornithine.
FAU - Hu, C A
AU  - Hu CA
AD  - Howard Hughes Medical Institute, Department of Pediatrics and Institute of
      Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore,
      Maryland 21205, USA.
FAU - Lin, W W
AU  - Lin WW
FAU - Obie, C
AU  - Obie C
FAU - Valle, D
AU  - Valle D
LA  - eng
SI  - GENBANK/AF056573
SI  - GENBANK/AF056574
SI  - GENBANK/U68758
SI  - GENBANK/U76542
GR  - 5RO1EY02948/EY/NEI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (DNA, Complementary)
RN  - 0 (Isoenzymes)
RN  - E524N2IXA3 (Ornithine)
RN  - EC 1.2.1.88 (1-Pyrroline-5-Carboxylate Dehydrogenase)
RN  - EC 1.2.1.88 (ALDH4A1 protein, human)
RN  - EC 1.5.- (Oxidoreductases Acting on CH-NH Group Donors)
SB  - IM
MH  - 1-Pyrroline-5-Carboxylate Dehydrogenase
MH  - Alternative Splicing
MH  - Amino Acid Sequence
MH  - Animals
MH  - CHO Cells
MH  - Cricetinae
MH  - DNA, Complementary/genetics
MH  - *Gene Expression Regulation, Enzymologic
MH  - Humans
MH  - Isoenzymes/genetics/metabolism
MH  - Mice
MH  - Molecular Sequence Data
MH  - Organ Specificity
MH  - Ornithine/antagonists & inhibitors/metabolism
MH  - Oxidoreductases Acting on CH-NH Group Donors/*genetics/metabolism
MH  - Sequence Alignment
MH  - Substrate Specificity
EDAT- 1999/02/26 00:00
MHDA- 1999/02/26 00:01
CRDT- 1999/02/26 00:00
PHST- 1999/02/26 00:00 [pubmed]
PHST- 1999/02/26 00:01 [medline]
PHST- 1999/02/26 00:00 [entrez]
AID - 10.1074/jbc.274.10.6754 [doi]
PST - ppublish
SO  - J Biol Chem. 1999 Mar 5;274(10):6754-62. doi: 10.1074/jbc.274.10.6754.