TY - JOUR
T1 - Molecular enzymology of mammalian Δ1-pyrroline-5-carboxylate synthase. Alternative splice donor utilization generates isoforms with different sensitivity to ornithine inhibition
AU - Hu, Chien An A.
AU - Lin, Wei Wen
AU - Obie, Cassandra
AU - Valle, David
PY - 1999/3/5
Y1 - 1999/3/5
N2 - Δ1-Pyrroline-5-carboxylate synthase (P5CS; EC not assigned), a mitochondrial inner membrane, ATP- and NADPH-dependent, bifunctional enzyme, catalyzes the reduction of glutamate to Δ1-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 γ- 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 γ-glutamyl kinase, and γ-glutamyl phosphate reductase-deficient strains of Saccharomyces cerevisiae and showed that they conferred the proline prototrophy. Additionally, we found expression of the murine putative PSCS 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 K(i) of ~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.
AB - Δ1-Pyrroline-5-carboxylate synthase (P5CS; EC not assigned), a mitochondrial inner membrane, ATP- and NADPH-dependent, bifunctional enzyme, catalyzes the reduction of glutamate to Δ1-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 γ- 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 γ-glutamyl kinase, and γ-glutamyl phosphate reductase-deficient strains of Saccharomyces cerevisiae and showed that they conferred the proline prototrophy. Additionally, we found expression of the murine putative PSCS 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 K(i) of ~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.
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U2 - 10.1074/jbc.274.10.6754
DO - 10.1074/jbc.274.10.6754
M3 - Article
C2 - 10037775
AN - SCOPUS:0033525863
SN - 0021-9258
VL - 274
SP - 6754
EP - 6762
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 10
ER -