Molecular enzymology of mammalian Delta(1)-pyrroline-5-carboxylate synthase - Alternative splice donor utilization generates isoforms with different sensitivity to ornithine inhibition

Delta(1)-Pyrroline-5-carboxylate synthase (P5CS; EC not assigned), a mitoch ondrial inner membrane, ATP- and NADPH-dependent, bifunctional enzyme, cata lyzes the reduction of glutamate to Delta(1)-pyrroline-5-carboxylate, a cri tical step in the de novo biosynthesis of proline and ornithine. We utilize d published plant P5CS sequence to search the expressed sequence tag data b ase and cloned two full-length human P5CS cDNAs differing in length by 6 ba se 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 tw o additional amino acids in the region predicted to be the gamma-glutamyl k inase active site of P5CS, The long form predominates in all tissues examin ed except gut. We also isolated the corresponding long and short murine P5C S transcripts. To confirm the identity of the putative P5CS cDNAs, we expre ssed both human forms in gamma-glutamyl kinase- and gamma-glutamyl phosphat e reductase-deficient strains of Saccharomyces cerevisiae and showed that t hey conferred the proline prototrophy. Additionally, we found expression of the murine putative P5CS cDNAs conferred proline prototrophy to P5CS-defic ient Chinese hamster ovary cells (CHO-K1). We utilized stable CHO-K1 cell t ransformants to compare the biochemical characteristics of the long and sho rt murine P5CS isoforms, We found that both confer P5CS activity and that t he short isoform is inhibited by L-ornithine with a K-i of similar to 0.25 mM. Surprisingly, the long isoform is insensitive to ornithine inhibition. Thus, the two amino acid insert in the long isoform abolishes feedback inhi bition of P5CS activity by L-ornithine.