REGULATION OF N-ARGININE DIBASIC CONVERTASE ACTIVITY BY AMINES - PUTATIVE ROLE OF A NOVEL ACIDIC DOMAIN AS AN AMINE BINDING-SITE

Peptide sequence analysis and cDNA cloning indicate that a previously described mouse arginine-specific dibasic cleaving enzyme (dynorphin c onverting enzyme) [Csuhai et al. (1995) Biochemistry 34, 12411] is the homologue of N-arginine dibasic convertase (NRDc) isolated from rat t estis [Chesneau et al. (1994) J. Biol. Chem. 269, 2056]. A mouse NRDc cDNA exhibited 98% amino acid identity with the rat cDNA. However, wit hin a 74 residue acidic stretch, this identity drops to 82%, Likewise, the corresponding acidic stretch of human NRDc is only 73% identical with that of rat NRDc. To reconcile previously observed kinetic differ ences between rat and mouse NRDc, the hydrolysis of peptide substrates by the rat, human, and mouse enzymes was compared using phosphate and Tris as buffers. Although the three NRDc's behaved similarly, Tris ha d a pronounced effect on the kinetics of peptide hydrolysis. With BAM- 8, alpha-neoendorphin, and dynorphin B as substrates, Tris increased K -M up to 40-fold with little change in V-max, while with dynorphin A o r somatostatin 28 as substrate, Tris caused a decrease in K-M of up to 100 fold, again with only a modest change in V-max. Other amines, inc luding the polyamines putrescine, spermidine, and spermine, all affect ed NRD convertase activity. It is proposed that amines bind to the aci dic stretch found in NRDc, and that quantitative differences in the se nsitivity to amines between the rat, mouse, and human enzymes can be a t least partially accounted for by differences in their acidic stretch . The role of polyamines as physiological modulators of N-arginine dib asic convertase is considered.