Kinetic analysis of spermine binding to NRD convertase

N-arginine dibasic convertase cleaves polypeptides between paired basic res idues containing the sequence Arg-Arg or Arg-Lys. The enzyme contains a lar ge anionic domain, which in the rat enzyme consists of 57 acidic residues o ut of a stretch of 76 amino acids. Polyamines modulate the activity of the enzyme presumably by binding at the anionic domain (Csuhai ct al. (1995) Bi ochemistry 34, 12411-12419). In this study a kinetic analysis of the effect of salts and amines, particularly the polyamine spermine, on the rat enzym e was studied, Simple salts were inhibitory with no apparent specificity fo r the anion or cation. Inhibition resulted in an increased K-m and a decrea sed V-max. Evidence that amines bind to an anionic domain was obtained by t he finding that N,N-bis [2-hydroxyethyl]-2-aminoethanesulfonic acid, which is structurally related to the inhibitory amine triethanolamine, is noninhi bitory. Inhibition exhibited a complex dependence on spermine concentration . The data fit a model in which enzyme-spermine and enzyme-(spermine), comp lexes are formed. A pH-independent K-d (similar to 0.1 mu M) was obtained f or enzyme-spermine formation, while enzyme-(spermine), formation was depend ent on pH; K-d at pH 6.5 = 1 mu M and a K-d at pH 8 = similar to 16 mu M. D irect binding of spermine was demonstrated by the ability of spermine to in crease the thermal stability of the enzyme. The concentration dependence fo r the spermine-induced increase in thermal stability fits a model in which formation of the enzyme-spermine complex is sufficient to account for the o bserved changes. (C) 1999 Academic Press.