Kinetic analysis of spermine binding to NRD convertase.
N-arginine dibasic convertase cleaves polypeptides between paired basic residues containing the sequence Arg-Arg or Arg-Lys. The enzyme contains a large anionic domain, which in the rat enzyme consists of 57 acidic residues out of a stretch of 76 amino acids. Polyamines modulate the activity of the enzyme presumably by binding at the anionic domain (Csuhai et al. (1995) Biochemistry 34, 12411-12419). In this study a kinetic analysis of the effect of salts and amines, particularly the polyamine spermine, on the rat enzyme was studied. Simple salts were inhibitory with no apparent specificity for the anion or cation. Inhibition resulted in an increased Km and a decreased Vmax. Evidence that amines bind to an anionic domain was obtained by the finding that N,N-bis [2-hydroxyethyl]-2-aminoethanesulfonic acid, which is structurally related to the inhibitory amine triethanolamine, is noninhibitory. Inhibition exhibited a complex dependence on spermine concentration. The data fit a model in which enzyme-spermine and enzyme-(spermine)2 complexes are formed. A pH-independent Kd ( approximately 0.1 microM) was obtained for enzyme-spermine formation, while enzyme-(spermine)2 formation was dependent on pH; Kd at pH 6.5 = 1 microM and a Kd at pH 8 = approximately 16 microM. Direct binding of spermine was demonstrated by the ability of spermine to increase the thermal stability of the enzyme. The concentration dependence for the spermine-induced increase in thermal stability fits a model in which formation of the enzyme-spermine complex is sufficient to account for the observed changes.
Csuhai
E
E
Department of Biochemistry, University of Kentucky, Lexington, USA.
Juliano
M A
MA
Juliano
L
L
Hersh
L B
LB
D017738:Alkanesulfonic Acids / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D004795:Enzyme Stability / Q000187:drug effects; D004983:Ethanolamines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008297:Male; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011489:Protein Denaturation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D051381:Rats; D012492:Salts / Q000494:pharmacology; D013096:Spermine / Q000378:metabolism* / Q000494:pharmacology; D013696:Temperature