Kinetic analysis of spermine binding to NRD convertase

Eva Csuhai, Maria Aparecida Juliano, Luiz Juliano, Louis B. Hersh

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

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 K(m) and a decreased V(max). 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 enzymespermine and enzyme- (spermine)2 complexes are formed. A pH-independent K(d) (~0.1 μM) was obtained for enzyme-spermine formation, while enzyme-(spermine)2 formation was dependent on pH; K(d) at pH 6.5 = 1 μM and a K(d) at pH 8 = ~16 μM. 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.

Original languageEnglish
Pages (from-to)291-300
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume362
Issue number2
DOIs
StatePublished - Feb 15 1999

Bibliographical note

Funding Information:
1This work was supported in Brazil by Fundac¸ão de Amparo Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This work was also supported in part by NIDA/NIH Grant DA02243. E. Cs. is a recipient of a NIDA/NIH postdoctoral fellowship DA05671.

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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