Exploration of subsite binding specificity of human cathepsin D through kinetics and rule‐based molecular modeling

Paula E. Scarborough, Ben M. Dunn, Kunchur Guruprasad, Chris Topham, Tom L. Blundell, Gary R. Richo, Gregory E. Conner

Research output: Contribution to journalArticle

97 Scopus citations

Abstract

The family of aspartic proteinases includes several human enzymes that may play roles in both physiological and pathophysiological processes. The human lysosomal aspartic proteinase cathepsin D is thought to function in the normal degradation of intracellular and endocytosed proteins but has also emerged as a prognostic indicator of breast tumor invasiveness. Presented here are results from a continuing effort to elucidate the factors that contribute to specificity of ligand binding at individual subsites within the cathepsin D active site. The synthetic peptide Lys-Pro-Ile-Glu-Phe*Nph-Arg- Leu has proven to be an excellent chromogenic substrate for cathepsin D yielding a value of k(cat)/K(m) = 0.92 x 10-6 s-1 M-1 for enzyme isolated from human placenta. In contrast, the peptide Lys-Pro-Ala-Lys- Phe*Nph-Arg-Leu and all derivatives with Ala-Lys in the P3-P2 positions are either not cleaved at all or cleaved with extremely poor efficiency. To explore the binding requirements of the S3 and S2 subsites of cathepsin D, a series of synthetic peptides was prepared with systematic replacements at the P2 position fixing either Ile or Ala in P3. Kinetic parameters were determined using both human placenta cathepsin D and recombinant human fibroblast cathepsin D expressed in Escherichia coli. A rule-based structural model of human cathepsin D, constructed on the basis of known three- dimensional structures of other aspartic proteinases, was utilized in an effort to rationalize the observed substrate selectivity.

Original languageEnglish (US)
Pages (from-to)264-276
Number of pages13
JournalProtein Science
Volume2
Issue number2
DOIs
StatePublished - Feb 1993

Keywords

  • aspartic proteinase
  • cathepsin D
  • chromogenic assay
  • kinetics
  • molecular modeling
  • substrate specificity
  • synthetic substrate

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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