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 journalArticlepeer-review

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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