Exploration of subsite binding specificity of human cathepsin D through kinetics and rule-based molecular modeling.
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 kcat/Km = 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.
Scarborough
P E
PE
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610.
Guruprasad
K
K
Topham
C
C
Richo
G R
GR
Conner
G E
GE
Blundell
T L
TL
Dunn
B M
BM
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D002402:Cathepsin D / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002863:Chromogenic Compounds / Q000737:chemistry / Q000378:metabolism*; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D011480:Protease Inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002863:Chromogenic Compounds; D009842:Oligopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D