Exploring the binding preferences/specificity in the active site of human cathepsin E.
Aspartic proteinases are produced in the human body by a variety of cells. Some of these proteins, examples of which are pepsin, gastricsin, and renin, are secreted and exert their effects in the extracellular spaces. Cathepsin D and cathepsin E on the other hand are intracellular enzymes. The least characterized of the human aspartic proteinases is cathepsin E. Presented here are results of studies designed to characterize the binding specificities in the active site of human cathepsin E with comparison to other mechanistically similar enzymes. A peptide series based on Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu was generated to elucidate the specificity in the individual binding pockets with systematic substitutions in the P5-P2, and P2'-P3' based on charge, hydrophobicity, and hydrogen bonding. Also, to explore the S2 binding preferences, a second series of peptides based on Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu was generated with systematic replacements in the P2 position. Kinetic parameters were determined for both sets of peptides. The results were correlated to a rule-based structural model of human cathepsin E, constructed on the known three-dimensional structures of several highly homologous aspartic proteinases; porcine pepsin, bovine chymosin, yeast proteinase A, human cathepsin D, and mouse and human renin. Important specificity-determining interactions were found in the S3 (Glu-13) and S2 (Thr-222, Gln-287, Leu-289, Ile-300) subsites.
Rao-Naik
C
C
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610, USA.
Guruprasad
K
K
Batley
B
B
Rapundalo
S
S
Hill
J
J
Blundell
T
T
Kay
J
J
Dunn
B M
BM
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry; D001665:Binding Sites; D020059:Cathepsin E; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D011485:Protein Binding; D012984:Software; D013056:Spectrophotometry, Ultraviolet; D013223:Statistics as Topic
D009842:Oligopeptides; D002403:Cathepsins; D016282:Aspartic Acid Endopeptidases; D020059:Cathepsin E