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 ce lls. 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 intracellula r enzymes. The least characterized of the human aspartic proteinases i s cathepsin E. Presented here are results of studies designed to chara cterize the binding specificities in the active site of human cathepsi n E with comparison to other mechanistically similar enzymes. A peptid e series based on Lys-Pro-Ala-Lys-PheNph-Arg-Leu was generated to elu cidate the specificity in the individual binding pockets with systemat ic substitutions in the P-5-P-2, and P-2'-P-3' based on charge, hydrop hobicity, and hydrogen bonding. Also, to explore the S-2 binding prefe rences, a second series of peptides based on Lys-Pro-ne-Glu-PheNph-Ar g-Leu was generated with systematic replacements in the P-2 position. Kinetic parameters were determined for both sets of peptides. The resu lts were correlated to a rule-based structural model of human cathepsi n E, constructed on the known three-dimensional structures of several highly homologous aspartic proteinases; porcine pepsin, bovine chymosi n, yeast proteinase A, human cathepsin D, and mouse and human renin. I mportant specificity-determining interactions were found in the S-3 (G lu-13) and S-2 (Thr-222, Gln-287, Leu-289, Ile-300) subsites. (C) 1995 Wiley-Liss, Inc.