title: Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2' and P3' variations in extended substrates.
abstract: We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S(4) and S(3) subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S(2)' and S(3)'. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P(4), P(3), P(2)' and P(3)' were made. The S(4) to S(2)' subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S(3)', indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S(4), S(3), S(2)' and S(3)' of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P(2)' respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P(3)'. Basic residues at P(3) and P(4) were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P(2)' (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His(111) (H111A) and His(110) (H110A) of cathepsin B led to an increase in k(cat) values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.
authors.affiliation: Department of Pharmacology, Instituto Butantan, 05503-900, São Paulo, Brazil.
authors.forename: F C
authors.initials: FC
authors.lastname: Portaro
authors.forename: A B
authors.initials: AB
authors.lastname: Santos
authors.forename: M H
authors.initials: MH
authors.lastname: Cezari
authors.forename: M A
authors.initials: MA
authors.lastname: Juliano
authors.forename: L
authors.initials: L
authors.lastname: Juliano
authors.forename: E
authors.initials: E
authors.lastname: Carmona
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002401:Cathepsin B / Q000737:chemistry* / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry; D010206:Papain / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D009842:Oligopeptides; D011994:Recombinant Proteins; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; C534280:Ctsl protein, rat; D010206:Papain