title: Isolation and characterization of a protease inhibitor from Acacia karroo with a common combining loop and overlapping binding sites for chymotrypsin and trypsin.
abstract: By using affinity and reversed-phase HPLC (RP-HPLC) chromatographies two chymotrypsin-trypsin inhibitors were isolated from seeds of Acacia karroo, a legume of the subfamily Mimosoideae. The primary structure of one of these inhibitors, named AkCI/1, was determined. The inhibitor consists of two polypeptide chains, 139 and 44 residues respectively, which are linked by a single disulfide bridge. The amino acid sequence of AkCI/1 is homologous to and showed more than 60% sequence similarity with other protease inhibitors isolated earlier from the group of Mimosoideae. AkCI/1 inhibits both chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4) in a 1:1M ratio with Ki values of 2.8 × 10(-12)M and 1.87 × 10(-12)M, respectively. The P1-P1' residues for trypsin were identified as Arg68-Ile69 by selective hydrolysis of the inhibitor at this site, with bovine trypsin and human trypsin IV. The cleavage did not affect the inhibition of trypsin, but fully abolished the chymotrypsin inhibitory activity of AkCI/1. This finding together with our studies on competition of the two enzymes for the same combining loop suggests that the same loop has to contain the binding sites for both proteases. The most likely P1 residue of AkCI/1 for chymotrypsin is Tyr67.
authors.affiliation: Department of Biochemistry, Eötvös Loránd University, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Patthy
authors.affiliation: Department of Biochemistry, Eötvös Loránd University, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary.
authors.forename: Tamás
authors.initials: T
authors.lastname: Molnár
authors.affiliation: Department of Biochemistry, Eötvös Loránd University, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary.
authors.forename: Pálma
authors.initials: P
authors.lastname: Porrogi
authors.affiliation: Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, PO Box 77 000, Port Elizabeth 6031, South Africa.
authors.forename: Ryno
authors.initials: R
authors.lastname: Naudé
authors.affiliation: Department of Biochemistry, Eötvös Loránd University, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary; Molecular Biophysics Research Group, Hungarian Academy of Sciences and Eötvös Loránd University, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary. Electronic address: graf@elte.hu.
authors.forename: László
authors.initials: L
authors.lastname: Gráf
mesh_terms: D000045:Acacia / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D006801:Humans; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D014357:Trypsin / Q000737:chemistry*
chemical_list: D010940:Plant Proteins; D011480:Protease Inhibitors; D002918:Chymotrypsin; D014357:Trypsin
keywords: Acacia karroo; Chymotrypsin; Combining loop; Kunitz-type protease inhibitor; Trypsin