caa a22 4500 397558066 CHVBK 20180308164808.0 cr unu---uuuuu 161202e199610 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021489 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021489 Substrate Specificities of Pepstatin-Insensitive Carboxyl Proteinases from Gram-Negative Bacteria [Elektronische Daten] [Masaaki Ito, Ben M. Dunn, Kohei Oda] Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, and Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, are the first and second examples of unique carboxyl proteinasea [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. The substrate specificities of PCP and XCP were studied using a series of synthetic chromogenic peptide substrates with the general structure, P5-P4-P3-P2-Phe-Nph-P2′-P3′ (P5, P4, P3, P2, P2′, P3′: a variety of amino acids, Nph is p-nitro-L-phenylalanine, and the Phe-Nph bond is cleaved). PCP and XCP were shown to hydrolyze a synthetic substrate, Lys-Pro-Ala-Leu-Phe-Nph-Arg-Leu, most effectively among 28 substrates. The kinetic parameters of this peptide for PCP were Km = 6.3 μM, kcat=51.4 s−1, and kcat/Km=8.16 μM−1·s−1. The kinetic parameters for XCP were Km=3.6 μM, kcat=52.2 s−1, and kcat 7equals;14.5 μM−1·s−1. PCP showed a stricter substrate specificity than XCP. That is, the specificity constant (kcat/Km) of each substrate for PCP was in general < 0.5 μM−1·s−1, but was drastically improved by the replacement of Lys by Leu at the P2 position. On the other hand, XCP showed a less stringent substrate specificity, with most of the peptides exhibiting reasonable kcat/Km values (> 1.0 μM−1·s−1). Thus it was found that the substrate specificities of PCP and XCP differ considerably, in spite of the high similarity in their primary structures. In addition, tyrostatin was found to be a competitive inhibitor for XCP, with a K1 value of 2.1 nM, as well as for PCP (K1=2.6nM). © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence carboxyl proteinase nationallicence Gram-negative bacteria nationallicence kinetic property nationallicence pepstatin nationallicence substrate specificity nationallicence Ito Masaaki Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology Sakyo-ku, Kyoto 606 aut Dunn Ben M. Department of Biochemistry and Molecular Biology, University of Florida Gainesville, FL 32610, USA aut Oda Kohei Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology Sakyo-ku, Kyoto 606 aut The Journal of Biochemistry Oxford University Press 120/4(1996-10), 845-850 0021-924X 120:4<845 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021489 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021489 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ito Masaaki Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology Sakyo-ku, Kyoto 606 aut NATIONALLICENCE 700 1- Dunn Ben M. Department of Biochemistry and Molecular Biology, University of Florida Gainesville, FL 32610, USA aut NATIONALLICENCE 700 1- Oda Kohei Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology Sakyo-ku, Kyoto 606 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/4(1996-10), 845-850 0021-924X 120:4<845 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford