TAILIEUCHUNG - Báo cáo khoa học: Processing, catalytic activity and crystal structures of kumamolisin-As with an engineered active site

Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad, Ser278-Glu78-Asp82, and a putative transition-state stabilizing residue, Asp164. In this study, the mutants D164N and E78H⁄D164N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. | ềFEBS Journal Processing catalytic activity and crystal structures of kumamolisin-As with an engineered active site Ayumi Okubo1 Mi Li2 3 Masako Ashida1 Hiroshi Oyama4 Alla Gustchina2 Kohei Oda4 Ben M. Dunn5 Alexander Wlodawer2 and Toru Nakayama1 1 Department of Biomolecular Engineering Graduate Schoolof Engineering Tohoku University Sendai Japan 2 Protein Structure Section Macromolecular Crystallography Laboratory NationalCancer Institute at Frederick MD USA 3 Basic Research Program SAIC-Frederick NationalCancer Institute at Frederick MD USA 4 Department of Applied Biology Faculty of Textile Science Kyoto Institute of Technology Japan 5 Department of Biochemistry and Molecular Biology University of Florida Gainesville FL USA Keywords active site autolysis catalytic mechanism serine proteases Correspondence T. Nakayama Department of Biomolecular Engineering Graduate School of Engineering Tohoku University 6-6-11 Aoba-yama Sendai 980-8579 Japan Fax Tel 81 22 795 7270 E-mail nakayama@ These authors contributed equally to this work Received 23 February 2006 revised 31 March 2006 accepted 10 April 2006 doi Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad Ser278-Glu78-Asp82 and a putative transition-state stabilizing residue Asp164. In this study the mutants D164N and E78H D164N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. Unlike the wild-type and D164N proenzymes which undergo instantaneous processing to produce their 37-kDa mature forms the expressed E78H D164N proenzyme exists as an equilibrated mixture of the nicked and intact forms of the precursor. X-ray crystallographic structures of the mature forms of the two mutants showed that in each of them the catalytic Ser278 makes direct hydrogen bonds with the side chain of Asn164. In .

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