caa a22 4500 475744640 CHVBK 20180406123509.0 cr unu---uuuuu 170329e20000201xx s 000 0 eng 10.1023/A:1007043530616 doi (NATIONALLICENCE)springer-10.1023/A:1007043530616 Some Molecular and Enzymatic Properties of a Homogeneous Preparation of Thiaminase I Purified from Carp Liver [Elektronische Daten] [Małgorzata Boś, Andrzej Kozik] A homogeneous preparation of thiaminase I (thiamine:base 2-methyl-4-aminopyrimidine-5-methenyl transferase, EC 2.5.1.2) was obtained from carp liver, for the first time from a nonbacterial source. Its molecular mass was 55 kDa by gel filtration and by SDS—PAGE regardless the presence of the reducing agent, indicating that the native enzyme consists of a single polypeptide chain. The determined sequence of 20 residues at the N-terminal of carp thiaminase I seemed to be unique. The enzyme was tested for ability to decompose a number of thiamine analogues. Even very extensive modifications of the thiazolium fragment were well tolerated, but around the pyrimidine fragment the active center seemed to exert steric restrictions against 1′ (N)- and 2′ (C)- atoms, while the 4′-amino group and untouched 6′-carbon atom were absolutely essential for the enzyme action. Numerous nucleophiles could be used by the enzyme as cosubstrates, aniline, pyridine, and 2-mercaptoethanol being the best among compounds tested. Protein chemical modification experiments indicated that histidine residues, carboxyl groups, and sulfhydryl groups may play specific roles in the thiaminase I-catalyzed reaction. Like in the bacterial enzyme, a sulfhydryl group may be a catalytically critical active-site nucleophile. The histidine residues and carboxyl groups may be essential for thiamine binding to the active site. Plenum Publishing Corporation, 2000 Thiaminase I nationallicence thiamine analogues nationallicence chemical modification nationallicence sulfhydryl groups nationallicence carp liver nationallicence Boś Małgorzata Department of Biochemistry, The Jan Zurzycki Institute of Molecular Biology, Jagiellonian University, 31-120, Kraków, Poland aut Kozik Andrzej Department of Biochemistry, The Jan Zurzycki Institute of Molecular Biology, Jagiellonian University, 31-120, Kraków, Poland aut Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/2(2000-02-01), 75-84 0277-8033 19:2<75 2000 19 10930 https://doi.org/10.1023/A:1007043530616 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1007043530616 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Boś Małgorzata Department of Biochemistry, The Jan Zurzycki Institute of Molecular Biology, Jagiellonian University, 31-120, Kraków, Poland aut NATIONALLICENCE 700 1- Kozik Andrzej Department of Biochemistry, The Jan Zurzycki Institute of Molecular Biology, Jagiellonian University, 31-120, Kraków, Poland aut NATIONALLICENCE 773 0- Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/2(2000-02-01), 75-84 0277-8033 19:2<75 2000 19 10930 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer