Some molecular and enzymatic properties of a homogeneous preparation of thiaminase I purified from carp liver.
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.
Boś
M
M
Department of Biochemistry, The Jan Zurzycki Institute of Molecular Biology, Jagiellonian University, Kraków, Poland.
Kozik
A
A
D019883:Alkyl and Aryl Transferases / Q000032:analysis / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002347:Carps / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology*; D015394:Molecular Structure; D008970:Molecular Weight; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013831:Thiamine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism
D000596:Amino Acids; D019883:Alkyl and Aryl Transferases; C022737:thiamin pyridinylase; D013831:Thiamine