title: Conversion of trypsin to a copper enzyme: tyrosinase/catechol oxidase by chemical modification.
abstract: New active sites can be introduced into naturally occurring enzymes by the chemical modification of specific amino acid residues in concert with genetic techniques. Chemical strategies have had a significant impact in the field of enzyme design such as modifying the selectivity and catalytic activity which is very different from those of the corresponding native enzymes. Thus, chemical modification has been exploited for the incorporation of active site binding analogs onto protein templates and for atom replacement in order to generate new functionality such as the conversion of a hydrolase into a peroxidase. The introduction of a coordination complex into a substrate binding pocket of trypsin could probably also be extended to various enzymes of significant therapeutic and biotechnological importance. The aim of this study is the conversion of trypsin into a copper enzyme: tyrosinase by chemical modification. Tyrosinase is a biocatalyst (EC.1.14.18.1) containing two atoms of copper per active site with monooxygenase activity. The active site of trypsin (EC 3.4.21.4), a serine protease was chemically modified by copper (Cu(+2)) introduced p-aminobenzamidine (pABA- Cu(+2): guanidine containing schiff base metal chelate) which exhibits affinity for the carboxylate group in the active site as trypsin-like inhibitor. Trypsin and the resultant semisynthetic enzyme preparation was analysed by means of its trypsin and catechol oxidase/tyrosinase activity. After chemical modification, trypsin-pABA-Cu(+2) preparation lost 63% of its trypsin activity and gained tyrosinase/catechol oxidase activity. The kinetic properties (K(cat), K(m), K(cat)/K(m)), optimum pH and temperature of the trypsin-pABA-Cu(+2) complex was also investigated.
authors.affiliation: Ege University, Faculty of Science, Biochemistry Department, Bornova, Izmir, Turkey. burcu.okutucu@ege.edu.tr
authors.forename: Burcu
authors.initials: B
authors.lastname: Okutucu
authors.forename: Ali
authors.initials: A
authors.lastname: Zeytunluoglu
authors.forename: Figen
authors.initials: F
authors.lastname: Zihnioglu
mesh_terms: D000818:Animals; D001550:Benzamidines / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D004156:Catechol Oxidase / Q000378:metabolism*; D003300:Copper / Q000737:chemistry*; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001550:Benzamidines; C006500:4-aminobenzamidine; D003300:Copper; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase; D014357:Trypsin