title: Kinetic analysis of semisynthetic peroxidase enzymes containing a covalent DNA-heme adduct as the cofactor.
abstract: The reconstitution of apo enzymes with DNA oligonucleotide-modified heme (protoporphyrin IX) cofactors has been employed as a tool to produce artificial enzymes that can be specifically immobilized at the solid surfaces. To this end, covalent heme-DNA adducts were synthesized and subsequently used in the reconstitution of apo myoglobin (aMb) and apo horseradish peroxidase (aHRP). The reconstitution produced catalytically active enzymes that contained one or two DNA oligomers coupled to the enzyme in the close proximity to the active site. Kinetic studies of these DNA-enzyme conjugates, carried out with two substrates, ABTS and Amplex Red, showed a remarkable increase in peroxidase activity of the DNA-Mb enzymes while a decrease in enzymatic activity was observed for the DNA-HRP enzymes. All DNA-enzyme conjugates were capable of specific binding to a solid support containing complementary DNA oligomers as capture probes. Kinetic analysis of the enzymes immobilized by the DNA-directed immobilization method revealed that the enzymes remained active after hybridization to the capture oligomers. The programmable binding properties enabled by DNA hybridization make such semisynthetic enzyme conjugates useful for a broad range of applications, particularly in biocatalysis, electrochemical sensing, and as building blocks for biomaterials.
authors.affiliation: Universität Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Strasse 6, 44227 Dortmund, Germany.
authors.forename: Ljiljana
authors.initials: L
authors.lastname: Fruk
authors.forename: Joachim
authors.initials: J
authors.lastname: Müller
authors.forename: Christof M
authors.initials: CM
authors.lastname: Niemeyer
mesh_terms: D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D003067:Coenzymes / Q000138:chemical synthesis / Q000378:metabolism*; D018736:DNA Adducts / Q000138:chemical synthesis / Q000378:metabolism; D006418:Heme / Q000138:chemical synthesis* / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D007700:Kinetics; D010544:Peroxidases / Q000138:chemical synthesis / Q000378:metabolism*; D015202:Protein Engineering
chemical_list: D001051:Apoenzymes; D003067:Coenzymes; D018736:DNA Adducts; D006418:Heme; D006735:Horseradish Peroxidase; D010544:Peroxidases