title: Site-directed mutagenesis of tobacco anionic peroxidase: Effect of additional aromatic amino acids on stability and activity.
abstract: Tobacco anionic peroxidase (TOP) is known to effectively catalyze luminol oxidation without enhancers, in contrast to horseradish peroxidase (HRP). To pursue structure-activity relationship studies for TOP, two amino acids have been chosen for mutation, namely Thr151, close to the heme plane, and Phe140 at the entrance to the active site pocket. Three mutant forms TOP F140Y, T151W and F140Y/T151W have been expressed in Escherichia coli, and reactivated to yield active enzymes. Single-point mutations introducing additional aromatic amino acid residues at the surface of TOP exhibit a significant effect on the enzyme catalytic activity and stability as judged by the results of steady-state and transient kinetics studies. TOP T151W is up to 4-fold more active towards a number of aromatic substrates including luminol, whereas TOP F140Y is 2-fold more stable against thermal inactivation and 8-fold more stable in the reaction course. These steady-state observations have been rationalized with the help of transient kinetic studies on the enzyme reaction with hydrogen peroxide in a single turnover regime. The stopped-flow data reveal (a) an increased stability of F140Y Compound I towards hydrogen peroxide, and thus, a higher operational stability as compared to the wild-type enzyme, and (b) a lesser leakage of oxidative equivalents from TOP T151W Compound I resulting in the increased catalytic activity. The results obtained show that TOP unique properties can be further improved for practical applications by site-directed mutagenesis.
authors.affiliation: Chemistry Faculty, Lomonosov Moscow State University, 119992 Moscow, Russia; Innovations and High Technologies MSU Ltd, 109559 Moscow, Russia. Electronic address: andrey.poloznikov@gmail.com.
authors.forename: A A
authors.initials: AA
authors.lastname: Poloznikov
authors.affiliation: Innovations and High Technologies MSU Ltd, 109559 Moscow, Russia; A.N. Bach Institute of Biochemistry, RAS, 119071 Moscow, Russia.
authors.forename: G S
authors.initials: GS
authors.lastname: Zakharova
authors.affiliation: Chemistry Faculty, Lomonosov Moscow State University, 119992 Moscow, Russia.
authors.forename: T A
authors.initials: TA
authors.lastname: Chubar
authors.affiliation: Chemistry Faculty, Lomonosov Moscow State University, 119992 Moscow, Russia; Innovations and High Technologies MSU Ltd, 109559 Moscow, Russia.
authors.forename: D M
authors.initials: DM
authors.lastname: Hushpulian
authors.affiliation: Chemistry Faculty, Lomonosov Moscow State University, 119992 Moscow, Russia; Innovations and High Technologies MSU Ltd, 109559 Moscow, Russia; A.N. Bach Institute of Biochemistry, RAS, 119071 Moscow, Russia.
authors.forename: V I
authors.initials: VI
authors.lastname: Tishkov
authors.affiliation: Chemistry Faculty, Lomonosov Moscow State University, 119992 Moscow, Russia; Burke Medical Research Institute, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, 785 Mamaroneck Ave, White Plains, NY 10605, USA.
authors.forename: I G
authors.initials: IG
authors.lastname: Gazaryan
mesh_terms: D024322:Amino Acids, Aromatic; D052160:Benzothiazoles / Q000378:metabolism; D055162:Biocatalysis; D004795:Enzyme Stability; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D058849:Protein Refolding; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000378:metabolism; D013696:Temperature
chemical_list: D024322:Amino Acids, Aromatic; D052160:Benzothiazoles; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D006861:Hydrogen Peroxide; D010544:Peroxidases; C085547:tobacco anionic peroxidase
keywords: Catalytic activity; Chemiluminescence; Recombinant tobacco peroxidase; Refolding; Site-directed mutagenesis