Kinetic studies of site-directed mutational isomalto-dextranase-catalyzed hydrolytic reactions on a 27 MHz quartz-crystal microbalance.
A quartz-crystal microbalance (QCM) technique was applied to analyze effects of site-directed mutagenesis of a glycosidase (isomalto-dextranase) on the hydrolysis mechanism of the substrate binding (k(on), k(off), and K(d)) and the catalytic process (k(cat)), separately, by using a dextran-immobilized QCM in buffer solution. D266N, D198N, and D313N mutants, which are predicted as critical residues of the isomalto-dextranase hydrolytic activity, dramatically decreased the apparent enzyme activity. The D266N mutant, however, did not change the substrate binding ability (K(d)), and the D198N and D313N mutants largely increased K(d) values due to the increase of k(off) and/or the decrease of k(on) values, as well as the negatively small k(cat) values. From these results, we estimate the reaction mechanism, in which Asp266 acts as only a general acid in the catalytic process, Asp198 acts as both nucleophile in the catalytic process and binding the substrate, and Asp313 acts as only the substrate binding.
Nihira
Takanori
T
Department of Biomolecular Engineering, Frontier Collaborative Research Center, Tokyo Institute of Technology and CREST, Japan Science and Technology Corporation, Yokohama 226-8501, Japan.
Mizuno
Masahiro
M
Tonozuka
Takashi
T
Sakano
Yoshiyuki
Y
Mori
Toshiaki
T
Okahata
Yoshio
Y
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D002384:Catalysis; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011791:Quartz; D013379:Substrate Specificity / Q000235:genetics
D003911:Dextrans; D004800:Enzymes, Immobilized; D011791:Quartz; D006026:Glycoside Hydrolases; C091051:glucan 1,6-alpha-isomaltosidase