or
Title | Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11 |
Authors | Byung Chul Oh; B S Chang; Kwan Hwa Park; Nam Chul Ha; Hyung Kwoun Kim; Byung Ha Oh; Tae Kwang Oh |
Publisher | American Chemical Society |
Issue Date | 2001 |
Citation | Biochemistry, vol. 40, no. 32, pp. 9669-9676 |
Keywords | amino acids; calcium; calorimetry; catalysis; ionization; mutagenesis; bacillus; amino acid; bacterial enzyme; inositol phosphate |
Abstract | The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes phytate (myoinositol hexakisphosphate, IP6) to less phosphorylated myo-inositol phosphates in the presence of Ca2+. In this report, we discuss the unique Ca2+-dependent catalytic properties of the phytase and its specific substrate requirement. Initial rate kinetic studies of the phytase indicate that the enzyme activity follows a rapid equilibrium ordered mechanism in which binding of Ca2+ to the active site is necessary for the essential activation of the enzyme. Ca2+ turned out to be also required for the substrate because the phytase is only able to hydrolyze the calcium-phytate complex. In fact, both an excess amount of free Ca2+ and an excess of free phytate, which is not complexed with each other, can act as competitive inhibitors. The Ca2+-dependent catalytic activity of the enzyme was further confirmed, and the critical amino acid residues for the binding of Ca2+ and substrate were identified by site-specific mutagenesis studies. Isothermal titration calorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding. The pH dependence of the Vmax and Vmax/Km consistently supported these observations by demonstrating that the enzyme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate. The C2+-dependent activation of enzyme and substrate was found to be different from other histidine acid phytases that hydrolyze metal-free phytate. |
ISSN | 0006-2960 |
Link | |
Appears in Collections | |
Registered Date |
2017-04-19 |
