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Title 

Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11

Authors 

Byung Chul OhB S ChangKwan Hwa ParkNam Chul HaHyung Kwoun KimByung Ha OhTae Kwang Oh

Publisher 

American Chemical Society

Issue Date 

2001

Citation 

Biochemistry, vol. 40, no. 32, pp. 9669-9676

Keywords 

amino acidscalciumcalorimetrycatalysisionizationmutagenesisbacillusamino acidbacterial enzymeinositol 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 

http://dx.doi.org/10.1021/bi010589u

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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