Phytase catalysis of dephosphorylation studied using isothermal titration calorimetry and electrospray ionization time-of-flight mass spectroscopy.

Autor: Kempapidis T; Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, S1 3JD, United Kingdom., Bradshaw NJ; Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, S1 3JD, United Kingdom., Hodges HE; Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, S1 3JD, United Kingdom., Cowieson AJ; DSM Nutritional Products, 4303, Kaiseraugst, Switzerland., Cameron DD; Department of Animal and Plant Science, University of Sheffield, Sheffield, S1 3JD, United Kingdom., Falconer RJ; Department of Chemical Engineering and Advanced Materials, University of Adelaide, Adelaide, SA, 5005, Australia. Electronic address: robert.falconer@adelaide.edu.au.
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2020 Oct 01; Vol. 606, pp. 113859. Date of Electronic Publication: 2020 Jul 29.
DOI: 10.1016/j.ab.2020.113859
Abstrakt: Phytases are important commercial enzymes that catalyze the dephosphorylation of myo-inositol hexakisphosphate (phytate) to its lower inositol phosphate (IP) esters, IP6 to IP1. Digestion of phytate by Citrobacter braakii 6-phytase deviates significantly from monophasic Michaelis-Menten kinetics. Analysis of phytate digestion using isothermal titration calorimetry (ITC) using the single injection method produced a thermogram with two peaks consistent with two periods of high enzyme activity. Continuous-flow electrospray ionization time-of-flight mass spectroscopy (ESI-ToF-MS) provided real-time analysis of phytase catalysis. It was able to show that the first two cleavage steps were rapid and concurrent but the third cleavage step from IP4 to IP3 was slow. The third (IP4 to IP3), fourth (IP3 to IP2) and fifth (IP2 to IP1) cleavages were effectively sequential due to the preferred association of the more phosphorylated species with the phytase catalytic site. This created a bottleneck during the cleavage of IP4 to IP3 until the point at which IP4 was exhausted and was followed by the rapid cleavage of IP3 to IP2, which was observed as the second peak in the ITC thermogram. This work illustrates the importance of an orthogonal approach when studying non-specific or complex enzyme catalyzed reactions.
(Copyright © 2020 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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