Rapid kinetic studies of PhyA from Selenomonas ruminantium, and a simplified means of production of a phosphate biosensor
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Date
2016
Authors
Smith, Dustin D.
University of Lethbridge. Faculty of Arts and Science
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Publisher
Lethbridge, Alta : University of Lethbridge, Dept. of Chemistry and Biochemistry
Abstract
Myo-inositol polyphosphates (IPs) are ubiquitous in nature and involved in various cellular events. Dephosphorylation of IPs by protein tyrosine phosphatase-like polyphosphatases (PTPLPs) occurs via a complex pathway, and the in vivo function of many of these enzymes remains unknown. In order to further our understanding of PTPLP catalyzed dephosphorylation of IPs; I present rapid kinetics studies of the representative PTPLP PhyA from Selenomonas ruminantium (PhyAsr). These studies revealed kinetic parameters of PhyAsr dimerization, and myo-inositol hexakisphosphate (IP6) binding to the homodimer. In addition to studying PhyAsr, I have developed a simplified methodology to produce a biosensor capable of detecting phosphate release in real-time. The phosphate biosensor is fluorescently labeled and utilizes Escherichia coli Phosphate-binding protein (PhoS). The results show that my proposed methodology yields a functional biosensor and is feasible for large-scale production. I envision this methodology to be versatile and useful for a large number of research applications where detection of free phosphate is required.
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Keywords
Biochemistry , Enzyme kinetics , Phosphate sensor , PTPLP , myo-inositol , Phosphatase , Stopped-flow , PhyA , PhoS , Phosphate release , Phosphate binding protein , Phytase