AMINO ACID SEQUENCE OF NUCLEOSIDE HYDROLASES FROM PLANTS
AMINO ACID SEQUENCE OF NUCLEOSIDE HYDROLASES FROM PLANTS
No Thumbnail Available
Date
2014-06-24
Authors
Ogorodnik, Kateryna V.
Journal Title
Journal ISSN
Volume Title
Publisher
Middle Tennessee State University
Abstract
Nucleoside hydrolases are key enzymes of the purine salvage pathways of various bacteria, yeast, parasitic protozoa, insects, fish, and plants. While the structures of nucleoside hydrolases from parasitic protozoans have been extensively studied, almost no structural information beyond subunit molecular weights is available for nucleoside hydrolases from plants.
Based on a nonlinear regression using the Michaelis-Menten equation, the Michaelis constants (Km) of inosine for yellow lupin adenosine nucleosidase and inosine nucleosidase were determined. A Km of 260 75 μM for inosine nucleosidase and 9820 13801 μM for adenosine nucleosidase was found. The high standard deviation of the Km for adenosine nucleosidase was due to the solubility limits of inosine preventing a complete kinetic analysis.
Three nucleoside hydrolases from plants, adenosine and inosine nucleosidase from yellow lupin, and adenosine nucleosidase from soybean, along with rihC from E. coli have been partially sequenced by mass spectrometry. After a final polishing step based on isoelectric focusing was performed, the enzymes were subjected to tryptic digestion followed by separation of the resulting peptides using reverse phase HPLC. The peptides were subjected to MS/MS and the sequence of selected peptides determined using PEAKS software.
Based on a nonlinear regression using the Michaelis-Menten equation, the Michaelis constants (Km) of inosine for yellow lupin adenosine nucleosidase and inosine nucleosidase were determined. A Km of 260 75 μM for inosine nucleosidase and 9820 13801 μM for adenosine nucleosidase was found. The high standard deviation of the Km for adenosine nucleosidase was due to the solubility limits of inosine preventing a complete kinetic analysis.
Three nucleoside hydrolases from plants, adenosine and inosine nucleosidase from yellow lupin, and adenosine nucleosidase from soybean, along with rihC from E. coli have been partially sequenced by mass spectrometry. After a final polishing step based on isoelectric focusing was performed, the enzymes were subjected to tryptic digestion followed by separation of the resulting peptides using reverse phase HPLC. The peptides were subjected to MS/MS and the sequence of selected peptides determined using PEAKS software.