Kinetic measurements of phosphoglucomutase by direct analysis of glucose-1-phosphate and glucose-6-phosphate using ion/molecule reactions and Fourier transform ion cyclotron resonance mass spectrometry.
A method for the direct determination of kinetic constants for phosphoglucomutase and its phosphorylated products is described. Fourier transform ion cyclotron resonance gas-phase ion/molecule reactions between trimethyl borate and glucose phosphate, phosphorylated at either the 1 or the 6 position, generate mass spectra distinguishable with regard to product ion distribution. A multicomponent quantification method is utilized to determine the composition of a binary mixture of the two positional isomers. Using this method, the conversion between glucose-1-phosphate and glucose-6-phosphate can be directly monitored without the use of coupling enzymes. The values of K(m) for glucose-1-phosphate and glucose-6-phosphate were determined using the substrate-velocity plot and the Haldane relationship, respectively. Values of V(max) for both the forward and the reverse directions were measured, and the equilibrium constant for the reversible reaction was determined using this methodology. Kinetic parameters measured correlate well with those obtained using traditional methods. The assay was demonstrated to be accurate and particularly convenient to determine kinetic constants for enzymatic systems that involve the interconversion of phosphorylated positional isomers.
Gao
Hong
H
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Leary
Julie A
JA
D002623:Chemistry Techniques, Analytical / Q000379:methods*; D005583:Fourier Analysis; D019298:Glucose-6-Phosphate / Q000032:analysis* / Q000378:metabolism; D005958:Glucosephosphates / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D010733:Phosphoglucomutase / Q000378:metabolism*
D005958:Glucosephosphates; D019298:Glucose-6-Phosphate; C031590:glucose-1-phosphate; D010733:Phosphoglucomutase