Kinetic characterization of methylthio-D-ribose-1-phosphate isomerase

Methylthio-D-ribose-1-phosphate (MTR1P) isomerase (MtnA) catalyzes the reversible isomerization of the aldose MTR1P into the ketose methylthio-D-ribulose 1-phosphate. It serves as a member of the methionine salvage pathway that many organisms require for recycling methylthio-D-adenosine, a byproduct of S-adenosylmethionine metabolism, back to methionine. MtnA is of mechanistic interest because unlike most other aldose–ketose isomerases, its substrate exists as an anomeric phosphate ester and therefore cannot equilibrate with a ring-opened aldehyde that is otherwise required to promote isomerization. To investigate the mechanism of MtnA, it is necessary to establish reliable methods for determining the concentration of MTR1P and to measure enzyme activity in a continuous assay. This chapter describes several such protocols needed to perform steady-state kinetics measurements. It additionally outlines the preparation of [³²P]MTR1P, its use in radioactively labeling the enzyme, and the characterization of the resulting phosphoryl adduct.