Methylthio-D-ribose-1-Phosphate Isomerase Uses a Novel Mechanism for Aldose–Ketose Isomerization

The enzyme-catalyzed interconversion of aldoses and ketoses historically involves one of two mechanisms, both of which require an aldehyde form of the substrate. Methylthio-D-ribose-1-phosphate (MTR1P) isomerase (MtnA), which functions in the methionine-salvage pathway, poses a challenge to this canon because its substrate cannot readily access such a form. In converting MTR1P to its ketose isomer, methylthio-D-ribulose 1-phosphate, MtnA must catalyze opening of the ribofuranose ring and hydrogen transfer between C-2 and C-1. Primary 2H and 13C kinetic isotope effects measured at these positions in MTR1P and ribose 1-phosphate indicate that these two events occur in a common and rate-limiting step with respect to kcat/KM. An inverse kinetic solvent viscosity effect and inverse solvent kinetic isotope effect were measured for this constant, implicating an open–closed protein conformational change after substrate binding and the participation of Cys160 as the catalytic base responsible for shuttling the proton between C-1 and C-2. An E2 elimination–tautomerization sequence is most consistent with these findings and represents a third mechanism for enzymatic aldose–ketose isomerization.