No abstract

Methylthio-d-ribose-1-phosphate (MTR1P) isomerase (MtnA) functions in the methionine salvage pathway by converting the cyclic aldose MTR1P to its open-chain ketose isomer methylthio-d-ribulose 1-phosphate (MTRu1P). What is particularly challenging for this enzyme is that the substrate’s phosphate ester prevents facile equilibration to an aldehyde, which in other aldose–ketose isomerases is known to activate the α-hydrogen for proton or hydride transfer between adjacent carbons. We speculated that MtnA could use covalent catalysis via a phosphorylated residue to permit isomerization by one of the canonical mechanisms, followed by phosphoryl transfer back to form the product. In apparent support of this mechanism, [32P]­MTR1P was found by SDS-PAGE and gel-filtration chromatography to radiolabel the enzyme. Susceptibility of this adduct to strongly acidic and basic pH and nucleophilic agents is consistent with an acyl phosphate. C160S and D240N, mutants of two conserved active-site residues, however, exhibited no difference in radiolabeling despite a reduction in activity of ∼107, leading to the conclusion that phosphorylation is unrelated to catalysis. Unexpectedly, prolonged incubations with C160S revealed up to 30% accumulation of radioactivity, which was identified by 31P and 13C NMR to be the result of a second adducta hemiketal formed between Ser160 and the carbonyl of MTRu1P. These results are interpreted as indirect support for a mechanism involving transfer of the proton from C-2 to C-1 by Cys160.