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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.
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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.
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- PAR ID:
- 10598328
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Methods in enzymology
- ISSN:
- 0076-6879
- Page Range / eLocation ID:
- 279 to 318
- Subject(s) / Keyword(s):
- Methionine salvage Isomerase Methylthio-D-ribose 1-phosphate Methylthio-D-ribose-1-phosphate isomerase Kinetics Covalent adduct Radioactive labeling o-Phenylenediamine
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Book Chapter:
- https://doi.org/10.1016/bs.mie.2023.03.015
