Mechanistic insights into isomerization reaction catalyzed by 5-methylthioribose-1-phosphate isomerase
Veeramachineni, Vamsee Mohan
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MtnA is an aldose-ketose isomerase which helps in the salvage of methionine, an essential amino acid in certain Bacteria (and in Eukarya). It catalyzes the inter conversion of 5-methylthioribose 1-phosphate (MTR-1-P) and 5-methylthioribulose 1-phosphate (MTRu-1-P), whose catalyzing mechanism is yet to be investigated in detail. MTR-1-P is a unique substrate which is locked in its circular conformation by the presence of phosphate group at the C1 position. The mechanism for the ring opening reaction cannot be explained in confidence through cis -enediol or hydride transfer mechanism owing to the presence of a phosphate group on the carbon involved in the transfer and due to the ability of the enzyme to act even in the absence of divalent metal ions respectively. In an effort to study the mechanism, three pathways involving (1) formation of oxocarbenium ion intermediate, (2) formation of covalently modified protein intermediate and (3) intramolecular phosphate transfer were proposed. In the current study, we used 32 P labeled MTR-1-P to understand the role of phosphate during catalysis. SDS-PAGE analysis indicate formation of an acylphosphate adduct upon incubation of isomerase with [ 32 P]MTR1P. Further analysis of labeled enzyme for the site of phosphorylation and its role in isomerization mechanism was unsuccessful owing to the very low levels of accumulation and instability of the phosphorylated enzyme adduct. Kinetic isotope effects on hydrogen transfer step were determined using [2- 2 H]MTR1P. D (V/K) and D V were found to be 3.9 and 1.4, respectively, for Bs MtnA and 2.0 and 1.3, respectively, for Hs MtnA. These results suggest that proton-/hydride-transfer step in isomerization is only partially rate limiting. Also, masked KIEs on k cat means a significant contribution from forward commitment, indicating a significantly slow product release step. Thus, KIE’s provided a valuable insight into the kinetic landscape of MtnA.