Substrate specificity and conformational activation mechanism of beta-phosphoglucomutase
Saltzberg, Daniel John
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Phosphate transfer is ubiquitous in nature, however the occurance of phosphomutases is rare. Their uniqueness can be attributed to the complex and malleable substrate recognition scheme that allows the enzyme to perform two similar, yet distinct, catalytic steps while maintaining strict fidelity for substrate versus water. The complexity of developing this mechanism is highlighted in that, while phosphomutase function has independently evolved in most larger phosphotransferase superfamilies, very little diversification of this function has developed. As such, phosphomutases provide a rich framework to study the intricate specificity mechanisms employed by enzymes. β-Phosphoglucomutase (bPGM) catalyzes the interconversion between β-glucose 1-phosphate (βG1P) and glucose 6-phosphate (G6P) via a β-glucose 1,6 bisphosphate (βG16P) intermediate. βPGM is in one of two subfamilies that have independently acquired phosphomutase activity within the ubiquitous Haloalkanoate Dehalogenase superfamily (HADSF) of phosphotransferases. The enzyme has been observed to undergo a large conformational change upon binding βG16P as well as a repositioning of the general acid/base catalyst residue Asp10. In addition, the mechanism involves cycling of the protonation state of Asp10, which requires a significant pKa shift. The importance of Asp10 and its activation of the enzyme have been discussed previously, however a clear understanding of the interplay between the conformational and catalytic activation mechanisms for βPGM has not been described. This work uses aqueous phase techniques, solution X-ray scattering and molecular dynamics, to probe the effect of individual ligand moieties on the conformational state of the enzyme and free energy molecular dynamics and electrostatic calculations determine the interplay between conformation, protonation and Asp10 activation. The results implicate a model where the ligand-induced conformational change is governed by the non-catalytic phosphate site, and this transition induces correct positioning of Asp10, which, in turn induces the pKa shift, forming the catalytically competent complex.