1 research outputs found
Molecular Differences between a Mutase and a Phosphatase: Investigations of the Activation Step in <i>Bacillus cereus</i> Phosphopentomutase
Prokaryotic phosphopentomutases (PPMs) are di-Mn<sup>2+</sup> enzymes
that catalyze the interconversion of α-d-ribose 5-phosphate
and α-d-ribose 1-phosphate at an active site located
between two independently folded domains. These prokaryotic PPMs belong
to the alkaline phosphatase superfamily, but previous studies of <i>Bacillus cereus</i> PPM suggested adaptations of the conserved
alkaline phosphatase catalytic cycle. Notably, <i>B. cereus</i> PPM engages substrates when the active site nucleophile, Thr-85,
is phosphorylated. Further, the phosphoenzyme is stable throughout
purification and crystallization. In contrast, alkaline phosphatase
engages substrates when the active site nucleophile is dephosphorylated,
and the phosphoenzyme reaction intermediate is only stably trapped
in a catalytically compromised enzyme. Studies were undertaken to
understand the divergence of these mechanisms. Crystallographic and
biochemical investigations of the PPM<sup>T85E</sup> phosphomimetic
variant and the neutral corollary PPM<sup>T85Q</sup> determined that
the side chain of Lys-240 underwent a change in conformation in response
to active site charge, which modestly influenced the affinity for
the small molecule activator α-d-glucose 1,6-bisphosphate.
More strikingly, the structure of unphosphorylated <i>B. cereus</i> PPM revealed a dramatic change in the interdomain angle and a new
hydrogen bonding interaction between the side chain of Asp-156 and
the active site nucleophile, Thr-85. This hydrogen bonding interaction
is predicted to align and activate Thr-85 for nucleophilic addition
to α-d-glucose 1,6-bisphosphate, favoring the observed
equilibrium phosphorylated state. Indeed, phosphorylation of Thr-85
is severely impaired in the PPM<sup>D156A</sup> variant even under
stringent activation conditions. These results permit a proposal for
activation of PPM and explain some of the essential features that
distinguish between the catalytic cycles of PPM and alkaline phosphatase