2 research outputs found
Solution NMR of a 463-Residue Phosphohexomutase: Domain 4 Mobility, Substates, and Phosphoryl Transfer Defect
Phosphomannomutase/phosphoglucomutase contributes to
the infectivity
of <i>Pseudomonas aeruginosa</i>, retains and reorients
its intermediate by 180°, and rotates domain 4 to close the deep
catalytic cleft. Nuclear magnetic resonance (NMR) spectra of the backbone
of wild-type and S108C-inactivated enzymes were assigned to at least
90%. <sup>13</sup>C secondary chemical shifts report excellent agreement
of solution and crystallographic structure over the 14 α-helices,
C-capping motifs, and 20 of the 22 β-strands. Major and minor
NMR peaks implicate substates affecting 28% of assigned residues.
These can be attributed to the phosphorylation state and possibly
to conformational interconversions. The S108C substitution of the
phosphoryl donor and acceptor slowed transformation of the glucose
1-phosphate substrate by impairing <i>k</i><sub>cat</sub>. Addition of the glucose 1,6-bisphosphate intermediate accelerated
this reaction by 2–3 orders of magnitude, somewhat bypassing
the defect and apparently relieving substrate inhibition. The S108C
mutation perturbs the NMR spectra and electron density map around
the catalytic cleft while preserving the secondary structure in solution.
Diminished peak heights and faster <sup>15</sup>N relaxation suggest
line broadening and millisecond fluctuations within four loops that
can contact phosphosugars. <sup>15</sup>N NMR relaxation and peak
heights suggest that domain 4 reorients slightly faster in solution
than domains 1–3, and with a different principal axis of diffusion.
This adds to the crystallographic evidence of domain 4 rotations in
the enzyme, which were previously suggested to couple to reorientation
of the intermediate, substrate binding, and product release
Solution NMR of a 463-Residue Phosphohexomutase: Domain 4 Mobility, Substates, and Phosphoryl Transfer Defect
Phosphomannomutase/phosphoglucomutase contributes to
the infectivity
of <i>Pseudomonas aeruginosa</i>, retains and reorients
its intermediate by 180°, and rotates domain 4 to close the deep
catalytic cleft. Nuclear magnetic resonance (NMR) spectra of the backbone
of wild-type and S108C-inactivated enzymes were assigned to at least
90%. <sup>13</sup>C secondary chemical shifts report excellent agreement
of solution and crystallographic structure over the 14 α-helices,
C-capping motifs, and 20 of the 22 β-strands. Major and minor
NMR peaks implicate substates affecting 28% of assigned residues.
These can be attributed to the phosphorylation state and possibly
to conformational interconversions. The S108C substitution of the
phosphoryl donor and acceptor slowed transformation of the glucose
1-phosphate substrate by impairing <i>k</i><sub>cat</sub>. Addition of the glucose 1,6-bisphosphate intermediate accelerated
this reaction by 2–3 orders of magnitude, somewhat bypassing
the defect and apparently relieving substrate inhibition. The S108C
mutation perturbs the NMR spectra and electron density map around
the catalytic cleft while preserving the secondary structure in solution.
Diminished peak heights and faster <sup>15</sup>N relaxation suggest
line broadening and millisecond fluctuations within four loops that
can contact phosphosugars. <sup>15</sup>N NMR relaxation and peak
heights suggest that domain 4 reorients slightly faster in solution
than domains 1–3, and with a different principal axis of diffusion.
This adds to the crystallographic evidence of domain 4 rotations in
the enzyme, which were previously suggested to couple to reorientation
of the intermediate, substrate binding, and product release