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Conformational cycling in beta-phosphoglucomutase catalysis: Reorientation of the beta-D-glucose 1,6-(bis) phosphate intermediate

Dai, JY ; Wang, LB ; Allen, KN ; Rådström, Peter LU and Dunaway-Mariano, D (2006) In Biochemistry 45(25). p.7818-7824
Abstract
Activated Lactococcus lactis beta-phosphoglucomutase (beta PGM) catalyzes the conversion of beta-D-glucose 1-phosphate (beta G1P) derived from maltose to beta-D-glucose 6-phosphate (G6P). Activation requires Mg2+ binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants k(cat) = 177 +/- 9 s(-1), K-m = 49 +/- 4 mu M for the substrate, beta G1P and K-m = 6.5 +/- 0.7 mu M for the activator beta-D-glucose 1,6-bisphosphate (beta G1,6bisP). The observed transient accumulation of [C-14]beta G1,6bisP (12% at similar to 0.1 s) in the single turnover reaction carried out with excess beta PGM (40 mu M) and limiting [C-14]beta G1P (5 mu M) and beta G1,6bisP (5 mu M)... (More)
Activated Lactococcus lactis beta-phosphoglucomutase (beta PGM) catalyzes the conversion of beta-D-glucose 1-phosphate (beta G1P) derived from maltose to beta-D-glucose 6-phosphate (G6P). Activation requires Mg2+ binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants k(cat) = 177 +/- 9 s(-1), K-m = 49 +/- 4 mu M for the substrate, beta G1P and K-m = 6.5 +/- 0.7 mu M for the activator beta-D-glucose 1,6-bisphosphate (beta G1,6bisP). The observed transient accumulation of [C-14]beta G1,6bisP (12% at similar to 0.1 s) in the single turnover reaction carried out with excess beta PGM (40 mu M) and limiting [C-14]beta G1P (5 mu M) and beta G1,6bisP (5 mu M) supported the role of beta G1,6bisP as a reaction intermediate in the conversion of the, G1P to G6P. Single turnover reactions of [C-14]beta G1,6bisP with excess, beta PGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the beta G1,6bisP binds to the active site in two different orientations (one positioning the C(1) phosphoryl group for reaction with Asp8, and the other orientation positioning the C(6) phosphoryl group for reaction with Asp8) with roughly the same efficiency. Single turnover reactions carried out with beta PGM, [C-14]beta G1P, and unlabeled beta G1,6bisP demonstrated complete exchange of label to the beta G1,6bisP during the catalytic cycle. Thus, the reorientation of the beta G1,6bisP intermediate that is required to complete the catalytic cycle occurs by diffusion into solvent followed by binding in the opposite orientation. Published X-ray structures of beta G1P suggest that the reorientation and phosphoryl transfer from beta G1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of beta G1,6bisP to beta G1P plus G6P was examined to evidence a significant stabilization of beta PGM aspartyl phosphate. (Less)
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publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemistry
volume
45
issue
25
pages
7818 - 7824
publisher
The American Chemical Society (ACS)
external identifiers
  • pmid:16784233
  • wos:000238386300014
  • scopus:33746895489
ISSN
0006-2960
DOI
10.1021/bi060136v
language
English
LU publication?
yes
id
67c9f11a-b19b-4aff-8d6a-0eeeb0766454 (old id 406311)
date added to LUP
2016-04-01 12:04:04
date last changed
2020-01-12 09:08:48
@article{67c9f11a-b19b-4aff-8d6a-0eeeb0766454,
  abstract     = {Activated Lactococcus lactis beta-phosphoglucomutase (beta PGM) catalyzes the conversion of beta-D-glucose 1-phosphate (beta G1P) derived from maltose to beta-D-glucose 6-phosphate (G6P). Activation requires Mg2+ binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants k(cat) = 177 +/- 9 s(-1), K-m = 49 +/- 4 mu M for the substrate, beta G1P and K-m = 6.5 +/- 0.7 mu M for the activator beta-D-glucose 1,6-bisphosphate (beta G1,6bisP). The observed transient accumulation of [C-14]beta G1,6bisP (12% at similar to 0.1 s) in the single turnover reaction carried out with excess beta PGM (40 mu M) and limiting [C-14]beta G1P (5 mu M) and beta G1,6bisP (5 mu M) supported the role of beta G1,6bisP as a reaction intermediate in the conversion of the, G1P to G6P. Single turnover reactions of [C-14]beta G1,6bisP with excess, beta PGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the beta G1,6bisP binds to the active site in two different orientations (one positioning the C(1) phosphoryl group for reaction with Asp8, and the other orientation positioning the C(6) phosphoryl group for reaction with Asp8) with roughly the same efficiency. Single turnover reactions carried out with beta PGM, [C-14]beta G1P, and unlabeled beta G1,6bisP demonstrated complete exchange of label to the beta G1,6bisP during the catalytic cycle. Thus, the reorientation of the beta G1,6bisP intermediate that is required to complete the catalytic cycle occurs by diffusion into solvent followed by binding in the opposite orientation. Published X-ray structures of beta G1P suggest that the reorientation and phosphoryl transfer from beta G1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of beta G1,6bisP to beta G1P plus G6P was examined to evidence a significant stabilization of beta PGM aspartyl phosphate.},
  author       = {Dai, JY and Wang, LB and Allen, KN and Rådström, Peter and Dunaway-Mariano, D},
  issn         = {0006-2960},
  language     = {eng},
  number       = {25},
  pages        = {7818--7824},
  publisher    = {The American Chemical Society (ACS)},
  series       = {Biochemistry},
  title        = {Conformational cycling in beta-phosphoglucomutase catalysis: Reorientation of the beta-D-glucose 1,6-(bis) phosphate intermediate},
  url          = {http://dx.doi.org/10.1021/bi060136v},
  doi          = {10.1021/bi060136v},
  volume       = {45},
  year         = {2006},
}