Use of silicate sol-gels to trap the R and T quaternary conformational states of pig kidney fructose-1,6-bisphosphatase.
Encapsulation of the homotetrameric pig kidney fructose-1,6-bisphosphatase (FBPase) in tetramethyl orthosilicate sol-gels was used to dramatically reduce the rate of the allosteric transition of the enzyme between the T and R allosteric states. When assayed in the absence of the allosteric inhibitor AMP, the enzyme encapsulated in the T-state exhibited little activity. The enzyme encapsulated in the R-state exhibited a 4-fold lower k(cat) and V(max) than the enzyme in solution, and the apparent K(m) for this enzyme was 350-fold higher than the corresponding value for the enzyme in solution. The [Mg(2+)](0.5) for the encapsulated enzyme was only 0.1 mM, compared to 0.54 mM for the normal enzyme. Magnesium activation, under both sets of conditions, was cooperative with a Hill coefficient of approximately 2. The activity of enzyme encapsulated in the R-state decreased to about 70% of initial activity within 1 min of adding AMP, it then decreased slowly to about 40% of initial activity over the following 7 h. Under the conditions tested, the encapsulated enzyme never became completely inactivated and AMP inhibition was no longer cooperative. For enzyme encapsulated in the T-state, activity was restored over approximately 7 h after removal of the AMP. The biphasic and slow responses to changing AMP levels suggest that encapsulated enzyme can be used to study the effects of local conformational changes distinct from the global quaternary conformational changes by slowing down the ability of the enzyme to carry out global rotations. The response to AMP exhibited by the encapsulated enzyme is consistent with the ability of AMP, at least partially, to directly influence the activity of the active site within each subunit.
McIninch
J K
JK
Department of Chemistry, Boston College, Merkert Chemistry Center, Chestnut Hill, MA 02467, USA.
Kantrowitz
E R
ER
D000249:Adenosine Monophosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent; D004800:Enzymes, Immobilized; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D005782:Gels; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D011487:Protein Conformation; D017640:Silicates / Q000737:chemistry; D013552:Swine
D002413:Cations, Divalent; D004800:Enzymes, Immobilized; D005782:Gels; D017640:Silicates; D000249:Adenosine Monophosphate; D006597:Fructose-Bisphosphatase; D008274:Magnesium