Glucose exchange and catalysis by two crystalline hexokinase x glucose complexes. Evidence for an obligatory ATP-dependent conformational change in catalysis.
Hexokinase PI x glucose crystals grown with radiolabeled glucose under conditions similar to those used for x-ray diffraction studies (Bennett, W.S., Jr., and Steitz, T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852) have been shown to contain 1 mol of tightly bound glucose. These crystals exchange all of this glucose in a single exponential process (kobs = 0.7 min-1). In the crystalline form they are, however, inactive and do not catalyze formation of any bound glucose-6-P, suggesting that lattice forces prevent catalysis. A new catalytically active E x glucose complex has been crystallized in the presence of glucose and ADP, a competitive inhibitor of ATP. These crystals readily lose ADP upon washing with concentrated (NH4)2SO4. They exchange all of the tightly bound glucose more slowly than the form grown in the absence of ADP (kobs = 0.05 min-1). Addition of MgATP to the suspension in ammonium sulfate results in rapid conversion of half of the bound glucose to bound glucose 6-phosphate followed by further reaction as products are released. This agrees with the previously measured equilibrium constant of unity for enzyme-bound phosphoryl transfer catalyzed in solution (Wilkinson, K.D., and Rose, I.A. (1979) J. Biol. Chem. 254, 12567-12572). These results indicate that the two E x glucose crystals are distinguished by a nucleotide-dependent conformational difference, which is stabilized by lattice forces. The active crystals allow the facile dissociation of the ADP used to induce the change. This conformational change appears to be pevented in the E x glucose crystals and to be necessary to produce the active ternary complex.
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D002384:Catalysis; D003461:Crystallography; D005947:Glucose / Q000378:metabolism*; D005958:Glucosephosphates / Q000494:pharmacology; D006593:Hexokinase / Q000378:metabolism*; D011487:Protein Conformation
D005958:Glucosephosphates; D000255:Adenosine Triphosphate; D006593:Hexokinase; D005947:Glucose