Abstract
The association-dissociation of yeast hexokinase has been re-examined and the size of the reacting form of the enzyme has been investigated under a variety of conditions. Both Sephacryl S-200 and high performance liquid chromatography on Bio-Sil have been employed with continuous effluent monitoring under reacting and nonreacting conditions. The reacting enzyme was monomeric under all conditions, suggesting that the dimer is not an important catalytic species in normal assays. The reacting enzyme remained as a monomer under the following conditions: 0.1-15 micrograms/ml loading concentration, from 30 to 100 mM ionic strength, with 2 mM citrate, with 50% D2O, and at 160 atm hydrostatic pressure. The dissociation constant for the nonreacting hexokinase was 22 (+/- 2) micrograms/ml (uncorrected for 5-fold dilution) in 100 mM triethanolamine, pH 6.5, and 25 degrees C. Glucose or MgATP had dissociative effects under all conditions studied, but MgATP was much less effective and only slightly more effective than an equivalent ionic strength. NaCl, between 30 and 80 mM, promoted dissociation, with a concomitant conformational change suggested by nonlinearity of log-log plots. The extent of dissociation with MgCl2 was slightly greater than an equivalent NaCl ionic strength and the shape of the association curves suggested the formation of an elongated dimer in the presence of MgCl2. The conclusion is made that hexokinase is monomeric under most assay conditions and that the dissociation is predominantly the result of the glucose interaction. High performance liquid chromatography has been shown to be a useful method of assessing the association state of enzymes under both reacting and nonreacting conditions.
Highlights
The enzyme has been investigated under a variety of Glucose has been shown by sedimentation and light scattering conditions.Both Sephacryl S-200 and high performance liquid chromatography on Bio-Si1 have been employed with continuous effluent monitoring under reacting and nonreactingconditions
Evidencewasalso extent of dissociation with MgClz wasslightly greater presented [10] from reacting enzyme sedimentation studies than an equivalent NaCl ionic strength and the shape that hexokinase exists predominantly in the monomeric state of the association curves suggested the formation of an under assayconditions but that the simultaneous presenocfe elongated dimer in the presence of MgClZ.The conclusion is made that hexokinase is monomeric under most assay conditions and that the dissociation is predominantly the result of the glucose interaction
The present studies with Sephacryl gel fitration and Bio-Sil size exclusion have confirmed that nonreacting hexokinase exists in a concentration-dependent, self-association-dissociation equilibrium of monomers and dimers
Summary
The enzyme has been investigated under a variety of Glucose has been shown by sedimentation and light scattering conditions.Both Sephacryl S-200 and high performance liquid chromatography on Bio-Si1 have been employed with continuous effluent monitoring under reacting and nonreactingconditions. Evidencewasalso extent of dissociation with MgClz wasslightly greater presented [10] from reacting enzyme sedimentation studies than an equivalent NaCl ionic strength and the shape that hexokinase exists predominantly in the monomeric state of the association curves suggested the formation of an under assayconditions but that the simultaneous presenocfe elongated dimer in the presence of MgClZ.The conclusion is made that hexokinase is monomeric under most assay conditions and that the dissociation is predominantly the result of the glucose interaction. - most extensively studied) exists in a monomer-dimer self- of the enzyme andstabilization of a moreactive enzyme association equilibrium (dimer M, 100,000).Dissociation is conformation [13].More recent experimentbsy Wilkinson and promoted by increases in ionic strength, pH, and temperature Rose [17] have suggested that stabilization of a dimeric form or by decreases in the enzyme concentration.Schulzeand in solution by ATP is deactivating and only at low enzyme. Wilkinson and Rose [17]
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