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Crystal structures of human hexokinase I reveal identical binding sites for phosphate and the 6-phosphoryl group of glucose 6-phosphate in proximity to Gly87, Ser88, Thr232, and Ser415, a binding site for the pyranose moiety of glucose 6-phosphate in proximity to Asp84, Asp413, and Ser449, and a single salt link involving Arg801between the N- and C-terminal halves. Purified wild-type and mutant enzymes (Asp84→ Ala, Gly87→ Tyr, Ser88→ Ala, Thr232→ Ala, Asp413→ Ala, Ser415→ Ala, Ser449→ Ala, and Arg801→ Ala) were studied by kinetics and circular dichroism spectroscopy. All eight mutant hexokinases have kcatandKmvalues for substrates similar to those of wild-type hexokinase I. Inhibition of wild-type enzyme by 1,5-anhydroglucitol 6-phosphate is consistent with a high affinity binding site (Ki= 50 μm) and a second, low affinity binding site (Kii=0.7 mm). The mutations of Asp84, Gly87, and Thr232listed above eliminate inhibition because of the low affinity site, but none of the eight mutations influence Kiof the high affinity site. Relief of 1,5-anhydroglucitol 6-phosphate inhibition by phosphate for Asp84→ Ala, Ser88→ Ala, Ser415→ Ala, Ser449→ Ala and Arg801→ Ala mutant enzymes is substantially less than that of wild-type hexokinase and completely absent in the Gly87→ Tyr and Thr232→ Ala mutants. The results support several conclusions. (i) The phosphate regulatory site is at the N-terminal domain as identified in crystal structures. (ii) The glucose 6-phosphate binding site at the N-terminal domain is a low affinity site and not the high affinity site associated with potent product inhibition. (iii) Arg801participates in the regulatory mechanism of hexokinase I.

Crystal structures of human hexokinase I reveal identical binding sites for phosphate and the 6-phosphoryl group of glucose 6-phosphate in proximity to Gly87, Ser88, Thr232, and Ser415, a binding site for the pyranose moiety of glucose 6-phosphate in proximity to Asp84, Asp413, and Ser449, and a single salt link involving Arg801 between the N- and C-terminal halves. Purified wild-type and mutant enzymes (Asp84 --> Ala, Gly87 --> Tyr, Ser88 --> Ala, Thr232 --> Ala, Asp413 --> Ala, Ser415 --> Ala, Ser449 --> Ala, and Arg801 --> Ala) were studied by kinetics and circular dichroism spectroscopy. All eight mutant hexokinases have kcat and Km values for substrates similar to those of wild-type hexokinase I. Inhibition of wild-type enzyme by 1,5-anhydroglucitol 6-phosphate is consistent with a high affinity binding site (Ki = 50 microM) and a second, low affinity binding site (Kii = 0.7 mM). The mutations of Asp84, Gly87, and Thr232 listed above eliminate inhibition because of the low affinity site, but none of the eight mutations influence Ki of the high affinity site. Relief of 1,5-anhydroglucitol 6-phosphate inhibition by phosphate for Asp84 --> Ala, Ser88 --> Ala, Ser415 --> Ala, Ser449 --> Ala and Arg801 --> Ala mutant enzymes is substantially less than that of wild-type hexokinase and completely absent in the Gly87 --> Tyr and Thr232 --> Ala mutants. The results support several conclusions. (i) The phosphate regulatory site is at the N-terminal domain as identified in crystal structures. (ii) The glucose 6-phosphate binding site at the N-terminal domain is a low affinity site and not the high affinity site associated with potent product inhibition. (iii) Arg801 participates in the regulatory mechanism of hexokinase I.

Mutation of Arg-15, Glu-19, Arg-22, and Thr-27 of porcine liver fructose-1,6-bisphosphatase was carried out by site-directed mutagenesis. These residues are conserved in all known primary sequences of mammalian fructose-1,6-bisphosphatase. On the basis of the crystal structure of the enzyme, Arg-15, Glu-19, and Arg-22 are located at the interface of the two dimers (C1-C2 and C3-C4), and Thr-27 is in the AMP binding site. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism (CD) spectrometry. No discernible differences were observed between the secondary structures of the wild-type and mutant forms of fructose-1, 6-bisphosphatase on the basis of CD data. Kinetic analyses revealed similar kcat values for mutants R15A, E19Q, R22K, and T27A of fructose-1,6-bisphosphatase; however, a 2-fold increase of kcat was observed with R22M compared with that of the wild-type enzyme. Small changes in Km values for fructose-1,6-bisphosphate were found in the five mutants. 4 6-fold decreases in Ki values for fructose 2,6-bisphosphate and 5-9-fold decreases in the binding affinity of Mg2+ relative to the wild-type enzyme were exhibited by R15A and E19Q. No alteration of Mg2+ cooperativity was found in the five mutants. Significant changes in Ki values for AMP were obtained in the case of R22K (30-fold) and T27A (1300-fold) with a Hill coefficient of 2.0. Replacement of Arg-22 with methionine, however, caused the total loss of AMP cooperativity without changing AMP affinity. Modeling of the mutant structures was undertaken in an attempt to define the functional role of Arg-22. These studies link specific interactions between subunits in fructose-1,6-bisphosphatase to observed properties of cooperativity.

The significance of interactions between AMP domains in recombinant porcine fructose-1,6-bisphosphatase (FBPase) is explored by site-directed mutagenesis and kinetic characterization of homogeneous preparations of mutant enzymes. Mutations of Lys42, Ile190, and Gly191 do not perturb the circular dichroism spectra, but have significant effects on ligand binding and mechanisms of cooperativity. The Km for fructose 1,6-bisphosphate and the Ki for the competitive inhibitor, fructose 2,6-bisphosphate, decreased by as much as 4- and 8-fold, respectively, in the Q32L, K42E, K42T, I190T, and G191A mutants relative to the wild-type enzyme. Q32L, unlike the other four mutants, exhibited a 1.7-fold increase in Kcat. Mg2+ binding is sigmoidal for the five mutants as well as for the wild-type enzyme, but the Mg2+ affinities were decreased (3-22-fold) in mutant FBPases. With the exception of Q32L (8-fold increase), the 50% inhibiting concentrations of AMP for K42E, K42T, I190T, and G191A were increased over 2,000-fold (>10 mM) relative to the wild-type enzyme. Most importantly, a loss of AMP cooperativity was found with K42E, K42T, I190T, and G191A. In addition, the mechanism of AMP inhibition with respect to Mg2+ was changed from competitive to noncompetitive for K42T, I190T, and G191A FBPases. Structural modeling and kinetic studies suggest that Lys42, Ile190, and Gly191 are located at the pivot point of intersubunit conformational changes that energetically couple the Mg2+-binding site to the AMP domain of FBPase.

The significance of interactions between AMP domains in recombinant porcine fructose-1,6-bisphosphatase (FBPase) is explored by site-directed mutagenesis and kinetic characterization of homogeneous preparations of mutant enzymes. Mutations of Lys42, Ile190, and Gly191do not perturb the circular dichroism spectra, but have significant effects on ligand binding and mechanisms of cooperativity. The Kmfor fructose 1,6-bisphosphate and the Kifor the competitive inhibitor, fructose 2,6-bisphosphate, decreased by as much as 4- and 8-fold, respectively, in the Q32L, K42E, K42T, I190T, and G191A mutants relative to the wild-type enzyme. Q32L, unlike the other four mutants, exhibited a 1.7-fold increase in Kcat. Mg2+binding is sigmoidal for the five mutants as well as for the wild-type enzyme, but the Mg2+affinities were decreased (3-22-fold) in mutant FBPases. With the exception of Q32L (8-fold increase), the 50% inhibiting concentrations of AMP for K42E, K42T, I190T, and G191A were increased over 2,000-fold (>10 mM) relative to the wild-type enzyme. Most importantly, a loss of AMP cooperativity was found with K42E, K42T, I190T, and G191A. In addition, the mechanism of AMP inhibition with respect to Mg2+was changed from competitive to noncompetitive for K42T, I190T, and G191A FBPases. Structural modeling and kinetic studies suggest that Lys42, Ile190, and Gly191are located at the pivot point of intersubunit conformational changes that energetically couple the Mg2+-binding site to the AMP domain of FBPase.

Mutants of hexokinase I (Arg539 --> Lys, Thr661 --> Ala, Thr661 --> Val, Gly534 --> Ala, Gly679 --> Ala, and Gly862 --> Ala), located putatively in the vicinity of the ATP binding pocket, were constructed, purified to homogeneity, and studied by circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and initial velocity kinetics. The wild-type and mutant enzymes have similar secondary structures on the basis of CD spectroscopy. The mutation Gly679 --> Ala had little effect on the kinetic properties of the enzyme. Compared with the wild-type enzyme, however, the Gly534 --> Ala mutant exhibited a 4000-fold decrease in kcat and the Gly862 --> Ala mutant showed an 11-fold increase in Km for ATP. Glucose 6-phosphate inhibition of the three glycine mutants is comparable to that of the wild-type enzyme. Inorganic phosphate is, however, less effective in relieving glucose 6-phosphate inhibition of the Gly862 --> Ala mutant, relative to the wild-type enzyme and entirely ineffective in relieving inhibition of the Gly534 --> Ala mutant. Although the fluorescence emission spectra showed some difference for the Gly862 --> Ala mutant relative to that of the wild-type enzyme, indicating an environmental alteration around tryptophan residues, no change was observed for the Gly534 --> Ala and Gly679 --> Ala mutants. Gly862 --> Ala and Gly534 --> Ala are the first instances of single residue mutations in hexokinase I that affect the binding affinity of ATP and abolish phosphate-induced relief of glucose 6-phosphate inhibition, respectively.

Mutants of hexokinase I (Arg539→ Lys, Thr661→ Ala, Thr661→ Val, Gly534→ Ala, Gly679→ Ala, and Gly862→ Ala), located putatively in the vicinity of the ATP binding pocket, were constructed, purified to homogeneity, and studied by circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and initial velocity kinetics. The wild-type and mutant enzymes have similar secondary structures on the basis of CD spectroscopy. The mutation Gly679→ Ala had little effect on the kinetic properties of the enzyme. Compared with the wild-type enzyme, however, the Gly534→ Ala mutant exhibited a 4000-fold decrease in kcatand the Gly862→ Ala mutant showed an 11-fold increase inKmfor ATP. Glucose 6-phosphate inhibition of the three glycine mutants is comparable to that of the wild-type enzyme. Inorganic phosphate is, however, less effective in relieving glucose 6-phosphate inhibition of the Gly862→ Ala mutant, relative to the wild-type enzyme and entirely ineffective in relieving inhibition of the Gly534→ Ala mutant. Although the fluorescence emission spectra showed some difference for the Gly862→ Ala mutant relative to that of the wild-type enzyme, indicating an environmental alteration around tryptophan residues, no change was observed for the Gly534→ Ala and Gly679→ Ala mutants. Gly862→ Ala and Gly534→ Ala are the first instances of single residue mutations in hexokinase I that affect the binding affinity of ATP and abolish phosphate-induced relief of glucose 6-phosphate inhibition, respectively.

Mutation of Arg-15, Glu-19, Arg-22, and Thr-27 of porcine liver fructose-1,6-bisphosphatase was carried out by site-directed mutagenesis. These residues are conserved in all known primary sequences of mammalian fructose-1,6-bisphosphatase. On the basis of the crystal structure of the enzyme, Arg-15, Glu-19, and Arg-22 are located at the interface of the two dimers (C1-C2 and C3-C4), and Thr-27 is in the AMP binding site. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism (CD) spectrometry. No discernible differences were observed between the secondary structures of the wild-type and mutant forms of fructose-1,6-bisphosphatase on the basis of CD data. Kinetic analyses revealed similar kcatvalues for mutants R15A, E19Q, R22K, and T27A of fructose-1,6-bisphosphatase; however, a 2-fold increase of kcatwas observed with R22M compared with that of the wild-type enzyme. Small changes in Kmvalues for fructose-1,6-bisphosphate were found in the five mutants. 4-6-fold decreases in Kivalues for fructose 2,6-bisphosphate and 5-9-fold decreases in the binding affinity of Mg2+relative to the wild-type enzyme were exhibited by R15A and E19Q. No alteration of Mg2+cooperativity was found in the five mutants. Significant changes in Kivalues for AMP were obtained in the case of R22K (30-fold) and T27A (1300-fold) with a Hill coefficient of 2.0. Replacement of Arg-22 with methionine, however, caused the total loss of AMP cooperativity without changing AMP affinity. Modeling of the mutant structures was undertaken in an attempt to define the functional role of Arg-22. These studies link specific interactions between subunits in fructose-1,6-bisphosphatase to observed properties of cooperativity.