Yeast hexokinase mutants.

Using yeast mutants, it is shown that growth on glucose occurs even in the absence of both hexokinase A and hexokinase B; fructose growth requires at least one of these two enzymes. Expression of hexokinase A and of glucokinase seem to be regulated.     

Yeast hexokinase: substrate-induced association--dissociation reactions in the binding of glucose to hexokinase P-II.

A method is described for the purification of native hexokinases P-I and P-II from yeast using preparative isoelectric focussing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer--dimer association--dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupired in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme-glucose complex was 3 X 10(-4) mol 1(-1) and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I=0.02 mol 1(-1), pH less than 7.5) was also independent of enzyme concentration (in the range 10-1000 mug ml-1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 X 10(-3) mol 1(-1) at pH 7.0 to 6 X 10(-3) at pH 6.5. Under intermediate conditions (pH 7.0, ionic strength=0.15 mol 1(-1)), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperatively (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.     

Glucokinase and hexokinase in pig erythrocytes.

We have shown different enzymatic activities responsible for the phosphorylation of glucose in pig erythrocytes. These activities were observed after partial purification from hemolyzed red cells. One of the enzymes involved is the hexokinase which is present in all tissues; the other is similar to hepatic glucokinase. We have determined the kinetic properties of these activities in hemolysates and in partially purified preparations. Their electrophoretic-migration characteristics were studied too.     

All hexokinase isoenzymes coexist in rat hepatocytes.

The cellular distribution of hexokinase isoenzymes, N-acetylglucosamine Kinase and pyruvate kinases in rat liver was studied. Hepatocytes and non-parenchymal cells with high viability and almost no cross-contamination were obtained by perfusion in situ of the liver with collagenase, with the use of an enriched cell-culture medium in all steps of cell isolation. Separation of hexokinase isoenzymes was done by DEAE-cellulose chromatography, and enzyme activities were measured by a specific radioassay. Cytosol from isolated hepatocytes contained high-affinity hexokinases A, B and C, in addition to hexokinase D. The last-mentioned represented about 95% of total glucose-phosphorylating activity. Only hexokinase A was found associated t the particulate fraction. Isolated non-parenchymal cells contained only hexokinases A, B and C. N-Acetylglucosamine kinase was measured with a specific radioassay and was found as a single enzyme form in both hepatocytes and non-parenchymal cells, with higher activities in the former. Pyruvate kinase isoenzyme L was present only in the hepatocytes and isoenzyme K only in the non-parenchymal liver cells, confirming that they are good cellular markers.     

Competitive inhibition of hexokinase isoenzymes by mercurials.

A difference in the mode of inhibition of hexokinase [EC 2.7.1.1] isoenzymes by p-chloromercuribenzenesulfonate was confirmed with respect to glucose between two Type I isoenzyme preparations purified from the kidney and spleen of rat. Essentially the same difference was observed when galactose was used as the substrate in place of glucose, as the kidney Type I isoenzyme was inhibited in a competitive manner while the spleen counterpart was inhibited in a non-competitive manner by sulfhydryl inhibitor. Both the Type I isoenzymes, however, were competitively inhibited by other mercurial sulfhydryl inhibitors, methyl and butyl mercuric chlorides. On the other hand, the Type II hexokinase isoenzymes purified from the muscle, heart, and spleen were all inhibited competitively by p-chloromercuribenzenesulfonate with respect to glucose. The mechanism of competitive inhibition of the hexokinase isoenzymes by sulfhydryl inhibitors was discussed in view of the difference in the mode of action of the mercurials with different isoenzymes.     

An essential arginyl residue in yeast hexokinase.

The inactivation of yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) by phenylglyoxal obeys pseudo first-order kinetics. Formation of a reversible enzyme-reagent complex prior to modification is suggested by the observed saturation kinetics. Loss of activity correlates with the incorporation of 1 mol of [14C]phenylglyoxal per mol 50 000 dalton subunit. No significant conformational change occurs concomitantly. Inactivation is attributable to modification of an arginyl residue. The pattern of protection by substrates and analogs favors an interaction of this essential residue with the terminal phosphoryl group of ATP or glucose 6-phosphate.     

Dissociation and catalysis in yeast hexokinase A.

1. The specific activity of yeast hexokinase A depends on the concentration of the protein in the solution being assayed. When a solution containing 13.5 mg of hexokinase A/ml is diluted 10--100-fold at various values of pH and temperature, there is a gradual decline in the specific activity of the enzyme until an equilibrium value is reached, which varies with the chosen experimental conditions. 2. The catalytic activity lost when hexokinase A (1 mg/ml) is incubated at 30degreesC is recovered by lowering the temperature to 25degreesC. 3. These concentration- and temperature-dependent phenomena are consistent with the existence of a monomer-dimer equilibrium in which the dimer alone is the catalytic form of the enzyme. 4. Glucose alone prevents the decline in specific activity of hexokinase A after dilution, but it does not re-activate dilute solutions solutions of the enzyme. It is concluded that glucose binds to both the dimer and the monomer and prevents both association and dissociation. 5. The progress curve describing the phosphorylation of glucose catalysed by hexokinase A does not attain a steady state. It is possible that dissociation of catalytically active dimers in a ternary complex with glucose and ATP (or glucose 6-phosphate and ADP) could explain the non-linearity of this progress curve.     

Hexose metabolism in pancreatic islets. Inhibition of hexokinase.

In islet homogenates, hexokinase-like activity (Km 0.05 mM; Vmax. 1.5 pmol/min per islet) accounts for the major fraction of glucose phosphorylation. Yet the rate of glycolysis in intact islets incubated at low glucose concentrations (e.g. 1.7 mM) sufficient to saturate hexokinase only represents a minor fraction of the glycolytic rate observed at higher glucose concentrations. This apparent discrepancy between enzymic and metabolic data may be attributable, in part at least, to inhibition of hexokinase in intact islets. Hexokinase, which is present in both islet and purified B-cell homogenates, is indeed inhibited by glucose 6-phosphate (Ki 0.13 mM) and glucose 1,6-bisphosphate (Ki approx. 0.2 mM), but not by fructose 2,6-bisphosphate. In intact islets, the steady-state content of glucose 6-phosphate (0.26-0.79 pmol/islet) and glucose 1,6-bisphosphate (5-48 fmol/islet) increases, in a biphasic manner, at increasing concentrations of extracellular glucose (up to 27.8 mM). From these measurements and the intracellular space of the islets, it was estimated that the rate of glucose phosphorylation as catalysed by hexokinase represents, in intact islets, no more than 12-24% of its value in islet homogenates.