Dissociation of yeast hexokinase by hydrostatic pressure

The pressure-induced dissociation of the isozymes P1 and P2 of hexokinase was investigated by studies of the spectral shift of the intrinsic protein fluorescence and by the fluorescence polarization of dansyl conjugates. The free energy of association of the monomers at atmospheric pressure, Katm, was -14.2 kcal mol-1 at 20 degrees C and -11.4 kcal mol-1 at 0 degrees C. The positive enthalpy indicates that the association of the monomers is entropy-driven, overcoming the negative enthalpy of hydration of the subunit interfaces. At 0 degrees C and 1 bar, glucose stabilizes the association by -1.1 kcal mol-1 and the binding of both adenosine 5'-(beta, gamma-methylenetriphosphate) (AMPPCP) and glucose by an even larger amount, -1.34 kcal mol-1. Paradoxically, adenosine 5'-triphosphate (ATP), or AMPPCP, in the absence of glucose destabilizes the association by +0.34 kcal mol-1, while adenosine 5'-diphosphate (ADP) stabilizes it by -0.6 kcal mol-1. Comparison of dV0, the apparent standard volume of association, at different pHs and temperatures indicates that its value (115-160 mL mol-1) is strongly dependent upon the ionization of a group at the subunit interface with a pK near neutrality. Under dissociating pressures, trypsin action results in permanent dissociation of the dimer, confirming earlier observations of Colowick by less direct methods. The P1 and P2 enzymes differ in Katm and dV0 and markedly so in the effects of salt upon the stability of the dimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Genetics of yeast hexokinase

Two independent isolates of Saccharomyces cerevisiae lacking hexokinase activity (EC 2.7.1.1) are described. Both mutant strains grow on glucose but are unable to grow on fructose, and contain two mutant genes hxk1 and hxk2 each. The mutations are recessive and noncomplementing. Genetic analysis suggests that these two unlinked genes hxk1 and hxk2 determine, independently of each other, the synthesis of hexokinase isozymes P1 and P2, respectively. hxk1 is located on chromosome VIR distal to met10, and hxk2 is on chromosome IIIR distal to MAL2. Of four hexokinase-positive spontaneous reversions, one is very tightly linked to hxk1 and the other three to the hxk2 locus. The reverted enzymes are considerably more thermolabile than the respective wild-type enzymes, and in one case show altered immunological properties. Data are presented which suggest that the hxk1 and hxk2 mutations are missense mutations in the structural genes of hexokinase P1 and hexokinase P2, respectively. These are presumably the only enzymes that allow S. cerevisiae to grow on fructose.     

Role of arginyl residues in yeast hexokinase PII.

Yeast hexokinase PII is rapidly inactivated (assayed at pH 8.0) by either butanedione in borate buffer or phenylglyoxal, reagents which are highly selective for the modification of arginyl residues. MgATP alone offers no protection against inactivation, consistent with low affinity of hexokinase for this nucleotide in the absence of sugar. Glucose provides slight protection against inactivation, while the combined presence of glucose and MgATP gives significant protection, suggesting that modified arginyl residues may lie at the active site, possibly serving to bind the anionic polyphosphate of the nucleotide in the ternary enzyme:sugar:nucleotide complex. Extrapolation to complete inactivation suggests that inactivation by butanedione correlates with the modification of 4.2 arginyl residues per subunit, and complete protection against inactivation by the combined presence of glucose and MgATP correlates with the protection of 2 to 3 arginyl residues per subunit. When the modified enzyme is assayed at pH 6.5, significant activity remains. However, modification by butanedione in borate buffer abolishes the burst-type slow transient process, observed when the enzyme is assayed at pH 6.5, to such an extent that after extensive modification the kinetic assays are characterized by a lag-type slow transient process. But even after extensive modification, hexokinase PII still demonstrates negative cooperativity with MgATP and is still strongly activated by citrate when assayed at pH 6.5.     

Autophosphorylation of yeast hexokinase PII

Autophosphorylation of hexokinase PII was studied using an enzyme purified from Saccharomyces cerevisiae. Incubation of this enzyme preparation with [gamma-32P]ATP and Mn2+ or Mg2+ gave a phosphoprotein of molecular mass 58,000 which corresponded to hexokinase PII. D-Xylose stimulated autophosphorylation of hexokinase PII. Dilution of hexokinase PII over a 10-fold concentration range did not change the specific activity of hexokinase PII autophosphorylation suggesting that it may occur by an intramolecular mechanism.     

Evidence for an essential glutamyl residue in yeast hexokinase.

Yeast hexokinase is rapidly inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate and nitrotyrosyl ethyl ester. Sugar substrates afford a partial protection, which is increased by the addition of ADP. Inactivation of the enzyme takes place concomitantly with the incorporation of 1 mol of nitrotyrosine per mol of 50 000-dalton subunit. Exhaustive proteolytic digestion of the modified protein and isolation of the nitrotyrosyl peptide by affinity chromatography, followed by electrophoresis, lead to the identification of the modified residue as a glutamyl residue. This modification of hexokinase occurs without gross conformational changes. The enzyme still binds its substrates, though binding of the nucleotides is perturbed. While the substrates afford a partial protection, they increase the incorporation of nitrotyrosine ethyl ester into the enzyme. This may be attributed to local conformational changes which their binding induces. It is concluded that a glutamyl residue is essential for yeast hexokinase activity and its catalytic function is discussed.