Fructose 1,6-diphosphate enhanced inactivation of yeast pyruvate kinase at 23°. Evidence for a stabilized dimer intermediate

Interactions of fructose 1,6-diphosphate, substrates, and monovalent cations with yeast pyruvate kinase monitored by changes in enzyme fluorescence

“…The effect appears to be due to both an increase in ionic strength since (CH3)4N+ (Figure 3) yields the same increasing trend in apparent Kd's for FDP although it does not function kinetically, and a specific K+ effect, since the extent of the increase in apparent Kb for FDP produced by (CH3)4N+ is not as great as by K+. A specific K+ effect is also supported by the report (Kuczenski and Suelter, 1971) that K+ and (CH3)4N+ do not exert equal stabilizing effects during the FDP-enhanced inactivation of the enzyme; K+ is a more effective stabilizer. The antagonistic effect of ionic strength on the binding of FDP is also observed in the catalytic assay using concentrations of ADP, PEP, FDP, and K+ which are below Km (0.5, 0.087, 0.027, and 10.0 mM, respectively) (R. T. 0 All runs were performed at 20°in 0.1 m Tris-HCl (pH 7.5).…”

M2 isozyme of pyruvate kinase from human kidney as the product of a separate gene: its purification and characterization

“…The data on the M2 isozyme, including the stability, are consistent with fructose 1,6-bisphosphate influencing a dimer-tetramer equilibrium in favor of the tetramer. This is in contrast to the effect of fructose 1,6-bisphosphate on the yeast enzyme where the dimer-tetramer equilibrium is altered in the direction of the dimer (Kuczenski and Suelter, 1971). Both the yeast and rat L isozyme of pyruvate kinase are made labile by fructose 1,6-bisphosphate while the M2 isozyme is stabilized suggesting that there are fundamental differences in the molecular response of the various isozymes to this organic phosphate even though the kinetic effect of the modulator in each instance is activation.…”

The preparation and properties of pyruvate kinase from yeast

“…On the basis of their studies of the cold-lability of yeast pyruvate kinase, Kuczenski & Suelter (1970a) suggested that the enzyme was not a homologous tetramer, but a dimer of dimers. This conclusion was reinforced by their later demonstration of the existence of a dimer intermediate in the dissociation promoted by fructose diphosphate at 23°C (Kuczenski & Suelter, 1971). The observation of fractional reactivity of the thiol groups reported here would be consistent with their proposal.…”

“…Therefore for it to be possible to observe less than four thiol groups reacting/mol of enzyme, either the four subunits are not equivalent in the molecule, or, less probably, there are four equivalent thiol groups at a site where steric hindrance prevents the entry of more than two iodoacetamide or thionitrobenzoate moieties. A heterologous tetramer has also been proposed by Kuczenski & Suelter (1971) to explain the results of their studies on the inactivation of the enzyme catalysed by fructose diphosphate. Similar instances have been documented by Levitzli et al (1971) in connexion with 'half-the-sites' reactivity.…”