The regulatory properties of yeast pyruvate kinase. Effects of NH4+ and K+ concentrations

Abstract

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied at 25 degrees C and pH 6.2 as a function of the concentrations of ADP, phosphoenolpyruvate, Mg2+ and either NH4+ or K+. The data were analysed by the exponential model for four substrates, obtained by extension of the model described by Ainsworth, Kinderlerer & Gregory [(1983) Biochem. J. 209, 401-411]. On that basis, it was concluded that NH4+ binding is almost non-interactive but leads to the appearance of positive interaction in the velocity response to increase in its concentration because of positive interactions with phosphoenolpyruvate and Mg2+. The data obtained with K+ lead to the same conclusions and differ only in suggesting that NH4+ is bound more strongly to the enzyme than is K+. Both data sets are used as the basis for a discussion of the substrate interactions of pyruvate kinase and it appears therefrom that the heterotropic interactions accord with what is known of the events that take place at the active site during catalysis. The paper also reports a determination of the dissociation constants for the NH4+ complexes with ADP and phosphoenolpyruvate and an examination of the simultaneous activation of pyruvate kinase by K+ and NH4+ ions.

Pyruvate kinase variant of fission yeast tunes carbon metabolism, cell regulation, growth and stress resistance

“…Notably, the A‐strain was also more resistant to thallium (I) acetate, which is highly toxic due to its similarity to potassium ions and binds to mammalian PYK with a stronger affinity, but weaker activating effect, than potassium (Kayne, ; Reuben & Kayne, ). The T‐strain, on the other hand, was more resistant to acriflavine, an antiseptic, and to EGTA, a chelator of bivalent cations (including Mg 2+ which activates S. cerevisiae PYK, Rhodes et al , ). We manually inspected dose–response curves (Appendix Fig S8) to select compounds for validation based on overall difference in maximum growth rate and consistency across concentrations.…”

Dichotomic Phylogenetic Tree of the Pyruvate Kinase Family

“…Position 117 is occupied by Glu in 121 sequences and by Lys, Ser, and Arg in 106, 2, and 1 sequence, respectively. The requirement for K ϩ by 21 pyruvate kinases that have Glu at position 117 has been examined, and the activity of all of them is K ϩ -dependent (8,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). The activities of eight pyruvate kinases that do not have Glu at that position have been characterized; in all cases the activity is K ϩ -independent.…”

“…In addition, it has been shown that K ϩ changes the kinetic mechanism of rabbit muscle pyruvate kinase and is involved in the acquisition of the active conformation of the enzyme (20 (21), a water molecule, and a phosphate oxygen of phosphoenolpyruvate analogs (22), or an oxygen from the ␥-phosphate of ATP (5). For a long time, it was thought that the absolute dependence of K ϩ was a common feature to all pyruvate kinases (8,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). However, as the number of characterized enzymes increased, it became apparent that the activity of several pyruvate kinases was K ϩ -independent, for example those from Escherichia coli (Type II) (39), Phycomyces blakesleeanus (40), Corynebacterium glutamicum (41), Zymomonas mobilis (42), Thermoproteus tenax (43), Synechococcus pcc 6301 (44), Archaeoglobus fulgidus, Pyrobaculum aerophilum, Aeropyrum pernix, and Thermotoga maritima (45).…”

The cooperative binding of fructose-1,6-bisphosphate to yeast pyruvate kinase.

“…The enzyme from yeast has been widely studied and its kinetics are well defined (Haeckel et al, 1968;Kinderlerer et al, 1986;Rhodes et al, 1986). The gene for yeast pyruvate kinase has been cloned and expressed to high levels in the yeast Saccharomyces cerevisiae and this allows easy purification of large amounts of the enzyme (Murcott et al, 1991).…”