Kinetic and Allosteric Consequences of Mutations in the Subunit and Domain Interfaces and the Allosteric Site of Yeast Pyruvate Kinase

130

Deficiency of human erythrocyte isozyme (RPK) is, together with glucose-6-phosphate dehydrogenase deficiency, the most common cause of the nonspherocytic hemolytic anemia. To provide a molecular framework to the disease, we have solved the 2.7 Å resolution crystal structure of human RPK in complex with fructose 1,6-bisphosphate, the allosteric activator, and phosphoglycolate, a substrate analogue, and we have functionally and structurally characterized eight mutants (G332S, G364D, T384M, D390N, R479H, R486W, R504L, and R532W) found in RPK-deficient patients. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. The mutations affect to a different extent thermostability, catalytic efficiency, and regulatory properties. These studies are the first to correlate the clinical symptoms with the molecular properties of the mutant enzymes. Mutations greatly impairing thermostability and/or activity are associated with severe anemia. Some mutant proteins exhibit moderate changes in the kinetic parameters, which are sufficient to cause mild to severe anemia, underlining the crucial role of RPK for erythrocyte metabolism. Prediction of the effects of mutations is difficult because there is no relation between the nature and location of the replaced amino acid and the type of molecular perturbation. Characterization of mutant proteins may serve as a valuable tool to assist with diagnosis and genetic counseling.

Section: Discussionsupporting

confidence: 87%

Structure and Function of Human Erythrocyte Pyruvate Kinase

Valentini¹,

Chiarelli²,

Fortin³

et al. 2002

130

“…Thus the replacements of the former four mutant enzymes seem to have caused either severe impairment in the ligation with a whole Glc-6-P molecule or damage on the structure necessary for the transmission of the allosteric signal arising from the binding of Glc-6-P. A similar observation has been reported previously (40) with a yeast pyruvate kinase of which the allosteric activator is Fru-1,6-P 2 . In this case, the replacement of Arg-459 participating in the binding with the phosphate group of Fru-1,6-P 2 to Gln caused complete desensitization to this effector, indicating that this Arg residue is also involved in allosteric signal transmission (41).…”

Section: Discussionmentioning

confidence: 84%

“…In fact, this case is very similar to that of glycogen phosphorylase from yeast. In this enzyme, the nonphosphorylated N-terminal peptide is extended to reach the catalytic site of the adjacent subunit to inhibit the enzyme, and when phosphorylated the N-terminal peptide folds back and the phosphate group is trapped at the allosteric inhibition site for Glc-6-P resulting deinhibition (41). Competition of the binding site between the phosphate group on Ser-15 and Glc-6-P was indeed demonstrated (49).…”

Section: Discussionmentioning

confidence: 97%

Maize Phosphoenolpyruvate Carboxylase

Takahashi-Terada

Kotera

Ohshima

et al. 2005

Phosphoenolpyruvate carboxylases (PEPC, EC 4.1.1.31) from higher plants are regulated by both allosteric effects and reversible phosphorylation. Previous x-ray crystallographic analysis of Zea mays PEPC has revealed a binding site for sulfate ion, speculated to be the site for an allosteric activator, glucose 6-phosphate (Glc-6-P) (Matsumura, H., Xie, Y., Shirakata, S., Inoue, T., Yoshinaga, T., Ueno, Y., Izui, K., and Kai, Y. (2002) Structure (Lond.) 10, 1721-1730). Because kinetic experiments have also supported this notion, each of the four basic residues (Arg-183, -184, -231, and -372 on the adjacent subunit) located at or near the binding site was replaced by Gln, and the kinetic properties of recombinant mutant enzymes were investigated. Complete desensitization to Glc-6-P was observed for R183Q, R184Q, R183Q/R184Q (double mutant), and R372Q, as was a marked decrease in the sensitivity for R231Q. The heterotropic effect of Glc-6-P on an allosteric inhibitor, L-malate, was also abolished, but sensitivity to Gly, another allosteric activator of monocot PEPC, was essentially not affected, suggesting the distinctness of their binding sites. Considering the kinetic and structural data, Arg-183 and Arg-231 were suggested to be involved directly in the binding with phosphate group of Glc-6-P, and the residues Arg-184 and Arg-372 were thought to be involved in making up the site for Glc-6-P and/or in the transmission of an allosteric regulatory signal. Most unexpectedly, the mutant enzymes had almost lost responsiveness to regulatory phosphorylation at Ser-15. An apparent lack of kinetic competition between the phosphate groups of Glc-6-P and of phospho-Ser at 15 suggested the distinctness of their binding sites. The possible roles of these Arg residues are discussed.

“…A similar modification of the kinetic parameters has been described for a point mutation in the Escherichia coli pyruvate kinase (38) precisely in a residue (Arg-271) located in the interface between the catalytic domain A and the regulatory domain C of FBP binding, suggesting that Ser-22 in Pyk1, located structurally in the same interface, might be the target for PKA phosphorylation. It is interesting to mention that yeast cells, containing a point mutation in a residue (R19Q) located in the vicinity of Ser-22 (Arg-19, contained in the consensus PKA phosphorylation sequence RRTS) from yeast Pyk1, do not grow in glucose as carbon source (39), indicating null or low catalytic activity for the Pyk1 from this mutant. The modification in the kinetic parameters observed, taken as a whole, suggest that the phosphorylated enzyme is more active; phosphorylation of rat liver pyruvate kinase by PKA, on the contrary, shifts the equilibrium between the active and inactive forms of the enzyme to favor the inactive form (40).…”

Section: Discussionmentioning

confidence: 99%

In Vivo and in Vitro Phosphorylation of Two Isoforms of Yeast Pyruvate Kinase by Protein Kinase A

Portela

Howell²,

Moreno

et al. 2002

Saccharomyces cerevisiae pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HATpk1⅐Bcy1) and to be phosphorylated in a cAMP-dependent process. Both glutathione S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated in vivo, in intact cells overexpressing the protein, or in vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK gene with an attenuated mutation (tpk1 w1 ). The effect of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels. Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mM. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6-bisphosphate it shows an n H value of 1.4, as compared with an n H of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity.