In pyruvate kinase

B30. B ianchi, P Terragna, C., Zappa, M., Alfinito, F and Zanella, A., Molecular characterization of L-PK gene in pyruvate kinase (PK) deficient Italian patients. Blood 84 (Suppl. 1), 14a (1994). 

The red blood cell has no mitochondria and is totally dependent on anaerobic glycolysis for ATP. In pyruvate kinase deficiency, the decrease in ATP causes the erythrocyte to lose its characteristic biconcave shape and signals its destruction in the spleen. In addition, decreased ion pumping by Na /K -ATPase results in loss of ion balance and causes osmotic fragility, leading to swelling and lysis. 

Figure 12-20 Equilibria in pyruvate kinase reaction as studied by 31P NMR at 40.3 MHz, pH 8.0,15°C. (A-C) Equilibria with low enzyme in levels 15% 2H20. (A) nP NMR spectrum of 1.5 ml of reaction mixture PEP, 13.3 mM ADP, 14.1 mM MgCl2, 20 mM potassium Hepes buffer, 100 mM KC, 50 mM without enzyme. (B) Equilibrium mixture after the addition of 1 mg of pyruvate kinase to the reaction mixture. (C) Equilibrium after the addition of potassium pyruvate (final concentration of 200 mM) to the sample of the spectrum in (B). (D,E) Equilibrium with enzyme concentrations in excess of the substrates. Sample volumes 1.1 ml with 10% 2H20. (D) Equilibrium mixture set up with enzyme (2.8 mM active sites) 2.8 mM PEP 2.4 mM ADP 5.7 mM MgCl2 100 mM potassium Hepes 100 mM KC1. (E) Spectrum after the addition of 50 pi of 400 mM EDTA (pH readjusted to 8.0) to the sample of spectrum D. The EDTA removes metal ions, stopping the catalytic reactions and sharpening the resonances. From Nageswara Rao et al.685...

A different problem results from deficiency of enzymes of glycolysis such as phosphofructokinase (see Box 20-D), phosphoglycerate rnutase, and pyruvate kinase. Lack of one isoenzyme of phosphoglycerate rnutase in muscle leads to intolerance to strenuous exercise/ A deficiency in pyruvate kinase is one of the most common defects of glycolysis in erythrocytes and leads to a shortened erythrocyte lifetime and hereditary hemolytic anemia.s... 

In view of these problems with Ni2+, Mna+ has been used as a probe for Mg2+ with some success. However, it should be noted that there is a difference in radius which may be manifested in different biochemical behaviour (Mn2+, 0.80 A Mg2+, 0.65 A). Thus Mn2+ has been used to probe the Mg2+ site in pyruvate kinase.95 While the Mg2+-activated enzyme is inhibited by Ca2+ and Li+, the Mn2+-activated enzyme is inhibited by Ca2+ and not by Li+. There are also differences in the catalytic and regulatory properties of the NAD+-specific malic enzyme of E. coli,104 depending upon whether the divalent activator is Mn2+ or Mg2+. It is necessary, therefore, to express a cautionary note. These two cations may act in slightly different ways to bring about a similar final result. In the second example it appears that the metal cofactors stabilize two different conformational states of the enzyme. 

The major end-products of carbohydrate metabolism in this species are lactate and succinate. The relative amount formed of each depends on the presence or absence of oxygen in the incubation medium, the presence or absence of glucose, and the presence or absence of fumarate. For example, anaerobiosis leads to an increase in lactate production, which is accompanied by a fall in the intracellular level of malate. Malate is an inhibitor of pyruvate kinase in M. expansa, so the fall in malate levels results in an increase in pyruvate kinase activity leading to a rise in lactate production. Conversely, under aerobic conditions, malate levels increase, pyruvate kinase activity is inhibited and lactate production is decreased (Fig. 5.5). 

In hexokinase deficiency, [BPG] is low and so oxygen binding increases and 02 is not released to the tissues properly. In pyruvate kinase deficiency, [BPG] is high and so oxygen binding is impaired and hemoglobin will not carry 02 to peripheral tissues. 

Lodato and Reed (1987) used epr spectroscopy with O-labelled substrates and inhibitors to analyse the binding of metal ions and substrates in pyruvate kinase, whose reaction is... 

The replacement of Mg by Mn allows distance measurements to be made from Mn to the site of NMR active monovalent cations. As discussed later, this allows a structure of the active site in pyruvate kinase to be established which shows that the phosphenolpyruvate substrate is linked to the enzyme surface through K (Figure 8). The activation of pyruvate by has been used as an analytical method for K" in the range 10 to 10 mol dm. ... 

Fl ire 2. Conformational changes in pyruvate kinase, induced by the binding of phosphoenolpynivate (A). 

FIGURE 4.16 Some a-helix and P-conformation arrangements in proteins, (a) P-barrel shape in pyruvate kinase, (b) Saddle shape in flavodoxin... 

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