Pyruvate kinase reaction

FIGURE 3.13 Phosphoenolpyruvate (PEP) is produced by the euolase reaction (hi glycolysis see Chapter 19) and hi turn drives the phosphorylation of ADP to form ATP in the pyruvate kinase reaction. 

FIGURE 19.29 A mechanism for the pyruvate kinase reaction, based on NMR and EPR studies by Albert Mildvan and colleagues. Phosphoryl transfer from phosphoenolpyrnvate (PEP) to ADP occurs in four steps (a) a water on the Mg ion coordinated to ADP is replaced by the phosphoryl group of PEP (b) Mg dissociates from the -P of ADP (c) the phosphoryl group is transferred and (d) the enolate of pyruvate is protonated. (Adapted from Mildvan, A., 1979. Advances in Eiizymology 49 103-126.)... 

The conversion of pyruvate into phosphoenolpyravate, which bypasses the pyruvate kinase reaction, requires two separate reactions carboxylation of pyruvate to... 

In the last step, pyruvate kinase transfers this residue to ADP. The remaining enol pyruvate is immediately rearranged into pyruvate, which is much more stable. Along with step [7] and the thiokinase reaction in the tricarboxylic acid cycle (see p. 136), the pyruvate kinase reaction is one of the three reactions in animal metabolism that are able to produce ATP independently of the respiratory chain. 

The first steps of actual gluconeogenesis take place in the mitochondria. The reason for this detour is the equilibrium state of the pyruvate kinase reaction (see p. 150). Even coupling to ATP hydrolysis would not be sufficient to convert pyruvate directly into phos-phoenol pyruvate (PEP). Pyruvate derived... 

The pyruvate kinase reaction is virtually irreversible under typical intracellular conditions, and the enzyme requires Mg or Mn in addition to the monovalent cation... 

Enzymes required to reverse the pyruvate kinase reaction... 

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...

The first step in the gluconeogenic direction involves the formation of phosphoenolpyruvate from pyruvate. Reversal of the pyruvate kinase reaction requires at least two ATP-to-ADP conversions. One means by which this is done is shown in figure 12.26. 

We stated that the equilibrium constant for the pyruvate kinase reaction is 106. Assume that the steady-state concentration of ATP is 2 mM and of ADP is 0.2 mM. Calculate the concentration ratio of pyruvate to PEP under these conditions. Does your calculation support or refute the assertion that the pyruvate kinase reaction is metabolically irreversible ... 

Given the ratio of ATP/ADP and the K q of 106, the equilibrium ratio of [Pyry]/[PEP] would be about 105. This calculation supports the metabolic irreversibility of the pyruvate kinase reaction. 

C, assuming that the pyruvate kinase reaction (see Fig. 13-13) is at equilibrium in the cell. 

The enzyme that catalyzes the conversion of PEP to pyruvate is pyruvate kinase. Liver pyruvate kinase is stimulated allosterically by fructose-1,6-diphosphate, AMP, ADP, and glyceraldehyde-3-phosphate. It is inhibited by alanine, ATP, NADH, and, more importantly, by cAMP- and Ca2 calmodulin-controlled phosphorylation. High blood glucagon levels thus inhibit the activities of both PFK II and pyruvate kinase in the liver through phosphorylation. Transcription of pyruvate kinase is also decreased by glucagon and increased by insulin. Muscle pyruvate kinase is not subject to cAMP or Ca2+ regulation. The pyruvate kinase reaction is practically irreversible. 

Pyruvate converted to PEP without using the pyruvate kinase reaction Formally, pyruvate is first converted to oxaloacetate, which is in turn converted to PEP. In the first reaction of this process P3mivate carboxylase adds carbon dioxide to pyruvate with the expenditure of one ATP equivalent of energy. Biotin, a carboxyl-group transfer cofactor in animals, is required by this enz)one ... 

The pyruvate kinase reaction has a large equilibrium constant because the initial product of pyruvate, the enol form, rearranges nonenzymatically to the favored keto form ... 

Because of the very high price of ATP, reaction (5.7) must be coupled with a regenerating system, the transfer of phosphate to ADP starting from the enol phosphate of pyruvic acid (an easily accessible and inexpensive phosphate), catalysed by the enzyme pyruvate kinase (reaction (5.8). In the same flask are mixed glucose, phosphoenolpyruvate, hexokinase, pyruvate kinase, and a catalytic quantity of ATP (about 1% mol) and the system produces D-glucose 6-phosphate until the phosphoenolpyruvate runs out. The kinases are easily accessible and, if they are immobilized on an insoluble support (see Section 10.4.1), they are reusable a certain number of times. In this way glucose 6-phosphate can be easily prepared on a 250 g scale (Poliak et al. 1977). 

Termonia and Ross (1981-1,2) developed a reaction scheme coupling phosphofructo-kinase and pyruvate kinase reactions by referring to experimental observations of known activations and inhibitions of enzymes by metabolites. By numerical analysis of the rate equations they confirmed the oscillations in the concentrations of fructose-6-phosphate, pyruvate, phosphoenolpyruvate, fructose 1,6-biphosphate, and ADP. 

The principle enzymes of glycolysis involved in regulation are hexokinase (reaction 1), phosphofructokinase (reaction 3), and pyruvate kinase (reaction 10) ... 

Phosphoenol pyruvate pyruvate. The third big step in the free-energy diagram is the pyruvate-kinase reaction, where ATP is formed from phosphoenol pyruvate. ATP inhibits pyruvate kinase, similar to the inhibition of PFK. Pyruvate kinase is also inhibited by acetyl-Coenzyme A, the product of pyruvate metabolism that enters the TCA cycle. Fatty acids also allosterically inhibit pyruvate kinase, serving as an indicator that alternative energy sources are available for the cell. 

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