Pyruvate kinase kinetics

CK catalyzes the reversible phosphorylation of creatine in the presence of ATP and magnesium. When creatine phosphate is the substrate, the resulting creatine can be measured as the ninhydrin fluorescent compound, as in the continuous flow Auto Analyzer method. Kinetic methods based on coupled enzymatic reactions are also popular. Tanzer and Gilvarg (40) developed a kinetic method using the two exogenous enzymes pyruvate kinase and lactate dehydrogenase to measure the CK rate by following the oxidation of NADH. In this procedure the main reaction is run in a less favorable direction. 

Pyruvate kinase (PK) is one of the three postulated rate-controlling enzymes of glycolysis. The high-energy phosphate of phosphoenolpyruvate is transferred to ADP by this enzyme, which requires for its activity both monovalent and divalent cations. Enolpyruvate formed in this reaction is converted spontaneously to the keto form of pyruvate with the synthesis of one ATP molecule. PK has four isozymes in mammals M, M2, L, and R. The M2 type, which is considered to be the prototype, is the only form detected in early fetal tissues and is expressed in many adult tissues. This form is progressively replaced by the M( type in the skeletal muscle, heart, and brain by the L type in the liver and by the R type in red blood cells during development or differentiation (M26). The M, and M2 isozymes display Michaelis-Menten kinetics with respect to phosphoenolpyruvate. The Mj isozyme is not affected by fructose-1,6-diphosphate (F-1,6-DP) and the M2 is al-losterically activated by this compound. Type L and R exhibit cooperatively in... 

L2. Lakomek, M Huppke, P Neubauer, B Pekrun, A., Winkler, H., and Schroter, W., Mutations in the R-type pyruvate kinase gene and altered enzyme kinetic properties in patients with hemolytic anemia due to pyruvate kinase deficiency. Ann. Hematol. 68,253-260 (1994). 

Treatment of 9-(/ -D-ribofuranosyluronic acid)adenine with diphenylphosphoro-chloridate and orthophosphate or tripolyphosphate yields (62) and (63), which, although unstable, inhibit rabbit AMP aminohydrolase and pyruvate kinase, respectively, with behaviour characteristic of active-site-specific reagents.98 Adenylate kinases from several sources are inactivated by iV6-[2- and 4-fluorobenzoyl]-adenosine-5 -triphosphates, with kinetics characteristic of active-site labelling, although these compounds were without effect on yeast hexokinase and rabbit pyruvate kinase.99... 

Facilitated diffusion within organisms takes place when carriers or proteins residing within membranes—ion channels, for instance—organize the movement of ions from one location to another. This diffusion type is a kinetic, not thermodynamic, effect in which a for the transfer is lowered and the rate of diffusion is accelerated. Facilitated diffusion channels organize ion movements in both directions, and the process can be inhibited both competitively and noncompetitively. It is known that most cells maintain open channels for K+ most of the time and closed channels for other ions. Potassium-ion-dependent enzymes include NaVK+ ATPases (to be discussed in Section 5.4.1), pyruvate kinases, and dioldehydratases (not to be discussed further). 

Effect of pyruvate kinase deficiency Pyruvate kinase deficiency accounts for 95 percent of all inherited defects in glycolytic enzymes. It is restricted to erythrocytes, and causes mild to severe chronic hemolytic anemia. Altered kinetics (for example, increased Km, decreased Vmax, etc.) most often account for the enzyme deficiency. 

Pyruvate kinase (PK) from F. hepatica closely resembles L-type PK from mammalian liver (Behm and Bryant, 1980). It shows cooperative kinetics with PEP, but in the presence of... 

Kahn A, Marie J, Garreau H, Sprengers ED. The genetic system of the L-type pyruvate kinase forms in man. Subunit structure, interrelation and kinetic characteristics of the pyruvate kinase enzymes from erythrocytes and liver. Biochim Biophys Acta 1978 523 59-74. 

C. The activity of regulatory enzymes such as fructose-1,6-bisphos-phatase, hexokinase, phosphofructokinase 1, and pyruvate kinase are frequently controlled by binding allosteric effectors. These allosteric enzymes usually exhibit sigmoidal kinetics. Lactate dehydrogenase is not controlled by allosteric effectors and therefore would be expected to exhibit Michaelis-Menten kinetics. 

Changes in the Kinetic Prqierties of the Enzyme Pyruvate Kinase as a Model Enzyme 161... 

Table 2. Kinetic and Regulatory Properties of Pyruvate Kinases ...

Table 3. Whelk Muscle Kinetic Constants of Pyruvate Kinase and Metabolite Levels in Aerobic Versus Anoxic Tissue ...

Table 4. Interactions Between pH and Temperature on the Kinetic Properties of Pyruvate Kinase from Whelk Muscle ...

Berlund, L. Humble, E. (1979). Kinetic properties of pyruvate kinase type A from kidney and type M from muscle. Arch. Biochem. Biophys. 195,347-361. 

Storey, K. B. (1985b). Kinetic and regulatory properties of pyruvate kinase isozymes from flight muscle and fat body of the cockroach, Periplaneta americana. J. Comp. Physiol. 155, 339-345. 

The geometry of a kinetically active complex of pyruvate kinase, Mn2+, K+, phosphate, and pyruvate which catalyzes the enolization of pyruvate has been elucidated by 13C and 31P NMR ((45) Fig. 4B)). The distances from Mn to the carboxyl and carbonyl carbon atoms of pyruvate (7.3 0.1 A) establish the substrate to be in the second coordination sphere, while the lower limit Mn2+ to phosphorus distance (4.5 0.4 A) suggests either a distorted inner sphere complex or the rapid averaging of 6% inner sphere complex with 94% second sphere complex. Mechanisms for pyruvate kinase, consistent with the geometries are given in Fig. 4 (15, 43, 45). 

This enzyme catalyzes a reaction that appears to be very homologous to that of pyruvate kinase. One similarity other than the proposed mechanism is that this enzyme also requires two cations to elicit the most active form of the enzyme. Although calculations of kinetic activation data indicate that both GDP and metal-GDP (and GTP and metal-GTP) can both serve as substrates for the enzyme, the function of GDPrree as substrate has not been demonstrated, and if so, the metal-GDP complex is the more active form (79). Unlike pyruvate kinase, this enzyme does not require a monovalent cation. Kinetic activation studies indicate that the enzyme-bound metal facilitates the interaction of the substrates phosphoenolpyruvate and nucleotide with the enzyme, but not the substrate CO2. High-resolution NMR studies of both phosphoenolpyruvate and the nucleotide have demonstrated that in contrast to the classic case of pyruvate kinase, the substrates appear to bind in the second coordination sphere of the bound cation. These results indicate a much different role in the activation processes. The activation appears to be modulated by metal-bound water molecules. This interaction appears to be with both the phosphate of phosphoenolpyruvate (80) and the y-phosphate of GTP (79) [as indicated in Fig. 6 (80).] Thus, in the activation of phosphoenolpyruvate, the metal must increase the cationic character of the water molecule with which the phosphate of the substrate interacts. 

A peptide from pyruvate kinase labeled with oADP (55) and one from ferre-doxin-NADP reductase labeled with oNADP (75) have been isolated and characterized. These are exceptions. Despite the large number of papers describing the kinetics of affinity labeling by periodate-oxidized nucleotides, there are very few reports of the identification of particular amino acids labeled by these reagents within determined peptide sequences. For enzyme products other than a Schiff base reducible by NaBHU, the instability of the products in the proteolytic digests of modified enzymes under conditions of peptide purification has precluded isolation of labeled peptides in most cases. 

Callens, M. and Opperdoes, F. R. (1992) Some kinetic properties of pyruvate kinase from Trypanosoma brucei. Mol. Biochem. Parasitol. 50 235-244. 

Several mechanistic explanations were proposed to explain deviations from Michaelis -Menten kinetics. Fo example for the synthesis of pyruvate the binding of one molecule of phosphoenol pyruvate (PEP) to pyruvate kinase increases the affinity with which pyruvate kinase binds subsequent molecules of phosphoenol pyruvate (Figure 6.22). 

Isani, G. Cattani, O. Carpene, E. Cortesi, P. Kinetic properties of liver and muscle pyruvate kinase of a marine teleost, sea bass (Dicentrarchus labrax L.).. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 107, 617-624 (1994)... 

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