Pyruvate kinase determination

Genetically-determined deficiency of G6PD is the most common cause of haemolysis arising from enzyme defects. Mutated glycolytic enzymes such as hexokinase, phosphofructokinase, aldolase and pyruvate kinase can also bring about haemolysis but the occurrence of these defects are much rarer than for G6PD deficiency (see Case N otes at the end of this chapter). 

Pyruvate kinase is a key enzyme in the important process of glycolysis, and its product, pyruvate, is passed into the Krebs cycle. As noted in Section 62.1.2.3.1, pyruvate requires two divalent cations and one monovalent cation for activity. These are usually Mg2+ and K+. The crystal structure of the enzyme from cat muscle has been determined to a resolution of 2.6 A.280... 

Creatine kinase was purified from rabbit muscle by the method of Kuby et al, (4). Rabbit muscle pyruvate kinase was purchased from Boehringer. Porcine muscle adenylate kinase was purchased from Sigma, and was further purified by gel filtration on Sephadex G-50. The enzymes were homogeneous as judged by their specific activities and by their migration as single components in sodium dodecyl sulfate gel electrophoresis. Proton NMR spectra at 250 MHz of 0.5-2.0 mM enzyme sites in 0 solution were obtained with a Bruker WM 250 MHz pulse FT spectrometer at 25°. At least 256 transients were accumulated over 8192 data points using 16 bit A/D conversion. Relaxation rates and histidine pK values were determined by standard NMR methods (5, 6),... 

Pyruvate Kinase. Six of the fourteen imidazole C-2 proton resonances and three imidazole C-4 proton resonances of this tetra-meric enzyme (Mr 237,000) were detected and their pK values determined. The substrate P-enolpyruvate selectively decreased the pK of one histidine (his(3)) by 0.4 units, from a value of 6.2 to 5.8, only in the presence of the cation activators Mg2+ and K+... 

These values are too short to be influenced significantly by Tr, the rotational correlation time of the enzyme-Gd3+ complex, or Tm, the mean residence time of water molecules in the first coordination sphere of the metal. Moreover, the minima in the plots of Tj p vs. Wj2 indicate that Tc must be dominated by Ts, the electron spin relaxation time. The Ts values for Gd + in this system are longer than most of those determined previously for Gd3+. The electron spin relaxation time for aqueous Gd3+ is (4-7) x 10 10s at 30 MHz (42), while values for Ts of (2-7) x 10 10s have been reported for complexes of Gd3+ with pyruvate kinase (37) and a value of 2.2 x 10- s has been found for a Gd 1"-lysozyme complex (36). Moreover, we have estimated a Tc of 6,8 x 10 10s for Gd + bound to parvalbumin.3 The long Gd3+ correlation times found in the present study are consistent with a poor accessibility of these Gd3+ sites to solvent water molecules. 

Most cestodes which have been investigated, however, conform to the second category, type 2, which is characterised by a C02-fixation step. Carbohydrate is degraded to the level of PEP by glycolysis, the steps involved being similar to those in mammalian tissue. At this point, the enzymes pyruvate kinase and phosphoenolpyruvate carboxykinase (PEPCK) compete for available substrate and a branch-point occurs (Fig. 5.4). The relative activities of these two enzymes determine the fate of the PEP and the subsequent types and amounts of end-products formed (see below). 

L(+)-Lactate dehydrogenase is specific for l(+)-lactate and does not react with d(—)-lactate. LDH is used in coupled enzyme assays, for example in the determination of ATPase (Penefsky and Bruist 1984), myokinase (Brolin 1983), and pyruvate kinase (Beutler 1971). It may also be used in the determination of lactate (Noll 1984), pyruvate (Lamprecht and Heinz 1984), and various other metabolites. 

The stereochemical consequence of [lsO]thiophosphoryl and [l60, nO, lsO]phos-phoryl group transfer catalyzed by seven phosphotransferases were simultaneously determined in the author s laboratory and in the laboratory of J.R. Knowles. The first to be completed was the demonstration of inversion by adenylate kinase however, prior to that glycerokinase, hexokinase and pyruvate kinase had been shown to catalyze [l80]thiophosphoryl group transfer with the same stereochemical consequences, either all with inversion or all with retention. Glycerokinase was later shown to catalyze both [l60, nO, l80]phosphoryl and [l80]thiophosphoryl group... 

The activity of the ATP-forming enzyme complex V is usually assessed by determining the reverse reaction ATP — ADP + Pi. The reaction is coupled to reactions catalyzed by pyruvate kinase (ADP + phosphoenolpyruvate —> pyruvate + ATP) and lactate dehydrogenase (pyruvate + NADH — lactate -F NAD+). This final reaction can be followed spectrophotometrically by measuring NADH at 340 nm. The activity of complex V (ATPase) can be derived from the rate of NADH conversion in the presence and absence of the specific complex V inhibitor oligomycin. 

The following method has been described for the assay of cyclic AMP [144, 145]. Cyclic AMP is converted to 5 -AMP with the aid of phosphodiesterase and then to ATP with myokinase (EC 2.V.4.3 ATP AMP phosphotransferase adenylate kinase) and pyruvate kinase (EC 2.7.1.40 ATP pyruvate phosphotransferase). ATP is measured by determining the orthophosphate which accumulates during incubation of ATP with a cycling system containing myosin, pyruvate kinase, and phosphoenol pyruvate. Alternately, the ATP is determined by its luminescent reaction with firefly luciferin and luciferase [145-147]. With a sensitivity to about 1 pmol/tube of cyclic AMP, this assay is almost as sensitive as the phosphorylase method, but with a linearity over three orders of magnitude, it is linear over a much wider range than the phosphorylase method. 

Determination of ADP and ATP by multiple enzymes in recycling systems pyruvate kinase and hexokinase co-immobilized on aminopropyl CPG, was demonstrated by Kirstein et al. [25]. In addition, a second reactor,with L-lactate dehydrogenase, lactate oxidase, and catalase, was used to increase the sensitivity from 6x 10 5 M with no recycling, to 2 x 10 6 M in the kinase bienzyme reactor,... 

The lactate oxidation catalyzed by LMO forms the basis of several other multienzyme electrodes (Fig. 86). The LDH-LMO sensor has also been used to assay the activity of alanine aminotransferase (ALAT, EC 2.6.1.2) and pyruvate kinase (PK, EC 2.7.1.40) (Weigelt 1987 Weigelt et al., 1988). The sample was added to the NADH-containing measuring solution and when the steady state signal for endogenous lactate and pyruvate was attained the substrates of the enzyme to be determined... 

Pyruvate kinase (PK) activity in hemolyzed erythrocytes has been determined by using an LDH-lactate monooxygenase sequence electrode (Weigelt et al., 1988). The enzymes were immobilized in gelatin and attached to an oxygen probe. Since the sample material contains only... 

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