Pyruvate kinase, activity determinations

Schematic representation of a lactate dehydrogenase (LDH) - lactate monooxygenase (LMO) sensor for the determination of pyruvate kinase activity. PEP = phosphoenolpyruvate.

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+... 

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). 

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 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... 

Fig. 87. Response curve of the sequential determination of lactate, pyruvate, and creatine kinase activity with a LMO-LDH-PK sensor. CP = creatine phosphate, lactate 3.4 mmol/1, pyruvate 245 pmol/1, CK 504 U/l.

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... 

It was found that the enzyme is specific for (/ )-ATPaS but does not react with (S)-ATPaS. As shown in Scheme 43, when (/ )-ATPatS and l70-acetate are used as substrates, the 170 from acetate will be incorporated into the pro-5 position of AMPS if the reaction proceeds with retention of configuration or into the pro-/ position if inversion occurs. To determine the configuration of the 170-labeled AMPS (compound type 4), it is converted to (S)-ATPoS by stereospecific phosphorylation at the pro-/ oxygen catalyzed by adenylate kinase, followed by a second phosphorylation catalyzed by pyruvate kinase (144,145). By such a conversion, 170 should be incorporated into the nonbridging position of (5)-ATPaS if the step of acetate activation proceeds with retention of configuration. On the other hand, 170 should be located at the P—O—P bridging... 

Fig. 7. Schematic drawing showing oxalate, ATP, and the two divalent cations bound at the active site of pyruvate kinase. M is the divalent cation at site 1 and M2 is the divalent cation at site II. Four oxygen ligands for M have been identified by EPR experiments with O labeling. Ligands X and X are contributed from the protein. The a,/3,y-tridentate coordination scheme for M2 was determined for Mn(II) at this site in one of the hybrid complexes formed in the presence of Cd(ll). Ligands Z, Z, and Z" are likely water molecules given the facile binding of the aquo-Cr "ATP species at this site (Si).

Fig. 22.12. Major sites of regulation in the glycolytic pathway. Hexokinase and phos-phofructokinase-1 are the major regulatory enzymes in skeletal muscle. The activity of pyruvate dehydrogenase in the mitochondrion determines whether pyruvate is converted to lactate or to acetyl Co A. The regulation shown for pyruvate kinase only occurs for the liver (L) isoenzyme.

N.m.r. has been used to study the active site of other zinc-containing enzymes. In bovine carbonic anhydrase there is a single zinc co-ordination site available for Cl interaction, and the latter can be inhibited by CN and acetazolamide. C1 Resonance has also been used to investigate the environmental diflFerences of the zinc in carbonic anhydrase isozymes and the activation of pyruvate kinase with zinc, and to determine the pJ values in cobalt(u)-carbonic anhydrase. It is suggested that, although the TpK of aquozinc is about 9, the environment of the zinc ion in carbonic anhydrase B greatly increases the tendency of a zinc-bound water molecule... 

Carbon for FAS could also be produced via ATP-citrate lyase. This activity, which will convert citrate to oxaloacetate plus acetyl-CoA, has been demonstrated in soybean extracts. But there is no current evidence for citrate transport into the plastid nor of localisation of this activity in the plctstid to support citrate cleaving enzyme as a source of carbon for FAS, at least for avocado mesoccirp plastids. Extracts of developing soybean also contain a NADP+-dependent medic enzyme.20 NAD+-dependent malic enzyme, which produces pyruvate ind Cctrbon dioxide from malate, is an enzyme specific to the mitochondrial matrix in higher pleints.21 The localisation of NADP+-malic enzyme in immature soybeans, eind the possibility of pyruvate production other than by pyruvate kinase, and the utilisation of this pyruvate in FAS, remain to be determined. 

Regulation of the Pyruvate Dehydrogenase Complex In animal tissues, the rate of conversion of pyruvate to acetyl-CoA is regulated by the ratio of active, phosphory-lated to inactive, unphosphorylated PDH complex. Determine what happens to the rate of this reaction when a preparation of rabbit muscle mitochondria containing the PDH complex is treated with (a) pyruvate dehydrogenase kinase, ATP, and NADH (b) pyruvate dehydrogenase phosphatase and Ca2+ (c) malonate. 

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