Yeast pyruvate kinase: essential lysine residues in the active site

1. Yeast pyruvate kinase was purified to near homogeneity and subjected to chemical modification by trinitrobenzenesulfonate and by P1, P2-bis (5' pyridoxal) diphosphate. 2. Labeled peptides were isolated and their amino acid composition was determined. 3. The results suggest that yeast pyruvate kinase has an essential lysine residue, and that this residue is in a location equivalent to an essential lysine described in the muscle enzyme. 4. Protection experiments indicate that this lysine is located at the nucleotide binding site.     

Monitoring active site alterations upon mutation of yeast pyruvate kinase using 205Tl+ NMR

The interaction of the monovalent cation with wild type (WT) yeast pyruvate kinase (YPK) and with the T298S, T298C, and T298A mutants was investigated by 205Tl+ NMR to monitor possible structural alterations at the active site by Thr-298 mutation. TlNO3 activates WT YPK with a kcat value similar to that obtained with KCl and an apparent Ka of 0.96 +/- 0.07 mm in the presence of Mn2+ and fructose 1,6-bisphosphate. With the three mutants, Tl+ is a better activator than is K+ based on kcat values. Tl+ activation and inhibition of YPK is affected by mutation of the active site Thr-298. The effect of Mn2+ on the 1/T value of 205Tl+1 in the presence of the WT and mutant YPK complexes was determined at 173 MHz (300 MHz, 1H) and 346 MHz (600 MHz, 1H). For each complex studied, 1/pT2p > 1/pT1p and 1/pT1p is frequency-dependent suggesting fast exchange conditions. The values of 1/pT1p differ for each mutant. A correlation time of 0.65 +/- 0.35 ns was estimated for the Mn2+-205Tl+ interaction. The Tl+-Mn2+ distances at the active site of YPK were calculated from the paramagnetic contribution of Mn2+ to 1/T1M of YPK-bound 205Tl+. The calculated Tl+-Mn2+ distance for the Thr-298 mutants is decreased by about 1 A from 6.0 +/- 0.2 A observed with WT. The results suggest conformational alterations at the active site of YPK where phosphoryl transfer occurs upon mutation of Thr-298. These conformational changes may, in part, explain the alteration in kcat and kcat/Km,PEP observed with the Thr-298 mutants.     

The preparation and properties of pyruvate kinase from yeast

A new method is described for the preparation of pyruvate kinase from yeast. This eliminates proteolysis during the preparation. The molecular weight of yeast pyruvate kinase is 215000, and it is composed of four subunits. Such properties of the enzyme as its extinction coefficient, cold-lability, thiol-group reactivity and binding of Mn(2+) ions are compared with those previously reported for yeast pyruvate kinase prepared by different methods. The specific activity is significantly higher than previously observed, but otherwise the enzyme is similar, apart from its molecular weight and Mn(2+)-binding characteristics, to preparations from Saccharomyces cerevisiae obtained in this laboratory (e.g. Fell et al., 1972, and references therein) and that of C. H. Suelter (e.g. Kuczenski & Suelter, 1971, and references therein), and is different from the enzyme isolated from Saccharomyces carlsbergensis by B. Hess and his co-workers (e.g. Wieker & Hess, 1972, and references therein).     

Immunological and kinetic properties of pyruvate kinase in rat pancreatic islets

Pyruvate kinase in rat pancreatic islets was characterized immunologically and kinetically. It is concluded that this activity is predominantly if not totally of the M₂ type.     

Effect of the chemical surface structure of functionalized polystyrene on the kinetic properties of immobilized yeast pyruvate decarboxylase]

The influence of the loading density of different functional groups and the length of suitable spacer structures on the kinetic properties of yeast pyruvate decarboxylase are investigated on identical polystyrene matrices. At constant concentrations of the fixed protein both the specific activity and the storage stability of the immobilized enzymes increase with increasing concentrations of the protein binding (C = O)-groups. pH-Optimum and K'M-value prove to be functions of the NH3+-content of the supports. Using four spacer resins with an equal content of spacer groups it could be shown that the optimum time of coupling as well as the maximum catalytical activity, storage stability and thermostability depend on the length of the spacer structures. On the other hand, the mobility of an ESR-marker fixed via the same spacers to the resin is not affected by the different spacer structures.     

Purification and kinetic properties of pyruvate kinase from Brochothrix thermosphacta.

Pyravate kinase (ATP: pyruvate 2-0 phosphotransferase E.C.2.7.1.40) was purified from Brochothrix thermosphacta. The enzyme is a homotetramer of monomer Mr 58,000. Fructose-1,6-bisphosphate stimulates activity and promotes hyperbolic kinetics although it is not essential for enzyme activity. The positive effect of fructose-1,6-bisphosphate on activity is repressed by inorganic phosphate which enhances cooperative kinetics. Unlike pyruvate kinases from other sources, the Brochothrix enzyme is uncompetitively inhibited by glucose-6-phosphate, although at high concentration. ATP is a strong inhibitor of pyruvate kinase and shifts the residual activity/pH profile towards more alkaline values.     

Crystal structure of the cytochrome P-450CAM active site mutant Thr252Ala.

The crystal structure of a cytochrome P-450CAM site-directed mutant in which the active site Thr252 has been replaced with an Ala (Thr252Ala) has been refined to an R factor of 0.18 at 2.2 A. According to sequence alignments (Nelson & Strobel, 1989), Thr252 is highly conserved among P-450 enzymes. The crystallographic structure of ferrous camphor- and carbon monoxide-bound P-450CAM (Raag & Poulos, 1989b) suggests that Thr252 is a key active site residue, forming part of the dioxygen-binding site. Mutation of the active site threonine to alanine produces an enzyme in which substrate hydroxylation is uncoupled from electron transfer. Specifically, hydrogen peroxide and "excess" water are produced instead of the product, 5-exo-hydroxycamphor. The X-ray structure has revealed that a local distortion in the distal helix between Gly248 and Thr252 becomes even more severe in the Thr252Ala mutant. Furthermore, a solvent molecule not present in the native enzyme is positioned in the dioxygen-binding region of the mutant enzyme active site. In this location, the solvent molecule could sterically interfere with and destabilize dioxygen binding. In addition, the active site solvent molecule is connected, via a network of hydrogen bonds, with an internal solvent channel which links distal helix residues to a buried Glu side chain. Thus, solvent protons appear to be much more accessible to dioxygen in the mutant than in the wild-type enzyme, a factor which may promote hydrogen peroxide and/or water production instead of substrate hydroxylation. On the basis of crystallographic and mutagenesis data, a proton delivery pathway involving residues Lys178/Arg186, Asp251, and Thr252 is proposed for wild-type P-450CAM. Coordinates of structures discussed in this paper have been submitted to the Brookhaven Protein Data Bank (Bernstein et al., 1977).     

Purification and kinetic properties of pyruvate kinase isoenzymes of Salmonella typhimurium.

Two forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) present in Salmonella typhimurium were purified to homogeneity from the same cultures by (NH4)2SO4 fractionation and gel filtration, anion-exchange and affinity chromatography. Mr values, subunit structure, amino acid composition and activity and stability conditions were determined for the two forms. Kinetic and regulatory properties of the two purified isoenzymes were studied.     

Mutagenesis of the active site lysine 221 of the pyruvate kinase from Bacillus stearothermophilus

Lysine 221 of the pyruvate kinase from Bacillus stearothermophilus was mutated to arginine, leucine, asparatic acid and cysteine. All the mutated enzymes were 10(4) to 10(5) times less active than the wild-type enzyme. The cysteine-free enzyme C9S/C268S, and the enzyme C9S/C268S/K221C, which possessed a unique sulfhydryl group at position 221, were prepared. The former had comparable activity to the wild-type enzyme and the latter was 10(4) times less active. These enzymes were denatured and renatured after aminoethylation. The C9S/C268S/K221C enzyme failed to regain its activity when renatured without aminoethylation; but when it was renatured after aminoethylation, it regained 4.5% of the activity of the C9S/C268S enzyme. This evidence suggests the importance of the Lys221 for the pyruvate kinase activity. The kinetic parameters of the S-aminoethylated C9S/C268S/K221C enzyme suggest that it has decreased affinity for phosphoenolpyruvate.     

Isoenzyme spectrum and kinetic properties of pyruvate kinase from the liver of thiamine-deficient rats

Thiamine-deficiency in animals induced by everyday subcutaneous administration of oxythiamine in a dose of 4, 40 and 100 mg/kg of weight for 10 days results in a decrease of the total activity of pyruvate kinase in the liver tissue and does not affect the mentioned index in the kidney and heart tissues. It is shown that as a result of the enzyme fractionation in the column with DEAE-cellulose the total activity of pyruvate kinase in the liver tissue of rats with thiamine-deficiency decreases due to L-isoform while the content of M-isoform remains unchanged. Thiamine deficiency does not affect kinetic characteristics of the L-isoform, extracted from the liver and this shows the absence of changes in the degree of phosphorylation of pyruvate kinase L-isoform under these conditions.     

The effect of storage on the kinetic properties of sheep hepatic pyruvate kinase

The kinetic properties of purified sheep hepatic pyruvate kinase change upon storage. Assayed at 0.5 mM fructose-1,6-diphosphate and 2 mM ADP, saturation of fresh enzyme with phosphoenolpyruvate is hyperbolic, with KPEP = 0.1 mM (pH 7.5, and 30 degrees C). Under similar conditions enzyme stored at -20 degrees C for 1 week or more yields a nonlinear Lineweaver-Burk plot for PEP. The data may be accounted for by the appearance of two enzymic forms with identical turnover numbers, but different KPEP (0.035 +/- 0.005 and 12.4 +/- 0.6 mM). Storage also increases the concentration of fructose-1,6-diphosphate required for maximal activation from nanomolar to millimolar levels. Assayed at 2 mM ADP and 2 mM PEP, the apparent KFDP is 10 mM. Preincubation of stored enzyme with PEP in the presence of mercaptoethanol leads to significant reversion to original kinetic properties. Available data suggest that the storage-dependent change in kinetic behavior rises from changes in subunit conformation and not from dissociation into subunits.     

Seasonal change and prolonged anoxia affect the kinetic properties of phosphofructokinase and pyruvate kinase in oysters

The effects of seasonal change, November versus July, and prolonged anoxia (96 h under N₂ gas) on the properties of phosphofructokinase and pyruvate kinase from five tissues (gill, mantle, hepatopancreas, phasic adductor, catch adductor) of the oyster, Crassostrea virginica, were investigated. Both enzymes showed tissue-specific and season-specific changes in kinetic properties; for pyruvate kinase this correlated with seasonal differences in enzyme elution patterns on hydroxylapatite chromatography. Kinetic properties of both enzymes in winter were consistent with primarily catabolic roles in glycolysis with responsiveness to cellular energy demands, whereas in summer these enzymes may be more closely regulated with respect to the biosynthetic and gluconeogenic functions of the tissues. Anoxia-induced changes in phosphofructokinase properties were relatively minor but anoxia stimulated changes in pyruvate kinase properties and elution profiles on hydroxylapatite in all tissues except mantle, with much greater effects seen for the enzyme from winter versus summer animals. For example, anoxia-induced changes in pyruvate kinase from winter gill included a fourfold rise in the substrate affinity constant for phosphoenolpyruvate, a sevenfold increase in the concentration of fructose-1,6-bisphosphate needed to activate the enzyme by 50%, and a 50% decrease in the concentration of L-alanine that inhibits activity by 50%. Changes in pyruvate kinase kinetics and hydroxylapatite elution patterns during prolonged anoxia are consistent with covalent modification of pyruvate kinase but contrary to results for many other mollusc species, anoxia exposure appears to induce a dephosphorylation of the enzyme. Accepted: 17 February 2000     

Isolation and sequence determination of an active site peptide of rabbit muscle pyruvate kinase

Rabbit muscle pyruvate kinase was inactivated by 2', 3'-dialdehyde ADP with the incorporation of one molecule of reagent per enzyme subunit. The inactivated protein was digested with trypsin after reduction and carboxymethylation. The labeled peptide was isolated by gel filtration and further purified by HPLC. The peptide was sequenced both by liquid-phase and gas-phase automatic Edman degradation. A 34-residue peptide was obtained. This peptide is identical to a tryptic peptide labeled with trinitrobenzenesulfonate, isolated and sequenced by Johnson et al. (Biochem. Biophys. Res. Commun. (1979) 90, 525-530) from bovine muscle pyruvate kinase. Available evidence suggests that dialdehyde ADP labels the enzyme at the same lysine in position 25 of the peptide, as found by Johnson et al. The high homology between the isolated peptide and regions of other pyruvate kinases from low to high eukaryotes supports the idea that this peptide is related to the enzyme active site.     

X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: roles of Thr252 and water in the active center

The structure-function relationship in cytochrome P450cam monooxygenase was studied by employing its active site mutant Thr252Ile. X-ray crystallographic analyses of the ferric d-camphor-bound form of the mutant revealed that the mutation caused a structural change in the active site giving an enlarged oxygen-binding pocket that did not contain any hydrophilic group such as the OH group of Thr and H(2)O. The enzyme showed a low monooxygenase activity of ca. 1/10 of the activity of the wild-type enzyme. Kinetic analyses of each catalytic step revealed that the rate of proton-coupled reduction of the oxygenated intermediate of the enzyme, a ternary complex of dioxygen and d-camphor with the ferrous enzyme, decreased to about 1/30 of that of the wild-type enzyme, while the rates of other catalytic steps including the reduction of the ferric d-camphor-bound form by reduced putidaredoxin did not change significantly. These results indicated that a hydrophilic group(s) such as water and/or hydroxyl group in the active site is prerequisite to a proton supply for the reduction of the oxygenated intermediate, thereby giving support for the operation of a proton transfer network composed of Thr252, Asp251, and two other amino acids and water proposed by previous investigators.     

Evolution of the functional properties of pyruvate kinase isozymes: pyruvate kinase L fromRana pipiens

Summary The regulatory properties of type L pyruvate kinase fromRana pipiens are intermediate between those of the mammalian K and L isozymes. As with mammalian type L, the levels of the frog isozyme are affected by the animal's nutritional state. The mammalian and amphibian isozymes show similar sensitivities to fructose 1,6-bisphosphate activation and amino acid inhibition. By contrast, the frog L isozyme shares several properties of the K class: ie. irreversible inactivation by oxidized glutathione and lack of response to a cyclic AMP stimulated phosphorylation. Furthermore, as for some mammalian K isozymes, frog type L shows a high PEP affinity and a low cooperativity of PEP binding.Insofar as the properties of this present day enzyme reflect those of its counterpart in the amphibian ancestor of higher vertebrates, our results suggest that at its first expression, the type L resembled the type K. Many important regulatory properties of the L isozyme, especially the sensitivity to phosphorylation, were acquired more recently perhaps in association with an increased importance of constant blood glucose.Abbreviations DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis(2-aminoethylether)-N,N-tetraacetic acid - FB fructose-1,6-bisphosphate - PEP phosphoenolpyruvate - PK pyruvate kinase