[66] Pyruvate kinase from muscle

Bücher, Theodor; Pfleiderer, G.

doi:10.1016/0076-6879(55)01071-9

Cited by 652 publications

(167 citation statements)

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“…Fractions of 9 drops each were collected. Cytochrome c was determined by the absorbance at 544 nm, haemoglobin at 420 nm, yeast alcohol dehydrogenase by the method ofVallee and Hoch [19], S-galactose dehydrogenase according to Wallenfels and Kurz [20] and pyruvate kinase by the procedure of Bucher and Pfleiderer [21]. The molecular weight of glutamate-semialdehyde dehydrogenase was calculated by the methods of Martin and h i e s [18] and Paetkau and Lardy [22].…”

Section: Sucrose-gradien T Ceri T Rifugat Ionmentioning

confidence: 99%

Proline Biosynthesis in Escherichia coli. Purification and Characterisation of Glutamate-Semialdehyde Dehydrogenase

Hayzer

Leisinger²

1982

Eur J Biochem

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Glutamate-semialdehyde dehydrogenase, catalysing the reduction in vivo of y-glutamyl phosphate to glutamate 5-semialdehyde in the pathway of proline biosynthesis in Escherichia coli, has been purified to homogeneity. High initial levels of the enzyme were achieved by using a multicopy ColEl-proA,B hybrid plasmid. The protein has a molecular weight of 1.89 x lo5 and consists of four identical subunits of molecular weight 4.7 x lo4 each. The pH optimum is 7.0 and the protein is stable for at least 10 min between pH 6.0-9.0 and for long periods at pH 7.0. It is rapidly inactivated at temperatures greatcr than 50 "C. The enzyme is very sensitive to inhibition byp-chloromercuribenzoate, copper and nickel ions.The synthesis of L-proline by Escherichia coli involves the reduction of L-glutamic acid to glutamate hemialdehyde, mediated by two consecutive enzymic steps. A y-glutamyl kinase (ATP : L-glutamate 5-phosphotransferase, EC 2.7.2.1 1) activates the y-carboxyl group of glutamate before its reduction to an aldehyde by NADPH-dependent glutamatesemialdehyde dehydrogenase [~-glutamate-5-semialdehyde : NADP+ oxidoreductase (phosphorylating), EC 1.2.1.41, or y-glutamyl-phosphate reductase]. Glutamate 5-semialdehyde undergoes spontaneous Schiff-base formation and cyclisation to L-1-pyrroline-5-carboxylic acid which is subsequently reduced to proline by an NADPH-dependent pyrroline-5-carboxylic acid reductase [L-proline : NAD(P) + 5-0x1-doreductase, EC 1.5.1.21. All three biosynthetic enzymes from Pseudomonas aeruginosa PA01 have been partially purified and characterized [1,2] whereas the first two of the E. coli biosynthetic enzymes have been subjected to investigation whilst in a very crude state [3,4]. Pyrroline-5-carboxylic acid reductase of E. coli, a highly active and readily measurable enzyme, has been partially purified and characterized [ 5 ] . The availability of pure samples of the first two enzymes of the pathway would enable a study to be made of the problems of the true nature of the activated intermediate in the pathway, generally assumed to by y-glutamyl phosphate, and whether there is aggregation between the kinase and glutamatesemialdehyde dehydrogenase. The function of such an enzyme complex would be to protect the glutamyl phosphate from a hostile nucleophilic and aqueous environment. To date these studies have been limited by the use of crude cell extracts but have provided suggestive evidence of the existence of an aggregate [6-81. This paper describes a method for the purification of the second enzyme in the proline biosynthetic pathway and its physical properties. MATERIALS AND METHODS Bacrerial StrainThe construction of Eschevichia coli strain CSH52/ pLC44-11 [ihi araA(lac pro)strA recA($J8Odlaci)(pLC44-1 I)] has been described by Hayzer and Leisinger [9].Large-Scale Fermentation of E. coli CSH52/pLC44-1 I A preculture of 300 ml of medium M63 [lo] plus glucose (0.5% w/v) in a 1-1 flask was grown overnight at 37°C with vigorous aeration. The entire amount was used to inocu1;lte 7 1 of the same med...

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Section: Sucrose-gradien T Ceri T Rifugat Ionmentioning

confidence: 99%

Proline Biosynthesis in Escherichia coli. Purification and Characterisation of Glutamate-Semialdehyde Dehydrogenase

Hayzer

Leisinger²

1982

Eur J Biochem

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“…Phosphoglycerate kinase (EC 2.7.2.3) was determined using the method of Suzuki & Imahori (1982), and phosphoglyceromutase (EC 5.4.2.1) and enolase (EC 4.2.1.11) by the method of Mulongoy & Elkan (1977). The pyruvate kinase (EC 2.7.1 -40) assay was modified from that of Bucher & Pfleiderer (1955), using 50 mMphosphate buffer (pH 7.9, 2 mM-ADP, 0.2 mM-NADH, 10 mM-MgC1, and 5 mM-phosphoenolpyruvate.…”

Section: Introductionmentioning

confidence: 99%

The Use of Activities of Carbon Catabolic Enzymes as a Probe for the Carbon Nutrition of Snakebean Nodule Bacteroids

1986

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In sugar-grown cells of cowpea Rhizobium strain NGR234 activities for enzymes of the EntnerDoudoroff and pentose phosphate pathways were present while the virtual absence of phosphofructokinase and fructose-bisphosphate aldolase indicated that the Embden-Meyerhof-Parnas pathway was unlikely to be significant. Invertase, fructokinase, glucose-6-phosphate dehydrogenase and the Entner-Doudoroff enzymes were present at only low activities in succinate grown cells, but were induced in sugar-grown cells. Isolated snakebean bacteroids contained very low activities of these four enzymes. Although C,-dicarboxylic acids exerted some repressive effect on induction of these enzymes, there was substantial enzyme activity induced in cells grown on sucrose plus a C, dicarboxylic acid. The data suggest that the peribacteroid membrane may be relatively impermeable to sugars and so dictate the carbon source(s) available to the bacteroids.

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“…0.1 IU/ml before use in kinetic experiments. Enzyme activity was measured at 25°C in the coupled lactate dehydrogenase assay, as described by Biicher and Pfleiderer [5]. The reaction mixture contained in a final volume of 1 ml: 0.01 M Tris-HC1, pH 8.0, at 25°C; 0.05 M KC1; 1 mM dithiothreitol; 0.1 mM NADH and 10 IU lactate dehydrogenase, dialysed against the buffer mentioned above.…”

Section: Methodsmentioning

confidence: 99%

Functional changes associated with the sequential transformation of L′4 into L4 pyruvate kinase

Sprengers

Staal

1979

Biochimica et Biophysica Acta (BBA) - Enzymology

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SummaryThe functional changes, associated with the sequential transformation of L:~ into L4 pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) were studied. L~ enzyme from human erythrocytes shows strong hysteretic behaviour: the initial rate of the enzyme preincubated with an unsaturating concentration of phosphoenolpyruvate is much higher than of the enzyme preincubated with ADP, at the same phosphoenolpyruvate concentration, although the 'final activity' (the activity of the linear part of the reaction progress curve) was the same in both cases. This phenomenon was observed both in the presence and absence of fructose 1,6-diphosphate. High concentrations of both 2÷ Mg~ree and MgATP 2-diminish the difference in initial rate, between the ADP and phosphoenolpyruvate preincubated enzymes: 2+ Mgfre e by stabilizing the phosphoenolpyruvate-induced form; ATPMg 2-by stabilizing the ADP-induced form. The magnitude of the difference in initial rates of the ADPor phosphoenolpyruvate-preincubated enzyme is a function of both substrates. L, pyruvate kinase(either from human liver or trypsin treated L~ enzyme) does not, or to a very slight extent, show such behaviour. L~L2 pyruvate kinase shows behaviour intermediate between L~ and L4 enzymes.A model is proposed to describe the kinetic behaviour of L~ and L4 enzymes.

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[66] Pyruvate kinase from muscle

Cited by 652 publications

References 12 publications

Proline Biosynthesis in Escherichia coli. Purification and Characterisation of Glutamate-Semialdehyde Dehydrogenase

Proline Biosynthesis in Escherichia coli. Purification and Characterisation of Glutamate-Semialdehyde Dehydrogenase

The Use of Activities of Carbon Catabolic Enzymes as a Probe for the Carbon Nutrition of Snakebean Nodule Bacteroids

Functional changes associated with the sequential transformation of L′4 into L4 pyruvate kinase

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