Bovine liver glutamate dehydrogenase. Equilibriums and kinetics of inactivation by pyridoxal

Piszkiewicz, Dennis; Smith, Emil L.

doi:10.1021/bi00800a030

Cited by 57 publications

(32 citation statements)

References 14 publications

(5 reference statements)

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“…This gave enzyme activities after reduction and dialysis that were in excellent agreement with those measured on samples taken straight from the inactivation incubation without reduction. 16,25,38] in which activity declines over about 20 min to a residual value, in this case 6 % of the starting activity, which then remains constant for several hours. This is known to be due to the modification of Lys-126 [10].…”

Section: Methodsmentioning

confidence: 95%

“…Given that PalP is selective for lysine residues, the reagent has also been found to be rather specific in two further senses: firstly, it frequently modifies only one or two lysine residues in a protein [4, [8][9][10][11]23], although most proteins have many lysine residues and these tend to be on the surface; secondly PalP may modify where pyridoxal, lacking the phosphate group, either does not (see, e.g., [3,4,11]) or else does so much less effectively [2,5,6,24]. Specificity of the first kind may be explained in terms of the unique environment of an individual lysine residue.…”

Section: Introductionmentioning

confidence: 99%

“…Specificity of the first kind may be explained in terms of the unique environment of an individual lysine residue. It has been suggested that the reactive lysine residues in ribonuclease A [11] and in bovine GDH [25] are more susceptible to attack by virtue of abnormally low pK values, perhaps reflecting a hydrophobic environment (but see [26] for discussion of the pHdependence of Schiff-base formation). Such factors alone, however, cannot explain specificity of the second kind; indeed such an environment might be thought to favour reaction by the uncharged pyridoxal.…”

Section: Introductionmentioning

confidence: 99%

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Is pyridoxal 5′-phosphate an affinity label for phosphate-binding sites in proteins?: The case of bovine glutamate dehydrogenase

Valinger

Engel

Metzler

1993

Biochemical Journal

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The effects of pyridoxal 5'-phosphate (PalP) on ox liver glutamate dehydrogenase (94% inactivation by 1.8 mM reagent at pH 7 and 25 degrees C) have been compared with those of three analogues, 5'-deoxypyridoxal (96% inactivation), pyridoxal 5'-sulphate (97%) and pyridoxal 5-methylsulphonate (94%), in order to establish whether PalP acts as an affinity label for this enzyme. Like PalP and unlike pyridoxal, which is a much less potent inactivator, none of the analogues has a free 5'-OH group to cyclize with the aldehyde function. The result with 5'-deoxypyridoxal shows that a negative charge, such as that of the phosphate group, is not required for efficient inactivation. With all four reagents, addition of an excess of cysteine or lysine led to 90-100% re-activation over 3-20 h. Dialysis also caused reactivation to a similar extent. A combination of 2.15 mM NADH, 1 mM GTP and 10 mM 2-oxoglutarate gave complete protection against PalP, but only partial protection against the analogues. 5'-Deoxypyridoxal still caused 20-25% inactivation in the presence of the protection mixture. Absorbance measurements after reduction with NaBH4 show the characteristic features of a reduced Schiff's base and allowed estimation of the extent of reaction. With all the reagents the protection mixture decreased incorporation by about 1 mol/mol, but levels of incorporation without protection varied from about 2 mol/mol for PalP up to about 5 mol/mol for 5'-deoxypyridoxal. The labelling at additional sites may explain the residual inactivation in the presence of potent protecting agents.

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Section: Methodsmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Is pyridoxal 5′-phosphate an affinity label for phosphate-binding sites in proteins?: The case of bovine glutamate dehydrogenase

Valinger

Engel

Metzler

1993

Biochemical Journal

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“…This seems to be more likely since the ratios of the various forms of coenzyme do not vary to a very large degree in vivo . With GDH, this communication is made evident by abrupt and striking changes in the circular dichroism and fluorescence spectra when GDH is half saturated (Bell and Dalziel, 1973) and from chemical modification studies demonstrating that the loss of enzymatic activity is extremely disproportional to the number of subunits modified (Piszkiewicz and Smith, 1971; Rasool et al, 1976; Syed and Engel, 1984). Therefore, it is clear that there is extensive inter-subunit communication in animal GDH and, as shown below, such communication is essential for catalysis and regulation.…”

Section: Homotropic and Heterotropic Regulation Of Gdhmentioning

confidence: 99%

The structure and allosteric regulation of glutamate dehydrogenase

Allen

et al. 2011

Neurochemistry International

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Glutamate dehydrogenase (GDH) has been extensively studied for more than 50 years. Of particular interest is the fact that, while considered by most to be a 'housekeeping' enzyme, the animal form of GDH is heavily regulated by a wide array of allosteric effectors and exhibits extensive inter-subunit communication. While the chemical mechanism for GDH has remained unchanged through epochs of evolution, it was not clear how or why animals needed to evolve such a finely tuned form of this enzyme. As reviewed here, recent studies have begun to elucidate these issues. Allosteric regulation first appears in the Ciliates and may have arisen to accommodate evolutionary changes in organelle function. The occurrence of allosteric regulation appears to be coincident with the formation of an 'antenna' like feature rising off the tops of the subunits that may be necessary to facilitate regulation. In animals, this regulation further evolved as GDH became integrated into a number of other regulatory pathways. In particular, mutations in GDH that abrogate GTP inhibition result in dangerously high serum levels of insulin and ammonium. Therefore, allosteric regulation of GDH plays an important role in insulin homeostasis. Finally, several compounds have been identified that block GDH-mediated insulin secretion that may be to not only find use in treating these insulin disorders but to kill tumors that require glutamine metabolism for cellular energy. Homotropic and heterotropic regulation of GDHGlutamate dehydrogenase (GDH) is found in nearly all living organisms and catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P) + as coenzyme (Hudson and Daniel, 1993). This homohexameric enzyme has subunits comprised of ~450 and ~500 amino acids in bacteria and animals, respectively. In eukaryotic organisms, GDH resides within the inner mitochondrial matrix where it catabolizes glutamate to feed 2-oxoglutarate to the Krebs cycle. Although there is some debate as to the directionality of the reaction, the high Km for ammonium in the reductive amination reaction seems to prohibit the reverse reaction under normal conditions in most organisms (Smith et al., 1975). Even in plants, recent 15 N incorporation studies in the presence of excess ammonium have shown that GDH functions in the oxidative deamination reaction (Aubert et al., 2001). However, some bacteria may use GDH rather than the normal glutamine synthetase-glutamate synthase (GS-GOGAT) pathway to fix nitrogen under high ammonia conditions (Kanamori et al., 1987). Under most in-vitro conditions, coenzyme * Corresponding author: tsmith@danforthcenter.org, Phone: (314) 587-1451, Fax: (314) 587-1551.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during...

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“…This implies that the p H dependence observed in this analysis is probably associated with the binding of glutamate and subsequent events occurring in the ternary complex. A role in substrate binding or catalysis has been ascribed to the reactive lysines 126 [14,15] and 27 [20] in ox liver glutamate dehydrogenase. The data in Fig.…”

Section: Discussionmentioning

confidence: 99%

A kinetic study of the oxidative deamination of L‐glutamate by Peptostreptococcus asaccharolyticus glutamate dehydrogenase using a variety of coenzymes

Hornby

Engel

1984

European Journal of Biochemistry

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1. The NAD + -specific glutamate dehydrogenase from Peptostreptococcus asaccharolyticus follows MichaelisMenten kinetics in contrast to the enzyme from several other sources, and thus gives linear double-reciprocal plots of initial-rate data.2. The initial-rate parameters have been determined for this bacterial glutamate dehydrogenase in the direction of oxidative deamination. The use of alternative coenzymes leads to some conclusions about the order of substrate addition.3. An investigation of the pH dependence of this reaction reveals that the binding of oxidised coenzyme is independent of pH over the range 6 -9.4. The kinetic data are consistent with an ordered addition of coenzyme prior to glutamate, the reverse of the mechanism derived with ox glutamate dehydrogenase in the presence of ADP.Peptostreptococcus asaccharolyticus is an exceptionally rich source of glutamate dehydrogenase, which catalyses the following reaction : L-Glutamate + NAD' + HzO 2-oxoglutarate + NH:The enzyme is highly specific for L-glutamate [l], although the coenzyme specificity is somewhat broader. This bacterial dehydrogenase shares some features with the corresponding mammalian enzymes : it is hexameric, with a subunit molecular mass of 49000 Da. However, it is not regulated by the purine nucleotides ADP or GTP, and double-reciprocal plots for oxidised coenzyme follow Michaelis-Menten kinetics. A major complicating factor in steady-state kinetic studies of ox liver glutamate dehydrogenase has been the superimposed cooperativity with respect to both NAD' and NADP' [2]. This problem of non-linearity has also arisen in the study of several other glutamate dehydrogenases (e.g. [3 -51). The opportunity to study a glutamate dehydrogenase bi-ter mechanism with simplified behaviour is afforded by the enzyme from P. asaccharolyticus. Several alternative coenzymes are used here to elucidate the order of substrate binding in the two-substrate direction (oxidative deamination of glutamate). This approach has also been used recently to study the same reaction with the ox liver enzyme in the presence of ADP [6]. In that study the results were consistent with an ordered mechanism, in which glutamate is the leading substrate.As a second approach, the pH dependence of the oxidative deamination of glutamate was examined with NAD' as the coenzyme. The initial-rate parameters were determined for seven pH values, and certain ratios of these parameters shed Abbreviations. AcPdAD+, acetylpyridine-adenine dinucleotide;Enzyme. Glutamate dehydrogenase (EC 1.4.1.2).dnNAD', deamino-NAD' , some light on the mechanism of the reaction. This investigation we believe, represents the first example of a detailed pH study with glutamate dehydrogenase from any source and is analogous to the work of Winer and Schwert [7] on lactate dehydrogenase, and Dickenson and Dickinson [8,9] on yeast alcohol dehydrogenase. MATERIALS AND METHODSThe details of bacterial growth and enzyme purification have been described elsewhere [I, 101. The enzyme was stored as a suspension in 80...

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Bovine liver glutamate dehydrogenase. Equilibriums and kinetics of inactivation by pyridoxal

Cited by 57 publications

References 14 publications

Is pyridoxal 5′-phosphate an affinity label for phosphate-binding sites in proteins?: The case of bovine glutamate dehydrogenase

Is pyridoxal 5′-phosphate an affinity label for phosphate-binding sites in proteins?: The case of bovine glutamate dehydrogenase

The structure and allosteric regulation of glutamate dehydrogenase

A kinetic study of the oxidative deamination of L‐glutamate by Peptostreptococcus asaccharolyticus glutamate dehydrogenase using a variety of coenzymes

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