Beef liver glutamate dehydrogenase: A study of the oxidation of various alternative amino acid substrates retaining the correct spacing of the two carboxylate groups

Hornby, D.; Engel, Paul C.; Hatanaka, Shin–ichi

doi:10.1016/0020-711x(83)90122-2

International Journal of Biochemistry

1983

DOI: 10.1016/0020-711x(83)90122-2

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Beef liver glutamate dehydrogenase: A study of the oxidation of various alternative amino acid substrates retaining the correct spacing of the two carboxylate groups

D. Hornby

Paul C. Engel

Shin–ichi Hatanaka

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Cited by 7 publications

(1 citation statement)

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“…The amino‐acid specificity of GDHs, wherever it has been examined in detail, is relatively strict [5–8]. The enzyme is intolerant of branched substrates, and for good catalytic efficiency, the side chain carboxyl must be present and in the correct position.…”

mentioning

confidence: 99%

Conversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site‐directed mutagenesis

Wang

Britton

Stillman

et al. 2001

European Journal of Biochemistry

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In earlier attempts to shift the substrate specificity of glutamate dehydrogenase (GDH) in favour of monocarboxylic amino-acid substrates, the active-site residues K89 and S380 were replaced by leucine and valine, respectively, which occupy corresponding positions in leucine dehydrogenase. In the GDH framework, however, the mutation S380V caused a steric clash. To avoid this, S380 has been replaced with alanine instead. The single mutant S380A and the combined double mutant K89L/S380A were satisfactorily overexpressed in soluble form and folded correctly as hexameric enzymes. Both were purified successfully by Remazol Red dye chromatography as routinely used for wild-type GDH.The S380A mutant shows much lower activity than wildtype GDH with glutamate. Activities towards monocarboxylic substrates were only marginally altered, and the pH profile of substrate specificity was not markedly altered. In the double mutant K89L/S380A, activity towards glutamate was undetectable. Activity towards L-methionine, L-norleucine and L-norvaline, however, was measurable at pH 7.0, 8.0 and 9.0, as for wild-type GDH.Ala163 is one of the residues that lines the binding pocket for the side chain of the amino-acid substrate. To explore its importance, the three mutants A163G, K89L/A163G and K89L/S380A/A163G were constructed. All three were abundantly overexpressed and showed chromatographic behaviour identical with that of wild-type GDH. With A163G, glutamate activity was lower at pH 7.0 and 8.0, but by contrast higher at pH 9.0 than with wild-type GDH. Activities towards five aliphatic amino acids were remarkably higher than those for the wild-type enzyme at pH 8.0 and 9.0. In addition, the mutant A163G used L-aspartate and L-leucine as substrates, neither of which gave any detectable activity with wild-type GDH. Compared with wild-type GDH, the A163 mutant showed lower catalytic efficiencies and higher K m values for glutamate/ 2-oxoglutarate at pH 7.0, but a similar k cat /K m value and lower K m at pH 8.0, and a nearly 22-fold lower S 0.5 (substrate concentration giving half-saturation under conditions where Michaelis -Menten kinetics does not apply) at pH 9.0.Coupling the A163G mutation with the K89L mutation markedly enhanced activity (100-1000-fold) over that of the single mutant K89L towards monocarboxylic amino acids, especially L-norleucine and L-methionine. The triple mutant K89L/S380A/A163G retained a level of activity towards monocarboxylic amino acids similar to that of the double mutant K89L/A163G, but could no longer use glutamate as substrate. In terms of natural amino-acid substrates, the triple mutant represents effective conversion of a glutamate dehydrogenase into a methionine dehydrogenase. Kinetic parameters for the reductive amination reaction are also reported.At pH 7 the triple mutant and K89L/A163G show 5 to 10-fold increased catalytic efficiency, compared with K89L, towards the novel substrates. In the oxidative deamination reaction, it is not possible to estimate k cat and K m separately, but for reductiv...

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mentioning

confidence: 99%

Conversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site‐directed mutagenesis

Wang

Britton

Stillman

et al. 2001

European Journal of Biochemistry

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Alteration of substrate regulation patterns in glutamate dehydrogenase by enzyme immobilization

O’Connor

Schütz

Bailey

1989

Biotech & Bioengineering

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Substrate regulation patterns were changed by covalent binding of bovine liver glutamate dehydrogenase via primary amino groups to CNBr- and CH-activated Sepharose 4B. Lineweaver-Burk plots show that the NAD activation region changed from being abrupt to elongated when the enzyme was immobilized to either support. The elongated region contains two inflection points and resembles substrate activation of several other allosteric oligomers. Glutamate induced varying degrees of abrupt activation in immobilized glutamate dehydrogenase and inhibited the native enzyme. This activation is characterized by an activation threshold, an increase in the apparent dissociation constant, and a correlation between the apparent rate constant and the degree of activation. These three features characterize other glutamate dehydrogenase systems.

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Kinetic studies of Ox-liver glutamate dehydrogenase oxidative deamination of two glutamate analogues, L-threo-γ-methylglutamate and L-α-amino-γ-nitraminobutyrate, in the presence of the allosteric effector ADP

Hornby¹,

Engel²,

Hatanaka³

1985

International Journal of Biochemistry

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Beef liver glutamate dehydrogenase: A study of the oxidation of various alternative amino acid substrates retaining the correct spacing of the two carboxylate groups

Cited by 7 publications

References 16 publications

Conversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site‐directed mutagenesis

Conversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site‐directed mutagenesis

Alteration of substrate regulation patterns in glutamate dehydrogenase by enzyme immobilization

Kinetic studies of Ox-liver glutamate dehydrogenase oxidative deamination of two glutamate analogues, L-threo-γ-methylglutamate and L-α-amino-γ-nitraminobutyrate, in the presence of the allosteric effector ADP

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