Crystal Structures of True Enzymatic Reaction Intermediates: Aspartate and Glutamate Ketimines in Aspartate Aminotransferase

Vladimir N. Malashkevich, Michael D. Toney, Johan N. Jansonius

Research output: Contribution to journalArticle

87 Citations (Scopus)

Abstract

The crystal structures of the stable, closed complexes of chicken mitochondrial aspartate aminotransferase with the natural substrates L-aspartate and L-glutamate have been solved and refined at 2.4- and 2.3-Å resolution, respectively. In both cases, clear electron density at the substrate-coenzyme binding site unequivocally indicates the presence of a covalent intermediate. The crystallographically identical environments of the two subunits of the α2 dimer allow a simple, direct correlation of the coenzyme absorption spectra of the crystalline enzyme with the diffraction results. Deconvolution of the spectra of the crystalline complexes using lognormal curves indicates that the ketimine intermediates constitute 76% and 83% of the total enzyme populations with L-aspartate and L-glutamate, respectively. The electron density maps accommodate the ketimine structures best in agreement with the independent spectral data. Crystalline enzyme has a much higher affinity for keto acid substrates compared to enzyme in solution. The increased affinity is interpreted in terms of a perturbation of the open/closed conformational equilibrium by the crystal lattice, with the closed form having greater affinity for substrate. The crystal lattice contacts provide energy required for domain closure normally supplied by the excess binding energy of the substrate. In solution, enzyme saturated with amino/keto acid substrate pairs has a greater total fraction of intermediates in the aldehyde oxidation state compared to crystalline enzyme. Assuming the only difference between the solution and crystalline enzymes is in conformational freedom, this difference suggests that one or more substantially populated, aldehydic intermediates in solution exist in the open conformation. Quantitative analyses of the spectra indicate that the value of the equilibrium constant for the open-closed conformational transition of the liganded, aldehydic enzyme in solution is near 1. The C4′ pro-S proton in the ketimine models is oriented nearly perpendicularly to the plane of the pyridine ring, suggesting that the enzyme facilitates its removal by maximizing σ-π orbital overlap. The absence of a localized water molecule near Lys258 dictates that ketimine hydrolysis occurs via a transiently bound water molecule or from an alternative, possibly more open, structure in which water is appropriately bound. A prominent mechanistic role for flexibility of the Lys258 side chain is suggested by the absence of hydrogen bonds to the amino group in the aspartate structure and the relatively high temperature factors for these atoms in both structures.

Original languageEnglish (US)
Pages (from-to)13451-13462
Number of pages12
JournalBiochemistry
Volume32
Issue number49
DOIs
StatePublished - 1993
Externally publishedYes

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Reaction intermediates
Aspartate Aminotransferases
Aspartic Acid
Glutamic Acid
Crystal structure
Enzymes
Crystalline materials
Substrates
Keto Acids
Coenzymes
Crystal lattices
Carrier concentration
Water
Mitochondrial Aspartate Aminotransferase
Electrons
Complement C4
ketimine
Molecules
Equilibrium constants
Deconvolution

ASJC Scopus subject areas

  • Biochemistry

Cite this

Crystal Structures of True Enzymatic Reaction Intermediates : Aspartate and Glutamate Ketimines in Aspartate Aminotransferase. / Malashkevich, Vladimir N.; Toney, Michael D.; Jansonius, Johan N.

In: Biochemistry, Vol. 32, No. 49, 1993, p. 13451-13462.

Research output: Contribution to journalArticle

Malashkevich, Vladimir N. ; Toney, Michael D. ; Jansonius, Johan N. / Crystal Structures of True Enzymatic Reaction Intermediates : Aspartate and Glutamate Ketimines in Aspartate Aminotransferase. In: Biochemistry. 1993 ; Vol. 32, No. 49. pp. 13451-13462.
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abstract = "The crystal structures of the stable, closed complexes of chicken mitochondrial aspartate aminotransferase with the natural substrates L-aspartate and L-glutamate have been solved and refined at 2.4- and 2.3-{\AA} resolution, respectively. In both cases, clear electron density at the substrate-coenzyme binding site unequivocally indicates the presence of a covalent intermediate. The crystallographically identical environments of the two subunits of the α2 dimer allow a simple, direct correlation of the coenzyme absorption spectra of the crystalline enzyme with the diffraction results. Deconvolution of the spectra of the crystalline complexes using lognormal curves indicates that the ketimine intermediates constitute 76{\%} and 83{\%} of the total enzyme populations with L-aspartate and L-glutamate, respectively. The electron density maps accommodate the ketimine structures best in agreement with the independent spectral data. Crystalline enzyme has a much higher affinity for keto acid substrates compared to enzyme in solution. The increased affinity is interpreted in terms of a perturbation of the open/closed conformational equilibrium by the crystal lattice, with the closed form having greater affinity for substrate. The crystal lattice contacts provide energy required for domain closure normally supplied by the excess binding energy of the substrate. In solution, enzyme saturated with amino/keto acid substrate pairs has a greater total fraction of intermediates in the aldehyde oxidation state compared to crystalline enzyme. Assuming the only difference between the solution and crystalline enzymes is in conformational freedom, this difference suggests that one or more substantially populated, aldehydic intermediates in solution exist in the open conformation. Quantitative analyses of the spectra indicate that the value of the equilibrium constant for the open-closed conformational transition of the liganded, aldehydic enzyme in solution is near 1. The C4′ pro-S proton in the ketimine models is oriented nearly perpendicularly to the plane of the pyridine ring, suggesting that the enzyme facilitates its removal by maximizing σ-π orbital overlap. The absence of a localized water molecule near Lys258 dictates that ketimine hydrolysis occurs via a transiently bound water molecule or from an alternative, possibly more open, structure in which water is appropriately bound. A prominent mechanistic role for flexibility of the Lys258 side chain is suggested by the absence of hydrogen bonds to the amino group in the aspartate structure and the relatively high temperature factors for these atoms in both structures.",
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N2 - The crystal structures of the stable, closed complexes of chicken mitochondrial aspartate aminotransferase with the natural substrates L-aspartate and L-glutamate have been solved and refined at 2.4- and 2.3-Å resolution, respectively. In both cases, clear electron density at the substrate-coenzyme binding site unequivocally indicates the presence of a covalent intermediate. The crystallographically identical environments of the two subunits of the α2 dimer allow a simple, direct correlation of the coenzyme absorption spectra of the crystalline enzyme with the diffraction results. Deconvolution of the spectra of the crystalline complexes using lognormal curves indicates that the ketimine intermediates constitute 76% and 83% of the total enzyme populations with L-aspartate and L-glutamate, respectively. The electron density maps accommodate the ketimine structures best in agreement with the independent spectral data. Crystalline enzyme has a much higher affinity for keto acid substrates compared to enzyme in solution. The increased affinity is interpreted in terms of a perturbation of the open/closed conformational equilibrium by the crystal lattice, with the closed form having greater affinity for substrate. The crystal lattice contacts provide energy required for domain closure normally supplied by the excess binding energy of the substrate. In solution, enzyme saturated with amino/keto acid substrate pairs has a greater total fraction of intermediates in the aldehyde oxidation state compared to crystalline enzyme. Assuming the only difference between the solution and crystalline enzymes is in conformational freedom, this difference suggests that one or more substantially populated, aldehydic intermediates in solution exist in the open conformation. Quantitative analyses of the spectra indicate that the value of the equilibrium constant for the open-closed conformational transition of the liganded, aldehydic enzyme in solution is near 1. The C4′ pro-S proton in the ketimine models is oriented nearly perpendicularly to the plane of the pyridine ring, suggesting that the enzyme facilitates its removal by maximizing σ-π orbital overlap. The absence of a localized water molecule near Lys258 dictates that ketimine hydrolysis occurs via a transiently bound water molecule or from an alternative, possibly more open, structure in which water is appropriately bound. A prominent mechanistic role for flexibility of the Lys258 side chain is suggested by the absence of hydrogen bonds to the amino group in the aspartate structure and the relatively high temperature factors for these atoms in both structures.

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