A Kinetic and Equilibrium Analysis of the Glutamic Oxaloacetate Transaminase Mechanism

Velick, Sidney F.; Vavra, John D.

doi:10.1016/s0021-9258(19)63406-x

Cited by 340 publications

(94 citation statements)

References 11 publications

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“…The 15 N chemical shift changes that we have observed for the histidine NH resonances in the pH range 5.5-8.5 are less than 2 ppm, indicating that all three active site histidines and the unidentified fourth one remain unprotonated at pH 5.5 and above and, therefore, have microscopic pK a values lower than 5.5. This is in agreement with the results of the 1 H NMR titration studies of porcine cytosolic AspAT (S. Tanase, personal communication) and previous observations that V max of the enzyme does not change in this pH range (Velick & Vavra, 1962;Kiick & Cook, 1983).…”

Section: Discussionsupporting

confidence: 93%

“…The yield of enriched protein was typically 35 mg per liter M9 medium. The activity of the purified enzyme was checked by the method of Velick and Vavra (1962) monitoring spectrophotometrically the production of oxalacetate. For k cat of the forward reaction we observed values of 215 ( 12 s -1 for the unlabeled enzyme and 223 ( 9 s -1 for the labeled enzyme, at 25 °C.…”

Section: Methodsmentioning

confidence: 99%

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Use of ¹H−¹⁵N Heteronuclear Multiple-Quantum Coherence NMR Spectroscopy To Study the Active Site of Aspartate Aminotransferase

et al. 1997

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Aspartate aminotransferase from Escherichia coli, an 88 kDa enzyme, was uniformly and selectively enriched with 15N and was studied by heteronuclear multiple-quantum coherence NMR spectroscopy in H2O. Good resolution was obtained for the downfield region (above 9.5 ppm chemical shift in the 1H dimension) for NH protons in the amide, indole, imidazole, and guanidinium group regions and several resonances were tentatively assigned. Two downfield resonances, at 12.6 and 11.36 ppm, appear to belong to oxygen- or sulfur-bound protons. The most downfield amide resonance at 11.78 ppm was assigned to the active site cysteine 192 whose peptide proton is 2.9 A away from the negatively charged carboxyl group of aspartate 199. Large downfield shifts (up to 1.15 ppm) of the indole NH resonance of the active site tryptophan 140 were observed upon binding of dicarboxylic inhibitors to the pyridoxal 5'-phosphate (PLP) form and of inorganic dianions to the pyridoxamine 5'-phosphate (PMP) form of the enzyme. We discuss these striking differences in the light of the available crystallographic data. Active sites of proteins, as well as specific inhibitory molecules, often contain negatively charged groups. These may be able to form hydrogen-bonds to NH groups and to shift the NH resonances downfield into a less crowded and therefore more readily observable region for many large proteins. Our approach, which makes use of both HMQC spectroscopy and NOE observations, should be widely applicable.

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Section: Discussionsupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Use of ¹H−¹⁵N Heteronuclear Multiple-Quantum Coherence NMR Spectroscopy To Study the Active Site of Aspartate Aminotransferase

et al. 1997

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“…High activities of AspAT and branched-chain aminotransferase were also detected in the cyanobacteria Anabaena cylindrica and Anacystis nidulans. [34][35][36] In cyanobacteria, the GS-GOGAT pathway, with a net yield of one glutamate molecule, represents the most important mode of use of 2-oxoglutarate, since cyanobacteria have an incomplete tricarboxylic acid cycle due to the lack of 2-oxoglutarate dehydrogenase and succinyl-CoA synthetase activities. 37) Therefore, those aminotransferase reactions with glutamate as amino donor, particularly the AspAT reaction, might be an important reaction for protein synthesis or many metabolic pathways.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Aspartate Aminotransferase from the CyanobacteriumPhormidium lapideum

Kim¹,

Ikegami²,

Nakaoka³

et al. 2003

Bioscience, Biotechnology, and Biochemistry

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Aspartate aminotransferase (AspAT) was purified to homogeneity from cell extracts of the non-N2-fixing cyanobacterium Phormidium lapideum. The NH2-terminal sequence of 25 amino acid residues was different from the sequences of the subfamily Ialpha of AspATs from eukaryotes and Escherichia coli, but it was similar to sequences of the subfamily Igamma of AspATs from archaebacteria and eubacteria. The enzyme was most active at 80 degrees C and was stable at up to 75 degrees C. Thermal inactivation (60-85 degrees C) of the enzyme followed first-order kinetics, with 2-oxoglutarate causing a shift of the thermal inactivation curves to higher temperatures. However, at 25 degrees C the kcat of P. lapideum AspAT was nearly equal to the values of AspATs from mesophilic organisms. The enzyme used L-aspartate and L-cysteine sulfinate as amino donors and 2-oxoglutarate as an amino acceptor. The Km values were 5.0 mM for L-aspartate, 5.7 mM for L-glutamate, 0.2 mM for 2-oxoglutarate, and 0.032 mM for oxaloacetate.

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“…The enzymatic activity for each a-keto acid was then tested by using Michaelis-Menten kinetics for the half reaction. 34 Initial velocities of the half reaction were measured at different concentrations of a-keto acids in the presence of excess Met. The conversion of each a-keto acid into the corresponding amino acid was monitored by measuring the decrease in absorbance of the a-keto acid at the absorption maximum.…”

Section: Resultsmentioning

confidence: 99%

Genetic incorporation of unnatural amino acids biosynthesized from α-keto acids by an aminotransferase

et al. 2014

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A Kinetic and Equilibrium Analysis of the Glutamic Oxaloacetate Transaminase Mechanism

Cited by 340 publications

References 11 publications

Use of ¹H−¹⁵N Heteronuclear Multiple-Quantum Coherence NMR Spectroscopy To Study the Active Site of Aspartate Aminotransferase

Use of ¹H−¹⁵N Heteronuclear Multiple-Quantum Coherence NMR Spectroscopy To Study the Active Site of Aspartate Aminotransferase

Characterization of Aspartate Aminotransferase from the CyanobacteriumPhormidium lapideum

Genetic incorporation of unnatural amino acids biosynthesized from α-keto acids by an aminotransferase

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A Kinetic and Equilibrium Analysis of the Glutamic Oxaloacetate Transaminase Mechanism

Cited by 340 publications

References 11 publications

Use of 1H−15N Heteronuclear Multiple-Quantum Coherence NMR Spectroscopy To Study the Active Site of Aspartate Aminotransferase

Use of 1H−15N Heteronuclear Multiple-Quantum Coherence NMR Spectroscopy To Study the Active Site of Aspartate Aminotransferase

Characterization of Aspartate Aminotransferase from the CyanobacteriumPhormidium lapideum

Genetic incorporation of unnatural amino acids biosynthesized from α-keto acids by an aminotransferase

Contact Info

Product

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About

Use of ¹H−¹⁵N Heteronuclear Multiple-Quantum Coherence NMR Spectroscopy To Study the Active Site of Aspartate Aminotransferase

Use of ¹H−¹⁵N Heteronuclear Multiple-Quantum Coherence NMR Spectroscopy To Study the Active Site of Aspartate Aminotransferase