Glutamic-Aspartic Transaminase

Jenkins, W. Terry; D'Ari, Linda

doi:10.1016/s0021-9258(18)96396-9

Cited by 85 publications

(27 citation statements)

References 15 publications

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“…Tryptophan 140 and Its Interaction with Dicarboxylates. Aspartate aminotransferases are known to bind dicarboxylate ions tightly (Jenkins, 1964;Jenkins & D'Ari, 1966;Michuda & Martinez-Carrion, 1969;Cheng et al, 1971;Ivanov et al, 1973;Bonsib et al, 1975;Jenkins & Fonda, 1985;Metzler, C. M., & Metzler, 1987). In a previous 1 H NMR study of AspAT from E. coli (Metzler, D. E., et al, 1994c) a sharp peak was seen to move from a position above 11 ppm downfield to 11.26 ppm upon titration of the enzyme with succinate.…”

Section: Discussionmentioning

confidence: 99%

“…Similarly, most in Vitro studies of the enzyme are carried out at relatively high ionic strength. AspAT is known to bind small monoanions and dianions such as Cl -, formate, acetate, sulfate, and phosphate (Jenkins & D'Ari, 1966;Bergami et al, 1968;Metzler, C. M., & Metzler, 1987) as well as dicarboxylates. Anions act as competitive inhibitors and are thought to participate in product displacement reactions (Jenkins & D'Ari, 1966;Cheng & Martinez-Carrion, 1972).…”

Section: Discussionmentioning

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: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

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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“…Apparently, at low pH values glutarate interacts through both its carboxylate groups with two positively charged groups on supernatant aspartate aminotransferase. However, at high pH values glutarate binds by only one carboxylate group (Jenkins and D'Ari, 1966). The two positively charged groups of aspartate aminotransferase that interact with glutarate must be closer together than the carboxyl binding sites on glutamate decarboxylase.…”

Section: Discussionmentioning

confidence: 99%

“…Competition between buffer anions and substrates or inhibitors has been observed previously with other enzymes. One of the positively charged sites on aspartate aminotransferase that interacts with the carboxylate group of glutarate is masked by the buffer anion which must be displaced by glutarate (Jenkins and D'Ari, 1966). Halide anions have been reported to inhibit mouse brain glutamate decarboxylase competitively with respect to the substrate (Susz et al, 1966).…”

Section: Discussionmentioning

confidence: 99%

Glutamate decarboxylase. Substrate specificity and inhibition by carboxylic acids

Fonda¹

1972

Biochemistry

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“…Enzyme.anion + ligand Enzyme.ligand + anion Indeed, when the concentration of the anion in solution is extrapolated to zero, the dissociation constant for glutarate is found to approach zero (Jenkins & D'Ari, 1966a). Therefore, the variation in affinity of the enzyme for any ligand can be a result of anion binding.…”

Section: Introductionmentioning

confidence: 99%

Aspartate aminotransferase reconstituted with N- and O-methylated vitamin B6 phosphates

Chen

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Eapoapo form of aspartate aminotransferase E.N-MePLP aspartate aminotransferase reconstituted with N-methylpyridoxal 5^-phosphate E.O-MePLP aspartate aminotransferase reconstituted with 0-methylpyridoxal 5'-phosphate kg a-ketoglutarate MDH malic dehydrogenase NADH the reduced form of g-nicotinamide adenine dinucleotide N-MePLP N-methylpyridoxal 5'-phosphate N-MePMP 0-methylpyridoxamine 5'-phosphate nmr nuclear magnetic resonance ORD optical rotary dispersion 0-MePLP 0-methylpyridoxal 5'-phosphate 0-MePMP 0-methylpyridoxamine 5'-phosphate PLP pyridoxal 5'-phosphate PMP pyridoxamine 5'-phosphate TEA.HCl triethanolamine hydrochloride Another attractive aspect of aspartate aminotransferase with respect to the study of coenzyme-protein interaction is the fact that the vitamin Bg cofactor can be removed readily from the protein. In its place, a selection of analogs of pyridoxal 5'-phosphate with various structural modifications can be incorporated. The apoenzyme reconstituted with these analogs displays different degrees of activity. The study of these apoenzyme-analog complexes can provide clues to the mechanisms of inter action between the coenzyme and various functional groups on the protein. Recently, the crystallization of both the mitochondrial and the cytoplasmic isoenzymes has sparked new excitement in the enzymology of transamination (Gehring et al., 1977; Arnone et al., 1977; Borisov et al., 1978). The three-dimensional structures of these proteins are now being determined by X-ray crystallography. The crystalline enzymes have been shown to be catalytically competent. Crystalline enzyme-substrate complexes of natural substrates and various inhibitory ligands as well as crystalline apoenzyme-analog complexes have been prepared (Metzler et al., 1978). Many of these possess linear dichroism and their polarized light absorption spectra have been recorded. Observations of these spectra together with the forthcoming knowledge of the three-dimensional structures of the proteins will provide a means of examining the inter action of the coenzyme with the protein at the atomic level.The study reported in this thesis was undertaken to further characterize two apoenzyme-analog complexes of aspartate aminotransferase.The N-and 0-methylated derivatives of pyridoxal 5'-phosphate have been incorporated into the enzyme (Furbish et al., 1969; Mora et al., 1972). Compounds 23 24 25 26 0.5-4.45 o.gs <2 min^ 'V/10 min^ 322,382,4084 3924 positive at 3804 380,42O4 3774 310(shoulder)4 310(shoulder)4 04 7.5II l4 <2 min to give band at 326 nm4 <2 min4 slow to give 335 nm4 V. s low4 5 min to give a small transient at 485 nm4 5 min, loss of 485 mn4 gave small band at 480 nmll

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Glutamic-Aspartic Transaminase

Cited by 85 publications

References 15 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

Glutamate decarboxylase. Substrate specificity and inhibition by carboxylic acids

Aspartate aminotransferase reconstituted with N- and O-methylated vitamin B6 phosphates

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Glutamic-Aspartic Transaminase

Cited by 85 publications

References 15 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

Glutamate decarboxylase. Substrate specificity and inhibition by carboxylic acids

Aspartate aminotransferase reconstituted with N- and O-methylated vitamin B6 phosphates

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Product

Resources

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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