Preparation of the phosphopyridoxamine form of the glutamic-aspartic transaminase

Jenkins, W. Terry; D'Ari, Linda

doi:10.1016/0006-291x(66)90656-5

Cited by 53 publications

(29 citation statements)

References 10 publications

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“…Construction of the PMP Form of AspAT. The PLP form of AspAT was converted to the PMP form by adding cysteine sulfinate, and the excess cysteine sulfinate was removed by Sephadex G-25 (Jenkins & D'Ari, 1966). Since PMP was completely dissociated from D222A and D222N during the G-25 chromatography, the PMP forms of these mutant enzymes were reconstituted by adding PMP to the apo-forms.…”

Section: Methodsmentioning

confidence: 99%

Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate

Yano

Kuramitsu²,

Tanase³

et al. 1992

Biochemistry

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Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP). This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis. The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme. In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH. The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme. Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme. While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate. In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme. The presteady-state reactions of these two mutant enzymes with [2-2H]aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that [kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H. (1990) Biochemistry 29, 5469-5476] for the wild-type enzyme. These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1. Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.

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Section: Methodsmentioning

confidence: 99%

Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate

Yano

Kuramitsu²,

Tanase³

et al. 1992

Biochemistry

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“…Direct observation of the pre-equilibrium of Scheme 1 is complicated by the ensuing slow shift of the absorbance maximum from 328nm to 360nm (X --Y). A close approximation to the pre-equilibrium was obtained in practice, however, in the presence of cysteinesulphinate, a substrate analogue that reacts rapidly and irreversibly with the aldimine form of aspartate transaminase giving pyruvate and the aminic form ofthe enzyme (Jenkins &D'Ari, 1966). As shown in Scheme 2, cysteinesulphinate can be used to recycle the aldimine form of the enzyme to the aminic species.…”

Section: Spectral Shiftfrom 332nm To 328nmmentioning

confidence: 68%

Reaction of the aminic form of aspartate transaminase with difluoro-oxaloacetate

Briley¹,

Eisenthal²,

Harrison³

et al. 1977

Biochemical Journal

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Addition of difluoro-oxaloacetate to the aminic form of aspartate transaminase causes a rapid shift of absorbance maximum of the enzyme from 332 nm to 328 nm, followed by a much slower shift to 360 nm corresponding to complete conversion of the aminic form of the enzyme into the aldimine form or a species with similar spectral parameters in rapid equilibrium with it. Kinetic analysis of both the initial fast reaction and the overall slow reaction by using repeated spectral scanning and stopped-flow techniques allows formulation of a basic reaction mechanism involving at least two intermediate enzyme complexes. Computer simulation of the progress curves of the initial fast reaction based on the suggested reaction mechanism gives kinetic parameters that are consistent with all the data obtained by other methods. A molecular reaction scheme involving a ketimine Schiff-base intermediate is proposed.

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“…Activity was assayed spectrophotometrically by using published procedures (Slebe & Martinez-Carrion, 1978). Apoenzyme was prepared according to Jenkins (Jenkins & D'Ari, 1966a). Apoenzyme obtained by using this method contains inorganic phosphate bound in the active site (Iriarte et al, 1985); phosphate was displaced by arsenate upon three dialysis against a hundredfold volume of 50 mM sodium arsenate, pH 6.8, followed by dialysis against 50 mM KC1 to remove the nonbound arsenate (Mattingly et al, 1982).…”

Section: Methodsmentioning

confidence: 99%

Ionization state of the coenzyme 5'-phosphate ester in cytosolic aspartate aminotransferase. A Fourier transform infrared spectroscopic study

Sanchez-Ruiz¹,

Martinez‐Carrion²

1986

Biochemistry

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In order to determine the ionization state of the 5'-phosphate of bound pyridoxal phosphate, a Fourier transform infrared spectroscopic study of cytosolic aspartate aminotransferase has been carried out. Dianionic and monoanionic phosphate monoesters give rise to two bands each in the infrared spectrum [Shimanouchi, T., Tsuboi, M., & Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498]. These bands can be identified in infrared spectra of the free coenzyme in solution. Due to interfering bands arising from the protein, only the band assigned to the symmetric stretching of the dianionic phosphate is observed in holoenzyme solutions. The integrated intensity of this band does not change with pH in the range 5.3-8.6, while for free pyridoxal phosphate, the integrated intensity of the same band changes with pH according to the pK value expected for the 5'-phosphate group in solution. Moreover, the value of the integrated intensity for the bound cofactor is close to the value given by free cofactor at pH 8-9. These results suggest that the 5'-phosphate of the bound cofactor remains mostly dianionic throughout the investigated pH range and disfavor other interpretations in terms of ionization of the phosphate group on the basis of the nuclear magnetic resonance 31P chemical shift-pH titration curve of holoenzyme [Schnackerz, K. D. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, E. A., Ed.) Part A, pp 195-208, Alan R. Liss, New York].(ABSTRACT TRUNCATED AT 250 WORDS)

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Preparation of the phosphopyridoxamine form of the glutamic-aspartic transaminase

Cited by 53 publications

References 10 publications

Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate

Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate

Reaction of the aminic form of aspartate transaminase with difluoro-oxaloacetate

Ionization state of the coenzyme 5'-phosphate ester in cytosolic aspartate aminotransferase. A Fourier transform infrared spectroscopic study

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