Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure

Kirsch, Jack F.; Eichele, Gregor; Ford, Geoffrey C.; Vincent, M. G.; Jansonius, Johan N.; Gehring, Heinz; Christen, Philipp

doi:10.1016/0022-2836(84)90333-4

Cited by 463 publications

(371 citation statements)

References 57 publications

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“…With regards to the co-factor-binding pocket in the active site of PLP-dependent enzymes, details of how the different proteins interact with the co-enzyme have been well-defined only in the cases for which high-resolution crystal structures have been determined (Kirsch et al, 1984;Hyde et al, 1988;McPhalen et al, 1992;Antson et al, 1993;Momany et al, 1995b;Toney et al, 1995). In particular, a glycine-rich loop has been recognized at the co-factor binding site in those enzymes for which crystal structures are available and has also been identified through site-directed mutagenesis in threonine and D-serine dehydratases (Dana et al, 1987;Marceau et al, 1990) and more recently in aminolevulinate synthase (Gong et al, 1995(Gong et al, , 1996.…”

Section: Discussionmentioning

confidence: 99%

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

et al. 1997

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Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzyme, the resultant C111A and C111 S mutant enzymes exhibit kc,, values of about 50% and 15%, respectively, at pH 6.8, while the K, values remain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (LJ-HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys-1 1 ]-+Ala mutant displays a reduced affinity for pyridoxal 5"phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D-Dopa and the bound co-enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pK, and pKb for the groups involved in catalysis were determined for the wild-type and the C l l l A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420 -+ 390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys-1 1 1 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Bom Voltattorni C. 1991. Eur JBiochern 201:393-397). Moreover, our findings provide evidence that Cys-111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active-site conformation.Keywords: active site; decarboxylase; pyridoxal 5"phosphate; site-directed mutagenesis The versatility of pyridoxal 5"phosphate (PLP) as a co-factor is demonstrated by the wide variety of reactions that enzymes dependent upon it catalyze. Although detailed catalytic mechanisms and information correlating structure and function have been reported for many PLP dependent enzymes, such information is conspicuously lacking for PLP dependent decarboxylases.Even though aromatic amino acid decarboxylases have been cloned from a number of mammalian (Ichinose et al., 1989;Tanaka et al., 1989;Kang & Joh, 1990;Taketoshi et al., 1990), insect (Eveleth et al., 1986, and plant sources (DeLuca et al., 1989;Kawalleck et al., 1993; Facchini & DeLuca, 1994)

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Section: Discussionmentioning

confidence: 99%

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

et al. 1997

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“…(1)] and TATase [Eq. (2)] reactions were monitored with the maleate dehydrogenase (MDH) 1,3 and the D-2-hydroxyisocaproate dehydrogenase (HDH) 38 coupled assays, respectively. Both coupling enzymes use NADH to reduce the a-ketoacid product of the aminotranferase reaction.…”

Section: Steady-state Kinetic Assaysmentioning

confidence: 99%

“…[3][4][5][6] Mutagenesis studies have shown that the specificity of these enzymes can be modulated through the modification of relatively few residues. [7][8][9][10][11] For example, the HEX 7 and SRHEPT 8 mutants, which behave as TATases in terms of substrate specificity (w-TATase active sites), were obtained from rational redesign and directed evolution of eAATase, respectively.…”

Section: Introductionmentioning

confidence: 99%

Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study

Deu

Kirsch

2011

Protein Science

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The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate-(AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.

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“…fold I-VII) and placed ALAS in the same fold as that of AAT (i.e., fold I). The X-ray crystal structure of Escherichia coli AAT revealed a conserved tyrosine residue (Y70) involved in cofactor PLP binding, which corresponds to the invariant Tyr-121 residue in murine erythroid ALAS (Kirsch et al, 1984;Smith et al, 1989).…”

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confidence: 99%

The role of tyrosine 121 in cofactor binding of 5‐aminolevulinate synthase

1998

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Abstract5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthesis in nonplant eukaryotes and some prokaryotes. It functions as a homodimer and requires pyridoxal 5"phosphate as an essential cofactor. Tyr-121 is a conserved residue in all known sequences of 5-aminolevulinate synthases. Further, it corresponds to Tyr-70 of Escherichia coli aspartate aminotransferase, which has been shown to interact with the cofactor and prevent the dissociation of the cofactor from the enzyme. To test whether Tyr-121 is involved in cofactor binding in murine erythroid 5-aminolevulinate synthase, Tyr-121 of murine erythroid 5-aminolevulinate synthase was substituted by Phe and His using site-directed mutagenesis. The Y121F mutant retained 36% of the wild-type activity and the K, value for substrate glycine increased 34-fold, while the activity of the Y 121 H mutant decreased to 5% of the wild-type activity and the K, value for glycine increased fivefold. The pKa, values in the pH-activity profiles of the wild-type and mutant enzymes were 6.41, 6.54, and 6.65 for wild-type, Y 121F, and Y 121H, respectively. The UV-visible and CD spectra of Y121F and Y 121H mutants were similar to those of the wild-type with the exception of an absorption maximum shift (420 + 395 nm) for the Y121F mutant in the visible spectrum region, suggesting that the cofactor binds the Y 121F mutant enzyme in a more unrestrained manner. Y121F and Y121H mutant enzymes also exhibited lower affinity than the wild-type for the cofactor, reflected in the K,, values for pyridoxal 5"phosphate (26.5,6.75. and 1.78 p M for Y 121F, Y 121H, and the wild-type, respectively). Further, Y12 IF and Y 121H proved less thermostable than the wild type. Taken together, these findings indicate that Tyr-121 plays a critical role in cofactor binding of murine erythroid 5-aminolevulinate synthase.

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Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure

Cited by 463 publications

References 57 publications

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study

The role of tyrosine 121 in cofactor binding of 5‐aminolevulinate synthase

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