Syncatalytic conformational changes in mitochondrial aspartate aminotransferases. Evidence from modification and demodification of Cys 166 in the enzyme from chicken and pig.

Gehring, Heinz; Christen, Philipp

doi:10.1016/s0021-9258(17)40817-9

Cited by 58 publications

(8 citation statements)

References 31 publications

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“…DISCUSSION Domain Closure in mAspAT K258H. The occurrence of two conformations of AspAT was predicted on the basis of solution studies (Birchmeier et al, 1973;Gehring & Christen, 1978) In solution, the unliganded enzyme exists in the open conformation. Upon binding of dicarboxylate ligands, the equilibrium is shifted toward the closed conformation.…”

Section: Resultsmentioning

confidence: 99%

“…They are identical to those of the wt mitochondrial enzyme. The equilibrium between the two conformations of mAspAT in solution can be monitored by the reaction of Cl66 with thiol reagents, which is faster by 1 order of magnitude in the closed form of the enzyme (Gehring & Christen, 1978). The reactivity of C166 is 1.5-2 times lower in the liganded forms of mAspAT K258H than in the corresponding complexes of the wild-type enzyme (Ziak et al, 1993).…”

Section: Resultsmentioning

confidence: 99%

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Structural Basis for the Catalytic Activity of Aspartate Aminotransferase K258H Lacking the Pyridoxal 5'-Phosphate-Binding Lysine Residue

Malashkevich¹,

Jäger²,

Ziak³

et al. 1995

Biochemistry

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Chicken mitochondrial and Escherichia coli aspartate aminotransferases K258H, in which the active site lysine residue has been exchanged for a histidine residue, retain partial catalytic competence [Ziak et al. (1993) Eur. J. Biochem. 211, 475-484]. Mutant PLP and PMP holoenzymes and the complexes of the latter (E. coli enzyme) with sulfate and 2-oxoglutarate, as well as complexes of the mitochondrial apoenzyme with N-(5'-phosphopyridoxyl)-L-aspartate or N-(5'-phosphopyridoxyl)-L-glutamate, were crystallized and analyzed by means of X-ray crystallography in order to examine how the side chain of histidine 258 can substitute as a general acid/base catalyst of the aldimine-ketimine tautomerization in enzymic transamination. The structures have been solved and refined at resolutions between 2.1 and 2.8 A. Both the closed and the open conformations, identical to those of the wild-type enzyme, were observed, indicating that the mutant enzymes of both species exhibit the same conformational flexibility as the wild-type enzymes, although in AspAT K258H the equilibrium is somewhat shifted toward the open conformation. The replacement of the active site K258 by a histidine residue resulted only in local structural adaptations necessary to accommodate the imidazole ring. The catalytic competence of the mutant enzyme, which in the forward half-reaction is 0.1% of that of the wild-type enzyme, suggests that the imidazole group is involved in the aldimine-ketimine tautomerization. However, the imidazole ring of H258 is too far away from C alpha and C4' of the coenzyme-substrate adduct for direct proton transfer, suggesting that the 1,3-prototropic shift is mediated by a water molecule. Although there is enough space for a water molecule in this area, it has not been detected. Dynamic fluctuations of the protein matrix might transiently open a channel, giving a water molecule fleeting access to the active site.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Structural Basis for the Catalytic Activity of Aspartate Aminotransferase K258H Lacking the Pyridoxal 5'-Phosphate-Binding Lysine Residue

Malashkevich¹,

Jäger²,

Ziak³

et al. 1995

Biochemistry

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“…Substrate Affinities and Domain Closure. The existence of two conformations of AATase was expected from solution studies (Birchmeier et al, 1973;Gehring & Christen, 1978) and was definitively demonstrated by the crystallographic determination of the three-dimensional structures of the open and closed conformations [see e.g. Christen and Metzler (1985), Picot et al (1991), andMcPhalen et al (1992a,b)].…”

Section: Discussionmentioning

confidence: 98%

Crystal structure of true enzymic reaction intermediates: Aspartate and glutamate ketimines in aspartate aminotransferase

1993

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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-A 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 alpha 2 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 sigma-pi 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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“…Secondly, a drastic conformational change leading to the rotation of the Schiff base plane and thereby to the protonation from the same direction as decarboxylation may occur after the initial decarboxylation step. Conformational changes that occur upon the substrate binding have been suggested for aspartate aminotransferase (Metzler et al, 1978;Gehring & Christen, 1978), tyrosine decarboxylase (Vederas et al, 1979), and tryptophanase (Vederas & Floss, 1980). However, a nearly 180°rotation after the formation of the anionic intermediate seems improbable because of necessity of an extremely large structural rearrangement.…”

Section: Discussionmentioning

confidence: 99%

Stereochemistry of meso-.alpha.,.epsilon.-diaminopimelate decarboxylase reaction: the first evidence for pyridoxal 5'-phosphate dependent decarboxylation with inversion of configuration

Asada¹,

Tanizawa²,

Sawada³

et al. 1981

Biochemistry

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The stereochemistry of the decarboxylation of meso-alpha,epsilon-diaminopimelate catalyzed by meso-alpha,epsilon-diaminopimelate decarboxylase (EC 4.1.1.20) of Bacillus sphaericus was determined by stereochemical analyses of [6-2H]-L-lysine produced by the reaction in D2O. The product [6-2H]-L-lysine was converted to levorotatory methyl 5-phthalimido[5-2H]valerate by the reactions not affecting the absolute configuration of the asymmetric carbon atom. By contrast, methyl 5-phthalimido[5-2H]valerate derived from [2,6-2H2]-L-lysine, which was produced from [2,6-2H2]diaminopimelate by decarboxylation in H2O, was dextrorotatory. The authentic methyl (R)-5-phthalimido[5-2H]valerate prepared from L-glutamate with glutamate decarboxylase was levorotatory. These results indicate that the meso-alpha,epsilon-diaminopimelate decarboxylase reaction proceeds in an inversion mode. The deuterium label in [6-2H]-L-lysine was fully conserved during the conversion into pelletierine through [1-2H]cadaverine by the stereospecific diamine oxidase reaction. Thus, the enzymatic decarboxylation of meso-alpha,epsilon-diaminopimelate occurs with inversion of configuration in contrast to the other amino acid decarboxylase reported so far.

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Syncatalytic conformational changes in mitochondrial aspartate aminotransferases. Evidence from modification and demodification of Cys 166 in the enzyme from chicken and pig.

Cited by 58 publications

References 31 publications

Structural Basis for the Catalytic Activity of Aspartate Aminotransferase K258H Lacking the Pyridoxal 5'-Phosphate-Binding Lysine Residue

Structural Basis for the Catalytic Activity of Aspartate Aminotransferase K258H Lacking the Pyridoxal 5'-Phosphate-Binding Lysine Residue

Crystal structure of true enzymic reaction intermediates: Aspartate and glutamate ketimines in aspartate aminotransferase

Stereochemistry of meso-.alpha.,.epsilon.-diaminopimelate decarboxylase reaction: the first evidence for pyridoxal 5'-phosphate dependent decarboxylation with inversion of configuration

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