Domain closure in mitochondrial aspartate aminotransferase

McPhalen, Catherine A.; Vincent, M. G.; Picot, Daniel; Jansonius, Johan N.; Lesk, Arthur M.; Chothia, Cyrus

doi:10.1016/0022-2836(92)90691-c

Cited by 185 publications

(143 citation statements)

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“…The approximately 30" forward rotation (in the view of Fig. 3) of the coenzyme is immediately apparent, and is similar to that observed experimentally with aspartate aminotransferase (Jansonius & Vincent, 1987;McPhalen et al, 1992;Jager et al, 1994) and proposed for DGD (Toney et al, 1995). This change in coenzyme orientation is brought about by the formation of an aldimine bond to GABA (displacing Lys 329), by the relief of steric interactions between P L P C4' and Lys 329 NE once this new bond is formed, and by the relaxation of contacts between the Val 300 side chain and the pyridine ring.…”

Section: Discussionsupporting

confidence: 85%

“…The active site region of the GABA-AT model is quite "open" compared to the closed form of AAT (McPhalen et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…The 3D-ID profiles (Bowie et al, 1991) for the GABA-AT and DGD models are very similar in form. Models for the obligatory external aldimine intermediates were built based on the structure of the holoenzyme, and on coenzyme rearrangements that have been observed experimentally in aspartate aminotransferase structures (Jansonius & Vincent, 1987;McPhalen et al, 1992;Jager et al, 1994). The complete holoenzyme model and the active site regions of the external aldimine models are available upon request to the authors.…”

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confidence: 99%

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Active site model for γ‐aminobutyrate aminotransferase explains substrate specificity and inhibitor reactivities

Toney

Pascarella²,

Biase³

1995

Protein Science

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A homology model for the pig isozyme of the pyridoxal phosphate-dependent enzyme y-aminobutyrate (GABA) aminotransferase has been built based mainly on the structure of dialkylglycine decarboxylase and on a multiple sequence alignment of 28 evolutionarily related enzymes. The proposed active site structure is presented and analyzed. Hypothetical structures for external aldimine intermediates explain several characteristics of the enzyme. In the GABA external aldimine model, the p r o 4 proton at C4 of GABA, which abstracted in the 1,3-azaallylic rearrangement interconverting the aldimine and ketimine intermediates, is oriented perpendicular to the plane of the pyridoxal phosphate ring. Lys 329 is in close proximity and is probably the general base catalyst for the proton transfer reaction. The carboxylate group of GABA interacts with Arg 192 and Lys 203, which determine the specificity of the enzyme for monocarboxylic w-amino acids such as GABA. In the proposed structure for the L-glutamate external aldimine, the a-carboxylate interacts with Arg 445. Glu 265 is proposed to interact with this same arginine in the GABA external aldimine, enabling the enzyme to act on w-amino acids in one half-reaction and on a-amino acids in the other. The reactivities of inhibitors are well explained by the proposed active site structure. The R and S isomers of &substituted phenyl andp-chlorophenyl GABA would bind in very different modes due to differential steric interactions, with the reactive S isomer leaving the orientation of the GABA moiety relatively unperturbed compared to that of the natural substrate. In our model, only the reactive S isomer of the mechanism-based inhibitor vinyl-GABA, an effective anti-epileptic drug known clinically as Vigabatrin, would orient the scissile C4-H bond perpendicular to the coenzyme ring plane and present the proton to Lys 329, the proposed general base catalyst of the reaction. The R isomer would direct the vinyl group toward Lys 329 and the C4-H bond toward Arg 445. The active site model presented provides a basis for site-directed mutagenesis and drug design experiments.Keywords: y-aminobutyrate aminotransferase; GABA; homology modeling; pyridoxal phosphate; Vigabatrin y-Aminobutyrate aminotransferase is a pyridoxal phosphatedependent homodimeric enzyme distributed widely in nature (Cooper, 1985, and references therein). It catalyzes, via a pingpong bi-bi kinetic mechanism, the reversible transfer of the y-amino group of GABA to a-ketoglutarate to yield succinic semialdehyde and L-glutamate, i.e.,

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Section: Discussionsupporting

confidence: 85%

“…The active site region of the GABA-AT model is quite "open" compared to the closed form of AAT (McPhalen et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Active site model for γ‐aminobutyrate aminotransferase explains substrate specificity and inhibitor reactivities

Toney

Pascarella²,

Biase³

1995

Protein Science

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mentioning

confidence: 99%

“…Direct experimental evidence for this proposal has been sparse. It includes the observations that the C R -CH 3 group of the competitive inhibitor 2-methylaspartate in the active site of aspartate aminotransferase is so oriented (12), that arginine decarboxylase is highly sensitive to the size of the substrate side chain (13), as well as the observed specificity in the tryptophan synthetase-catalyzed R-proton exchange of amino acids (14). The variation of reactivity with substrate size for several reactions is presented here and, along with data validating the binding subsite model, provides direct kinetic evidence that, in DGD at least, orientation of a bond to C R orthogonal to the plane of the pyridine ring greatly activates it toward either transamination or decarboxylation.…”

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confidence: 99%

Reactions of Alternate Substrates Demonstrate Stereoelectronic Control of Reactivity in Dialkylglycine Decarboxylase

1998

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Kinetic and product analyses of the reactions of dialkylglycine decarboxylase with several alternative substrates are presented. Rate constants for the reactions of amino and keto acids of several substrates decrease logarithmically with increasing side-chain size. Conversely, k cat for L-amino acid decarboxylation increases with side-chain size. These and other data confirm a proposed model for three binding subsites in the active site. In this model, bond making and breaking in both the decarboxylation and transamination half-reactions occurs at the "A" subsite, which maintains the scissile bond aligned with the p orbitals of the conjugated aldimine and thus maximizes stereoelectronic effects. This strongly supports the proposal by Dunathan (Proc. Natl. Acad. Sci. U.S.A. 55,[712][713][714][715][716]) that PLP-dependent enzymes can largely control reaction specificity by specific orientation about C R in the external aldimine intermediate. The "B" subsite can accept either an alkyl or a carboxylate group, while the "C" subsite accepts only small alkyl groups. This model predicts the existence of nonproductive binding modes for amino acids, which is proposed to be the ultimate origin of the k cat increase with side-chain size for L-amino acid decarboxylation. The specificity of the 2-aminoisobutyrate decarboxylation half-reaction toward oxidative decarboxylation is very high (<1 in 10 5 turnovers yields nonoxidative decarboxylation). The origin of this specificity is explored with the reactions of amino-and methylaminomalonate. These substrates exhibit high yields of nonoxidative decarboxylation, providing support for a model in which the interaction between a carboxylate group in the B subsite and Arg406 is a prerequisite to proton donation to and removal from C R .Dialkylglycine decarboxylase (DGD) 1,2 from Pseudomonas cepacia is a PLP dependent enzyme that catalyzes the oxidative decarboxylation of 2,2-dialkylglycines, yielding CO 2 , a ketone, and the PMP form of the enzyme (Scheme 1). The catalytic cycle is completed by transamination of an R-keto acid, preferentially pyruvate (1-3). The overall reaction catalyzed by DGD is shown below. The unusual ability of DGD to catalyze rapidly two classical PLPdependent reactions has rekindled an interest in this enzyme.The X-ray structure of DGD has been determined to 2.1-Å resolution (4, 5). The fold of the enzyme is roughly similar to the well-studied aspartate aminotransferase (6), which is a member of evolutionary subgroup I of aminotransferases (7). DGD is a representative member of evolutionary subgroup II (8), and its fold is closely similar to the other known structures of subgroup II enzymes (9, 10). The active site structure of DGD is similar to that of aspartate aminotransferase in that the interactions made between coenzyme and protein are highly conserved (5).Toney et al. (5) proposed a structurally based model of the active site of DGD in which there are three binding subsites (A-C) that differ in their functional group specificities. One of t...

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Structural principles governing domain motions in proteins

Hayward

1999

Proteins

127

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With the use of a recently developed method, twenty-four proteins for which two or more X-ray conformers are known have been analyzed to reveal structural principles that govern domain motions in proteins. In all 24 cases, the domain motion is a rotation about a physical axis created through local interactions both covalent and noncovalent. In many cases, two or more mechanical hinges separated in space create a stable hinge axis for precise control of the domain closure. The terminal regions of alpha-helices and beta-sheets have been found to act as mechanical hinges in a significant number of cases. In some cases, the two terminal regions of neighboring strands of a single beta-sheet can create a hinge axis, as can the two termini of a single alpha-helix. These two structures have been termed the "double-hinged beta-sheet" and "double-hinged alpha-helix," respectively. A flexible loop that attaches one domain to another and through which the effective hinge axis passes is another construct that is used to create a hinge. Noncovalent interactions between segments remote along the polypeptide chain can also form hinges. In addition alpha-helices that preserve their hydrogen bonding structure when bent have been found to behave as mechanical hinges. It is suggested that these alpha-helices act as a store of elastic energy that drives the closing of domains for rapid capture of the substrate. If the repertoire of possible interdomain structures is as limited as this study suggests, the dynamic behavior of proteins could soon be predicted using bioinformatics techniques. Proteins 1999;36:425-435.

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Domain closure in mitochondrial aspartate aminotransferase

Cited by 185 publications

References 54 publications

Active site model for γ‐aminobutyrate aminotransferase explains substrate specificity and inhibitor reactivities

Active site model for γ‐aminobutyrate aminotransferase explains substrate specificity and inhibitor reactivities

Reactions of Alternate Substrates Demonstrate Stereoelectronic Control of Reactivity in Dialkylglycine Decarboxylase

Structural principles governing domain motions in proteins

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