Kinetic isotope effect studies on aspartate aminotransferase: evidence for a concerted 1,3 prototropic shift mechanism for the cytoplasmic isozyme and L-aspartate and dichotomy in mechanism

Aspartate aininotransferase (mitochondrial isoenzyme from chicken) has been found to racemize very slowly dicarboxylic amino acid substrates in the presence of their cognate 0x0 acids [Kochhar, S. & Christen, P. (1988) Eur. J . Biochern. 175, 433-4381. Tyrosine, phenylalanine and alanine are racemized at the same rate although they undergo the transamination reaction 3-5 orders of magnitude more slowly than the dicarboxylic substrates. Similarly, the truncated enzyme aspartate aminotransferase-(27/32 -410) catalyzes the racemization at the same rate as the native enzyme, while its rate of transamination is decreased to 3% of that of the native enzyme. Apparently, the ratelimiting step in racemization is not immediately linked to the transamination cycle. Decreasing the water concentration in the reaction medium by adding methanol at 0°C drastically reduces the rate of racemization without affecting the rate of transamination. On the basis of these and additional kinetic data and the model of the three-dimensional structure of the active site, we conclude that a water molecule is responsible for the protonation of Ccr of the coenzyme-substrate intermediate from the wrong side. The diffusion of the water molecule into the interior of the enzyme appears to be the rate-limiting step in aspartate-aminotransferase-catalyzed racemization.The crucial chemical event in enzymic transamination is the tautomerization of the aldimine to the ketimine intermediate (for reviews on the mechanism of enzymic transamination, see Kirsch et al., 1984;Arnone et a]., 1985;. In the reverse half-reaction of enzymic transamination, i.e. from the pyridoxamine to the pyridoxal form of the enzyme, Ca of the substrate moiety of the coenzyme substrate adduct is protonated from the siside; the subsequent transimination of the aldimine intermediate yields the L-amino acid product. Protonation from the opposite side will result in a u-amino acid product. Recently we have found that in the case of aspartate aminotransferase protonation from the wrong side is a very rare event occurring once in 3 x lo7 and 7 x lo6 transamination cycles for the C4 and C 5 dicdrboxylate substrates, respectively. The rate of aspartate-aminotransferase-catalyzed racemization is thus only one to two orders of magnitude faster than that of the equivalent non-enzymic reaction of coenzyme and substrate (Kochhar and Christen, 1988

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“…The solvent isotope effect of 2.5 for enzymic transamination is close to values previously reported (Pfister et al, 1985;Julin and Kirsch, 1989).…”

Section: Resultssupporting

confidence: 90%

Mechanism of racemization of amino acids by aspartate aminotransferase

Kochhar

Christen

1992

European Journal of Biochemistry

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“…This would avoid formation of the quinonoid intermediate altogether. Analogous concerted transition states have been proposed for nonenzymatic model transaminations (30) and for aspartate aminotransferase (31).…”

Section: Discussionmentioning

confidence: 90%

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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“…Phenylpyruvic acid, and 2-oxo-4-methylpentanoic acid were obtained from Sigma. L-4-methylphenylalanine was from Schweizerhall, Inc. [a-'H]-~,~-aspartate (Julin & Kirsch, 1989) was a gift from M.D. Toney and D.A.…”

Section: Methodsmentioning

confidence: 99%

The use of natural and unnatural amino acid substrates to define the substrate specificity differences ofescherichia coliaspartate and tyrosine aminotransferases

et al. 1995

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The tyrosine (eTATase) and aspartate (eAATase) aminotransferases of Escherichia coli transaminate dicarboxylic amino acids with similar rate constants. However, eTATase exhibits -102-104-fold higher second-order rate constants for the transamination of aromatic amino acids than does eAATase. A series of natural and unnatural amino acid substrates was used to quantitate specificity differences for these two highly related enzymes. A general trend toward lower transamination activity with increasing side-chain length (extending from aspartate to glutamate to a-aminoadipate) is observed for both enzymes. This result suggests that dicarboxylate ligands associate with the two highly related enzymes in a similar manner. The high reactivity of the enzymes with L -A s~ and L-GIu can be attributed to an ion pair interaction between the side-chain carboxylate of the amino acid substrate and the guanidin0 group of the active site residue Arg 292 that is common to both enzymes. A strong linear correlation between side-chain hydrophobicity and transamination rate constants obtains for n-alkyl side-chain amino acid substrates with eTATase, but not for eAATase. The present kinetic data support a model in which eAATase contains one binding mode for all classes of substrate, whereas the active site of eTATase allows an additional mode that has increased affinity for hydrophobic amino acids.Keywords: aspartate aminotransferase; kinetics; substrate specificity; tyrosine aminotransferase Escherichia coli aspartate aminotransferase (eAATase; EC 2.6.1.1) and tyrosine aminotransferase (eTATase; EC 2.6.1.5) are PLP-containing transaminases that catalyze the reversible interconversion of amino acids and their corresponding a-keto acids. The bound cofactor acts as a transient amino group carrier, shuttling between the pyridoxal phosphate and pyridoxamine phosphate forms. The transamination reaction is described by a ping-pong bi-bi mechanism (Equation lA,B): with the side-chain carboxylate group of aspartate or glutamate (Kirsch et al., 1984;Malashkevich et al., 1993). Mutational stud-

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Kinetic isotope effect studies on aspartate aminotransferase: evidence for a concerted 1,3 prototropic shift mechanism for the cytoplasmic isozyme and L-aspartate and dichotomy in mechanism

Cited by 52 publications

References 45 publications

Mechanism of racemization of amino acids by aspartate aminotransferase

Mechanism of racemization of amino acids by aspartate aminotransferase

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

The use of natural and unnatural amino acid substrates to define the substrate specificity differences ofescherichia coliaspartate and tyrosine aminotransferases

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