Mechanism of racemization of amino acids by aspartate aminotransferase

Abstract

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

Aspartate racemase, generating neuronal D-aspartate, regulates adult neurogenesis

“…In a genomic sequence analysis, we were unable to find candidate mammalian DR genes based on homology to serine racemase or bacterial aspartate racemases, which are either PLP-independent or PLP-dependent enzymes (14,15). Vacca and coworkers (16)(17)(18) demonstrated that glutamate-oxalacetate transaminase (GOT) can generate small amounts of D-aspartate in the process of transaminating Laspartate to L-glutamate, and that formation of D-aspartate is augmented in GOT mutants, wherein histidine replaces tryptophan-140 and lysine replaces arginine-292. Tryptophan-140 normally prevents access of a proton donor, such as a water molecule, which can effectuate racemization (Fig.…”

Substitution of Apolar Residues in the Active Site of Aspartate Aminotransferase by Histidine

“…An increase in the rate of racemization is only observed with alanine as substrate. Apparently, the protonating water molecule (Kochhar and Christen, 1992) finds easier access to the re side of Ca in the case of alanine than of dicarboxylic or aromatic substrates. The side chain of alanine binds less tightly and fails to induce the movement of the small domain that closes the active-site cleft (Kirsch et al, 1984;Sandmeier, E., unpublished).…”

“…This slow side reaction occurs only once during lo7 transamination cycles (chicken mitochondrial AspAT) and is due to the reprotonation of the coenzyme-substrate adduct at Ca from the re instead of the si side (Kochhar and Christen, 1988). No ionizable groups are located at the re side; a transient water molecule appears to be responsible for the protonation at Ca from this side, its diffusion into the active-site pocket being the rate-limiting step of AspAT-catalyzed racemization (Kochhar and Christen, 1992).…”

The Investigation of Zinc-Dependent Enzymes in Pregnant Women and their Correlation to Zinc Deficiency

“…The kit provides a rapid, simple, sensitive, and reliable test suitable for high throughput activity assay of ALT with a detection limit of 10mU per well [14].…”