The reaction of ornithine aminotransferase with ornithine

Abstract

Rat liver ornithine aminotransferase is found to exchange the pro-S hydrogen on the 5-carbon atom of ornithine exclusively, thus showing that the mammalian enzyme transfers the 3-amino group and not the a-amino group as has been demonstrated with the plant enzyme [Mestichelli, Gupta & Spenser (1979) J. Biol. Chem. 254, The enzyme also transfers the a-amino group of glutamate and the kinetics of the half reactions between the enzyme and both amino acids are compared. Rate and dissociation constants for both half reactions are determined.Accounts of the metabolism of ornithine in standard textbooks (e.g. Mahler & Cordes, 1966;Lehninger, 1975) indicate that transamination of this diamino acid entails loss of the 3-amino group and not the a-amino group. The enzyme responsible, ornithine 5-aminotransferase (L-ornithine: 2-oxoacid aminotransferase, EC 2.6.1.13), also interconverts 2-oxoglutarate and glutamate efficiently in the second half of the complete Ping-Pong mechanism. It seemed curious that although ornithine has an a-amino group configurationally identical with that of L-glutamate the enzyme should act exclusively on the 3-amino group even though at pH values near neutrality the a-amino group (pK = 8.7) is more reactive than the 3-amino group (pK = 10.8).Mestichelli et al. (1979) provided evidence that the metabolic route from ornithine to proline in plants occurs via loss of the a-amino group, not the 3-amino group, and they pointed out that much of the evidence indicating 3-amino group transfer depended on the occurrence of a reaction between the product and 2-aminobenzaldehyde to give a yellow colour. The product of transamination of either group is a cyclic internal imine; Al-pyrroline-5-carboxylate if the 3-amino group is transferred and Al-pyrroline-2-carboxylate if the a-amino group is transferred. As pointed out by Mestichelli et al. (1979), both compounds react similarly with aminobenzaldehyde.Because transamination proceeds via labilization of a proton on the relevant carbon atom, the amino group transferred may be identified by determining which proton is exchanged by the enzyme in deuterated solvent. The present paper identifies the exchangeable ornithine proton. In addition the equilibria and kinetics of the separate half reactions with ornithine and glutamate are compared. Experimental Enzyme preparationOrnithine aminotransferase was prepared by a method based on that described by Peraino et al. (1969) but with the following modifications.(1) The amount of the enzyme present in rat liver is increased several-fold when the animals are fed on a high-protein diet. Because of the high cost of the commercial 60% casein laboratory diet used by Peraino et al. (1969) the rats in the present study were fed for four days on dry textured soya bean protein (Temptein Soya Protein Meat-Like Chunks, Brooke Bond Oxo Catering Services Division, Croydon, Surrey, U.K.).(2) The method of Peraino et al. (1969) includes a step in which the preparation is heated to 55 0C. We experienced occasional severe losses at ...

Determinants of Substrate Specificity in ω-Aminotransferases

“…An analysis of the kinetics of the separate half-reactions of wild-type rat kidney Orn-AT with ornithine, glutamate, and ketoglutarate has been reported earlier (3). In the present work, similar analyses were conducted on recombinant wild-type human Orn-AT to provide a reliable basis for determining the effects of the mutations.…”

“…between pyridoxaldimine and pyridoxamine forms by means of two coupled half-reactions (2,3). The half-reaction converting ketoglutarate to glutamate is the same for both enzymes as well as for the majority of other aminotransferases.…”

“…For this reason, the enzymes are known as "-aminotransferases" (4). In the case of GABA-AT, the amino group transferred is the only amino group in the substrate molecule, whereas Orn-AT specifically selects the -amino group of ornithine despite the presence of a second amino group on C␣ with the same configuration as that in glutamate (3).…”

5-Fluoromethylornithine, an irreversible and specific inhibitor of l-ornithine:2-oxo-acid aminotransferase

“…Studies of the mechanism of inactivation have not yet been carried out, but the available information supports the view that 5-FMOrn is an enzyme-activated irreversible inhibitor of OAT. One may assume that the reactions involved in the inactivation of OAT by 5-FMOrn are analogous to those suggested by Bey et al (1981) for the inactivation of GABA-T by some w-fluoromethyl derivatives of ,J-alanine and 4-aminobutyrate, because OAT and GABA-T are enzymes with comparable mechanism (Williams et al, 1982;Metcalf, 1979). It is therefore not surprising that some inactivators of GABA-T are also potent inactivators of OAT Yasuda et al, 1980;Jones et al, 1983).…”

Stereochemistry of the reaction catalysed by 2‐aminoethylphosphonate aminotransferase

“…Thus, we have demonstrated the removal of the p r o 4 hydrogen at the prochiral C2 carbon atom of 2-aminoethylphosphonic acid in F! aeruginosa as most of the examples cited in the literature, such as: bacterial [6] and mammalian [7] yaminobutyrate aminotransferases, bacterial L-lysine &-aminotransferase [8], bacterial [7] and mammalian [9] L-ornithine S-aminotransferase, remove exclusively the p r o 4 hydrogen from the prochiral m-carbon atom of amino donors. However, the labilization of the pro-R hydrogen was exceptionally found for the m-amino acid: pyruvate aminotransferase of Pseudomonas sp.…”