Aromatic amino acid aminotransferase (ArAT) from Escherichia coli was overexpressed in E. coli cells, purified, and characterized. The enzyme was similar to aspartate aminotransferase (AspAT) of E. coli in many aspects, such as gross protein structure and spectroscopic properties. The reactions of pyridoxal 5'-phosphate-form ArAT with amino acids and pyridoxamine 5'-phosphate-form ArAT with oxo acids were investigated using stopped-flow spectrophotometric techniques. The kinetic parameters for these "half" reactions could excellently explain the ArAT-catalyzed overall transamination reactions at pH 8.0. Reactions of ArAT with aspartate and tryptophan which had been deuterated at position 2 showed isotope effects of 2.5 and 6.0 in the kcat values of the half-reactions, showing that the proton-transfer step is at least partially rate-limiting for these reactions. ArAT and AspAT showed overlapping substrate specificity. Both ArAT and AspAT were active toward dicarboxylic substrates. ArAT showed, however, 10(3)-fold higher activity toward aromatic substrates than AspAT. This high activity toward aromatic substrates was in part ascribed to the active site hydrophobicity of ArAT, which was suggested to be about 1.4 times as large as that of AspAT. In addition to dicarboxylic substrate analogs, aromatic substrate analogs such as carboxylic acids, 2-methyl amino acids, and 3-hydroxy amino acids caused characteristic changes in the absorption spectra of ArAT, while these aromatic analogs did not significantly change the spectra of AspAT. In particular, the erythro-3-hydroxy analogs of phenylalanine and aspartate caused a prominent absorption of ArAT at around 500 nm, which is generally ascribed to the accumulation of quinonoid intermediates. The threo forms of these 3-hydroxy analogs acted as substrates for ArAT. The erythro and threo forms of 3-hydroxyaspartate reacted with AspAT similarly as they reacted with ArAT; however, both forms of 3-phenylserine were poor substrates for AspAT, although phenylalanine was a fairly good substrate for AspAT. The observations on the two erythro-3-hydroxy amino acids show the similar orientation of these analogs in the active site of ArAT, probably through a hydrogen-bonding network involving the hydroxy groups of the analogs and Tyr70, and suggest that the aromatic binding pocket is near or even overlaps the side-chain-carboxylate-binding site for dicarboxylic substrates.

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INTRODUCTIONIt has been shown that there are circadian variations in gastrointestinal function including gastric acid secretion, gastrointestinal motility, gastric mucosal blood flow, gastric epithelial cell turnover and gastric mucosal prostaglandin production. [1][2][3][4][5][6] With regard to circadian variation in gastric mucosal injury, Olson et al. 7 reported that acidified aspirin or absolute ethanol caused significantly more gastric mucosal lesions when administered in the light than in the dark phase. In contrast, Ventura et al. 8 reported that the highest acid secretory capacity and greatest degree of gastric mucosal damage produced by exogenously applied butyric acid was observed during the dark period in the rat gastric mucosa Ussing-chamber model. Waldrop et al. 9 demonstrated that significantly less severe ulcer formation occurred as a result of cervical spinal cord transection when rats developed the lesion at the onset of light (08.00 h) than at any other sample time. Thus, although rats show circadian variation in their susceptibility to gastric mucosal injury, results are variable among experimental models and conditions. The pineal gland secretory product, melatonin, is well known to have circadian and antistress SUMMARYBackground: We previously demonstrated the circadian variation of water-immersion restraint stress (WRS)-induced gastric mucosal lesions in rats. Aim: To investigate the roles of melatonin and prostaglandin in the gastric mucosa in circadian modulation of WRS. Methods: Fasted rats were subjected to 4-h WRS during both the diurnal and nocturnal phases of a light/dark cycle. Mucosal lesions, serum melatonin concentrations, mucosal generation of prostaglandin E 2 (PGE 2 ) and mucosal gene expressions of cyclooxygenase (COX)-1 and -2 were evaluated. Results: Lesion area after 4-h stress during the dark phase was significantly smaller than that in light-phase controls. Serum melatonin concentration in control rats during the light phase was significantly increased 4 h

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