Mode of Inhibition of Ornithine Aminotransferase by L-Canaline1
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J. Biochem. 83, 201-206 (1978) Mode of Inhibition of Ornithine Aminotransferase by L-Canaline1 Kiyoaki KITO, Yukihiro SANADA, and Nobuhiko KATUNUMA Department of Enzyme Chemistry, Institute for Enzyme Research , School of Medicine, Tokushima University, Tokushima , Tokushima 770 Received for publication, June 29 , 1977 The mechanism of inhibition of ornithine aminotransferase [EC 2.6.1.13] by L-canaline (ƒ¿-amino-ƒÁ-amino-oxybutyric acid) was investigated. Spectral changes of pyridoxal 5' - phosphate in ornithine aminotransferase on addition of L-canaline showed that L-canaline formed an oxime-type compound with pyridoxal 5'-phosphate that had the same spectra as the compound formed on addition of hydroxylamine to the holoenzyme. Kinetic studies indicated that hydroxylamine was a reversible noncompetitive inhibitor, whereas L-canaline was an irreversible inhibitor of ornithine aminotransferase. Other analogs, such as ƒÂ-aminovaleric acid and ƒ¿-N-acetyl-L-ornithine, also reacted with the pyridoxal 5'-phosphate of the enzyme, but these compounds were competitive inhibitors with respect to L-ornithine. L-Canaline and hydroxylamine also reacted with pyridoxal 5'-phosphate in pig heart aspartate amino transferase [EC 2.6.1.1] to produce an oxime, but both of them were reversible and noncompeti tive inhibitors of the enzyme. The Ki value of hydroxylamine for ornithine arninotransferase was 4.3 x 10-7 M and those of L-canaline and hydroxylamine for aspartate aminotransferase were 1.7 x 10-4 M and 2.2 x 10-5 M, respectively. Previously we reported (1) that L-canaline, a struc concentration of L-canaline. These findings sug tural analog of L-ornithine was a most powerful gest that there may be some special relation be inhibitor of ornithine aminotransferase. Concen tween L-canaline and the coenzyme pyridoxal trations as low as 10-7 M of L-canaline inhibited 5'-phosphate. Rahiala et al. (2) also examined ornithine aminotransferase but had almost no effect the absorption spectra of pyridoxal 5'-phosphate on pig heart aspartate aminotransferase. Rahiala alone and when mixed with L-canaline or cyclo et al. (2) compared the effects of L-canaline on serine, and suggested that inhibition of the pyri several enzymes involved in ornithine metabolism; doxal enzyme by L-canaline was due to its formation it is interesting that ornithine transcarbamylase of an oxime with pyridoxal 5'-phosphate. How [EC 2.1.3.3], which is not a pyridoxal enzyme, was ever, the inhibitory effects of L-canaline on pyri not inhibited, whereas ornithine decarboxylase doxal enzymes have not been elucidated at the [EC 4.1.1.7], which requires pyridoxal 5'-phos molecular enzyme level. Accordingly, using large phate as a coenzyme, was inhibited by a high amounts of crystalline ornithine aminotransferase and several analogs of L-ornithine, such as ƒ¿-N ' This work was supported by a Grant-in-Aid for acetyl-L-ornithine, ƒÂ-amino-valeric acid and L Scientific Research from the Ministry of Education, - norvaline, we studied the mechanism of inhibition Science and Culture of Japan (No. 138014). by L-canaline by examining spectral changes of Vol. 83, No. 1, 1978 201 202 K . KITO, Y. SANADA, and N. KATUNUMA pyridoxal 5'-phosphate in ornithine aminotrans ornithine aminotransferase was taken as 180,000 ferase on addition of these compounds . We also (8) and that of aspartate aminotransferase as 93,000 compared the modes of action of L-canaline and (9) in all calculations. Absorption spectra were hydroxylamine on pig heart aspartate aminotrans recorded using quartz cells of 1-cm light path in a ferase and on ornithine aminotransferase , to Hitachi 124 spectrophotometer equipped with a examine the role of apo-protein in the specific 0 to 0.2 absorbance unit expanded-scale slide on inhibition of ornithine aminotransferase by L a Hitachi 056 recorder. The reaction temperature - canaline. The present paper shows that the was controlled using a COOLNICS apparatus inhibition is due to oxime formation between the (model CTR-220, Yamato Scientific Co.). Ab aldehyde group of pyridoxal 5'-phosphate in sorption spectra in the presence of various effectors ornithine aminotransferase and the ƒÁ-aminooxy were recorded immediately after addition of the group of L-canaline. The reason for the spe effectors at 25°C. cificity of the inhibition of ornithine aminotrans ferase by L-canaline is discussed. RESULTS MATERIALS AND METHODS Formation of a Complex between L-Canaline and Pyridoxal 5'-Phosphate Bound to Ornithine L-Canaline and ƒ¿-N-acetyl-L-ornithine were ob Aminotransferase-L-Canaline has one a-amino tained from Sigma Chemical Co ., and ƒÂ-amino and one r-amino group, and to see which one valeric acid and L-norvaline from Nakarai Chemical reacts with the aldehyde group of pyridoxal 5' Industry Ltd. Pyridoxal 5'-phosphate and 6 - phosphate, we examined the absorption spectra of benzamido-4-methoxy-m-toluidine dizaonium zinc Schiff bases in ornithine aminotransferase after chloride were kindly supplied by Chugai Phar addition of L-canaline or other substrate analogs . maceutical Co. All other chemicals used were Previous work in this laboratory (4) showed that commercial products of the highest grade available . the pyridoxal 5'-phosphate form of ornithine A homogeneous crystalline preparation of ornithine aminotransferase had a maximum at 420 nm and aminotransferase from rat liver was obtained by that addition of L-ornithine resulted in formation the method of Sanada et al. (3) . Ornithine of the pyridoxamine 5'-phosphate form enzyme aminotransferase activity was assayed by the with a decrease in absorption at 420 nm and an method of Matsuzawa et al. (4). In kinetic increase at 330 nm. As shown in Fig . 1, addition experiments, reactions were carried out in 20 mm of 1 mm ƒ¿-N-acetyl-L-ornithine or ƒÂ-aminovaleric L-ornithine, 10 mm ƒ¿-ketoglutarate and 10-5 M acid to 2 ƒÊM enzyme in 0.05 M potassium phosphate pyridoxal 5'-phosphate in 0.05 M potassium phos buffer, pH 8.0, resulted in the same spectral shift phate buffer, pH 8.0. One unit of enzyme activity as addition of L-ornithine , although the changes was defined as the amount producing one ƒÊmole were slower than those with L-ornithine . Under of pyrroline-5-carboxylate per minute at 37•Ž . Pig the same conditions, addition of 1 MM L-norvaline heart cytoplasmic aspartate aminotransferase was did not cause any spectral change , but addition prepared by the method of Jenkins et al. (5) with of hydroxylamine to the enzyme caused formation an additional step of Sephadex G-100 gel filtration . of the oxime, as in the reaction of hydroxylamine Aspartate aminotransferase was assayed by the with free pyridoxal 5'-phosphate reported by method of Babson et al. (6) using 6-benzamido -4 Rahiala et al. (2). L-Canaline caused the same - methoxy-m-toluidine diazonium zinc chloride . spectral changes as hydroxylamine , except that ( The reaction mixture contained 20 MM L-aspartate , unlike the oxime of hydroxylamine) L -canaline 5 mm ƒ¿-ketoglutarate and 10-5 M pyridoxal 5' gave no peak at 320 nm. These absorption spectra - phosphate in 0.05 M potassium phosphate buffer, could not be due to formation of a complex between pH 8.0. One unit of enzyme activity was defined L-canaline or hydroxylamine and free pyridoxal as the amount producing one ƒÊmole of oxalo 5'-phosphate in the enzyme preparation . This was - acetate per minute at 37°C. Protein concentrations ruled out by passing the reaction mixture through were determined spectrophotometrically by the Sephadex G-25 equilibrated with 0 .05 M potassium method of Kalcker (7). The molecular weight of phosphate buffer, pH 8.0 , and showing that the J. Biochem. INHIBITION OF ORNITHINE AMINOTRANSFERASE BY L-CANALINE 203 eluate had the same absorption spectra as before. pH 6.0 and 8.0. These results show that the Thus L-canaline and hydroxylamine combine di ƒÁ-aminooxy group of L-canaline reacted with rectly with pyridoxal 5'-phosphate attached to the pyridoxal 5'-phosphate of ornithine aminotrans enzyme. The absorption spectrum of ornithine ferase to form an oxime, and that the 5-amino aminotransferase did not change between pH 6.0 group of ƒ¿-N-acetyl-L-ornithine or ƒÂ-aminovaleric and 8.0 (8) and formation of an oxime with L acid reacted to yield the pyridoxamine 5'-phosphate canaline or hydroxylamine also occurred between form of the enzyme. However, the a-amino group of L-norvaline or L-canaline did not react with the aldehyde group of pyridoxal 5'-phosphate bound to ornithine aminotransferase, because if it had, the absorption spectrum of the pyridoxamine 5'-phosphate form of the enzyme would have appeared. Kinetics of the Reactions of Ornithine Amino transferase with -Canaline and Other Analogs - The mechanisms of enzyme inhibition by L-canaline and hydroxylamine were similar. Next we com pared their reactivities with ornithine aminotrans ferase by kinetic studies. Lineweaver-Burk plots (data not shown) showed that both L-canaline and hydroxylamine were typical noncompetitive inhi bitors with respect to L-ornithine or ƒ¿-keto Fig. 1. Absorption spectra of ornithine aminotrans glutarate. To decide whether these inhibitors are ferase after additions of various effectors. 2 x 10-6 M reversible or irreversible, we plotted Vmax against Ornithine aminotransferase in 0.05 M potassium phos the amount of enzyme, as shown in Fig. 2. The phate buffer, pH 8.0 alone, - and with 1 MM L-orni lines for hydroxylamine had less steep slopes than thine, -; ƒ¿-N-acetyl-L-ornithine, ---; ƒÂ-amino the control line, but passed through the origin; valeric acid, ----; L-norvaline, ----- ; hydroxylamine, - --; and L-canaline, -. the lines for L-canaline had the same slope as the Fig. 2.