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. Kinetic analyses of the reactions of ornithine aminotransferase with L-canaline and hydroxylamine. Left; hydroxylamine was added at concentrations of 0 M (O), 10-7 M (•), 5 x 10-7 M (0), and 10-6 M (,L). Right; L-canaline was

added at concentrations of 0 M (0), 5 x 10-8 M (0), 10_1 M (0), and 5 x 10-' M

(,L). Vmax values are expressed as ƒÊmoles of pyrroline 5-carboxylate formed per minute. [E] is expressed as micrograms of enzyme protein added to the reaction mixture.

Vol. 83, No. 1, 1978 204 K . KITO, Y. SANADA, and N. KATUNUMA

control line but intersected the horizontal axis . absorption spectrum of aspartate aminotransferase These data indicate that L-canaline is an irreversible changed with pH, as reported by Jenkins et at. (5), inhibitor whereas hydroxylamine is a reversible but formation of an oxime with hydroxylamine or noncompetitive inhibitor of ornithine aminotrans L-canaline also occurred at between pH 4.4 and ferase. The Ki value for hydroxylamine was 8.0. Figure 4 shows kinetic results on the reactions 4.3 x 10-1 M. of L-canaline and hydroxylamine with aspartate Furthermore, the effects of ƒ¿-N-acetyl-L aminotransferase. In contrast to their effects with - ornithine and ƒÂ-aminovaleric acid were investigated . ornithine aminotransferase, both L-canaline and Both of them showed typical competitive inhibition hydroxylamine were reversible noncompetitive in with respect to L-ornithine . The Ki values for hibitors; the Ki values for L-canaline and by a -N-acetyl -L-ornithine and ƒ¿-aminovaleric acid were 8.8 mm and 17.7 mm , respectively. These values were about ten times higher than the K . value for L-ornithine (8) . Formation of a Complex between L-canaline

and Fyridoxal S' phosphate Bound to Aspartate

Aminotransferase-The other pyridoxal enzyme , pig heart aspartate aminotransferase was inhibited

by a rather high concentration of L-canaline (1) . For comparison, we examined its mode of inhi

bition by L-canaline. The spectral shift of pyri

doxal 5'-phosphate bound to aspartate aminotrans ferase on addition of L-canaline is shown in Fig . 3. The spectral change on addition of L-canaline was

the same as that on addition of hydroxylamine Fig. 3. Absorption spectra of aspartate aminotrans to the enzyme. This suggests that L-canaline ferase after addition of L-canaline or hydroxylamine . formed an oxime with pyridoxal 5'-phosphate in 2.0 x 10-5 M Aspartate aminotransferase in 0 .05 M potas aspartate aminotransferase , as it did with pyridoxal sium phosphate, pH 4.4 alone, -; and with I mm 5'-phosphate in ornithine aminotransferase . The L-canaline, -; or 1 mm hydroxylamine , -.

Fig. 4. Kinetic analyses of the reactions of aspartate aminotran sferase with L-canaline and hydroxylamine. Left; hydroxylamine was added at concentr ati ons of 0 M(0), 10-° M(•), 5 x 10-5 M(A) , and 10-4 M (O). Right; L-canaline was added at concentrations of 0 M(0) , 5 x 10-5 M (s ), 10-4 M (A ), and 2 x 10-4 M (p). Vmax values are expressed as [moles of oxaloacetate formed per minute xpressed as micrograms of enzyme protein added to the r . [E]is e eaction mixture.

J. Biochem. INHIBITION OF ORNITHINE AMINOTRANSFERASE BY L-CANALINE 205

droxylamine were 1.7 x 10-4 M and 2.2 x 10-5 M, aminotransferase. The Ki values for hydroxyl respectively. These values were about a hundred amine of 4.3 x 10-1 M with ornithine aminotrans times the Ki value for hydroxylamine in ornithine ferase and 2.2 x 10-5 M with aspartate aminotrans aminotransferase. This suggests that the reactivity ferase were very different, suggesting that the of pyridoxal 5'-phosphate in ornithine aminotrans reactivity of pyridoxal 5'-phosphate is much higher ferase is much higher than that of pyridoxal 5' in ornithine aminotransferase than in aspartate - phosphate in aspartate aminotransferase. aminotransferase. The reason why L-canaline specifically binds

DISCUSSION irreversibly to pyridoxal 5'-phosphate in ornithine aminotransferase is probably that it is structurally

In this work we found that the aldehyde group of similar to the substrate, L-ornithine. The a-amino

pyridoxal 5'-phosphate in ornithine aminotrans groups of L-canaline and L-norvaline do not bind ferase can react with the ƒÂ-amino group of L to pyridoxal 5'-phosphate in ornithine amino - ornithine, ƒ¿-N-acetyl-L-ornithine or ƒÂ-aminovaleric transferase, but Strecker (10) reported that DL

acid, or the ƒÁ-aminooxy group of L-canaline, but - norvaline decreased Vmax and increased Km for not with the a-amino group of L-canaline or the substrate. Katunuma et al. (1) found that

L-norvaline. Reaction of the a-amino group of branched-chain amino acids inhibited ornithine

L-canaline or L-norvaline with the aldehyde group aminotransferase. Matsuzawa (11) reported that

of pyridoxal 5'-phosphate in ornithine amino L-valine inhibited ornithine aminotransferase com

transferase would have resulted in conversion of petitively with respect to L-ornithine, but that the

the pyridoxal 5'-phosphate form of the enzyme to absorption spectrum of the enzyme in either the

the pyridoxamine 5'-phosphate form, but the latter pyridoxal 5'-phosphate or pyridoxamine 5'-phos

was not detected. phate form did not change on addition of L-valine

Both L-canaline and hydroxylamine reacted or other branched-chain amino acids. Further

with the aldehyde group of pyridoxal 5'-phosphate more, he showed that the carboxyl group was

of the enzyme to produce an oxime; however, essential and that the amino group was com

with hydroxylamine the pyridoxal 5'-phosphate of plementary in the inhibition of ornithine amino transferase by branched-chain amino acids. the enzyme must remain intact because hydroxyl Strecker (10) also found that many other structural amine is a reversible noncompetitive inhibitor, analogs of the substrate, namely L-canavanine, whereas with L-canaline the pyridoxal 5'-phosphate ƒ¿-ketovalerate, ƒÁ-aminobutyrate, ƒ¿-ketoisovalerate, is irreversibly converted to an oxime-type derivative. ƒ¿-ketocaptoate, cadaverine, putrescine, and Both these compounds inhibit ornithine amino ƒ¿- ketobutyrate, inhibited ornithine aminotransferase. transferase at the level of the coenzyme, inactivat These findings suggest that the ƒ¿-carboxyl group ing pyridoxal 5'-phosphate of the enzyme. Other and the a-amino group of substrates or inhibitors analogs, such as ƒ¿-N-acetyl-L-ornithine and are important for binding these substrates or ƒÂ- aminovaleric acid, inhibit the enzyme at the sub inhibitors to the enzyme. Similarly, in this work strate level, showing typical competitive inhibition we found that the ƒÁ-aminooxy group of L-canaline with respect to the substrate, L-ornithine. These formed an oxime with the aldehyde group of the two types of inhibition have very different Kl

values and the particular inhibitory mode of L pyridoxal 5'-phosphate in the enzyme, while the a-amino and carboxyl groups probably became - canaline is one reason why L-canaline inhibits the attached to the apoprotein at the active site of enzyme at such a low concentration. ornithine aminotransferase. The cooperative func We examined the specificity of L-canaline by tions of these three sites of L-canaline seem to comparing its effects on two pyridoxal enzymes, make the inhibition irreversible. Recently, Beeler ornithine aminotransferase and aspartate amino and Churchich (12) investigated the reactions of transferase in kinetic experiments. Hydroxylamine L-canaline with free pyridoxal 5'-phosphate and was a reversible noncompetitive inhibitor of both with the pyridoxal enzyme, cystathionase, and enzymes, whereas L-canaline was a reversible showed that L-canaline attacked the Schiff base noncompetitive inhibitor of aspartate aminotrans linkage of cystathionase several times faster than ferase, but an irreversible inhibitor of ornithine

Vol. 83, No. 1, 1978 206 K. KITO, Y. SANADA, and N. KATUNUMA it attacked the aldehyde group of free pyridoxal 2. Rahiala, E-L., Kekomaki, M., Janne, J., Raina, A., 5'-phosphate: the kinetic constant for the reaction & Rdiha, N.C.R. (1971) Biochim. Biophys. Acta of L-canaline with pyridoxal 5'-phosphate of 227,337-343 cystathionase was more than 3,700-1¥min-1. 3. Sanada, Y., Suemori, I., & Katunuma, N. (1970) Pyridoxal 5'-phosphate bound to ornithine amino Biochim. Biophys. Acta 220, 42-50 transferase is also much more reactive than free 4. Matsuzawa, T., Katsunuma, T., & Katunuma, N. (1968) Biochem. Biophys. Res. Commun. 32,151-166 pyridoxal 5'-phosphate with L-canaline, and the 5. Jenkins, W.T., Yphantis, D.A., & Sizer, IN. (1959) kinetic constant was 1,045-1¥s-1 (unpublished data). J. Biol. Chem. 234, 51-57 The present paper shows that L-canaline 6. Babson, A.L., Shapiro, P.P., Williams, P.A.R., & forms an oxime with highly reactive pyridoxal Philips, G.E. (1962) Clin. Chim. Acta 7, 199-205 5'-phosphate in ornithine aminotransferase and 7. Kalcker, H.M. (1947) J. Biol. Chem. 167, 461-475 that this results in specific inhibition of ornithine 8. Sanada, Y., Shiotani, T., Okuno, E., & Katunuma, aminotransferase by a much lower concentration N. (1976) Eur. J. Biochem. 69, 507-515 of L-canaline than those required to inhibit other 9. Feliss, N. & Martinez-Carrion, M. (1970) Biochem. pyridoxal enzymes. Biophys. Res. Commun. 40, 932-940 10. Strecker, H.J. (1965) J. Biol. Chem. 240, 1225-1230 11. Matsuzawa, T. (1974) J. Biochem. 75, 601-609 REFERENCES 12. Beeler, T. & Churchich, J.E. (1976) J. Biol. Chem. 1. Katunuma, N., Matsuda, Y., & Tomio, I. (1964) 251,5267-5271 J. Biochem. 56, 499-503

J. Biochem.