The Effect of Carboxylates and Halides on L-Lysine 6-Aminotransferase-Catalyzed Reactions

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1 /. Biochem. 95, (1984) The Effect of Carboxylates and Halides on L-Lysine 6-Aminotransferase-Catalyzed Reactions Tohru YOSHIMURA, Katsuyuki TANIZAWA, Hidehiko TANAKA, and Kenji SODA Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611 Received for publication, August 1, 1983 L-Lysine:2-oxoglutarate 6-aminotransferase catalyzes very slow transamination between L-alanine and 2-oxoglutarate. A high concentration of anions such as formate, acetate and halides greatly accelerated this transamination without affecting the affinity of the enzyme for L-alanine. In contrast, the anions strongly inhibited the normal L-lysine 6-transamination in a competitive manner with L-lysine and in a non-competitive manner with 2-oxoglutarate. This result suggests that the enzyme has an anion binding site which normally binds the carboxyl group of L-lysine. The binding of halides or carboxylates to this site probably induces a conformational change of the enzyme, and results in the inhibition of L-lysine 6-transamination, and in the stimulation of L-alanine transamination. Treatment of the enzyme with an arginine-specific dicarbonyl reagent, phenylglyoxal, led to a loss of the enzyme activity for L-lysine. The activity for L-alanine was not affected, but the stimulating effect of anions on L-alanine transamination was impaired. Thus, it is suggested that an arginine residues) plays an important role in the anion binding site. L-Lysine 6-aminotransferase [L-lysine: 2-oxoglutarate 6-aminotransferase, EC ] catalyzes the transfer of the terminal amino group of L- lysine to 2-oxoglutarate, producing zp-piperideine- 6-carboxylate, the intramoleculary dehydrated form of 2-aminoadipic-5-semialdehyde, and L- glutamate (1-3). The pro-s hydrogen at the C-6 carbon of L-lysine is stereospecifically abstracted in this reaction (4). The enzyme (molecular weight (Mr), 116,) purified to homogeneity from Flavobacterium lutescens (= Achromobacter liquidium) Abbreviations: Mr, molecular weight; pyridoxal-p, pyridoxal 5'-phosphate; pyndoxamine-p, pyridoxamine 5'-phosphate. IFO 384 is an oligomeric enzyme composed of one each of four non-identical subunits, A (Mr, 24,), B x (Mr, 28,), B, (Mr, 28,), and C (Mr, 45,) (5). Of the two molecules of bound cofactor, pyridoxal 5'-phosphate (pyridoxal-p), the one which absorbs at 415 nm, is bound to subunit B, and participates in the catalytic action of the enzyme (5, 6). Recently, we have observed that the catalytic efficiency and substrate specificity of this enzyme are markedly affected by the binding of one of the substrate pairs to the active site of the enzyme (Yagi, T., Yoshimura, T., Tanizawa, K., Misono, H., & Soda, K., manuscript in preparation), L- AJanine, L-glutamate, and L-phenylalanine, all of Vol. 95, No. 2,

56. T. YOSHIMURA, K. TANIZAWA, H. TANAKA, and K. SODA which are very poor substrates in the overall reaction (2, 3), can be good amino donors in a half reaction with the enzyme-bound pyridoxal-p.

2 56. T. YOSHIMURA, K. TANIZAWA, H. TANAKA, and K. SODA which are very poor substrates in the overall reaction (2, 3), can be good amino donors in a half reaction with the enzyme-bound pyridoxal-p. This discrepancy between the half and overall reactions leads to the suggestion that the enzyme has two substrate-binding sites which correspond to a- amino acids (or a-keto acids) and co-amino acids. To obtain further information on the substratebinding sites and their functions, we have studied the effect of monovalent anions on the reactivity of L-lysine 6-aminotransferase. We here show that the enzyme has an anion binding site which influences the reactivity of enzyme for L-lysine and other substrate L-amino acids by interacting with the carboxyl group of L-lysine or effective anions. Data are also presented which suggest that an arginine residue(s) serves as the anion binding site. EXPERIMENTAL PROCEDURES MaterialsL-Lysine 6-aminotransferase was purified to homogeneity from F. lutescens IFO 384 as described previously (2, 7). Lactate dehydrogenase (rabbit muscle) was obtained from Sigma; phenylglyoxal monohydrate was from Aldrich; pyridoxal-p and 2-oxoglutarate were from Nakarai Chemicals, Kyoto; and amino acids were from Ajinomoto Co., Tokyo. o-aminobenzaldehyde was prepared by reduction of o-nitrobenzaldehyde according to the method of Smith and Opie (8). MethodsL-Lysine 6-aminotransferase activity was measured by determination of glutamate with a Hitachi 835 high-speed amino acid analyzer, or by determination of /d 1 -piperideine-6-carboxylate with o-aminobenzaldehyde as described previously (2). The overall transamination between L-alanine and 2-oxoglutarate was determined by measurement of the amount of pyruvate or L-glutamate formed. The rate of pyruvate formation was determined by following the decrease in the absorbance at 34 nm in a coupled system containing lactate dehydrogenase and NADH. Detailed reaction conditions are described in the legends to tables and figures. Spectrophotometric measurements were carried out in a Union SM-41 spectrophotometer. RESULTS Effect of Monovalent Anions on Alanine Transamination by L-Lysine 6-AminotransferaseAs reported previously (J), L-lysine 6-aminotransferase catalyzes the transamination between L-alanine and 2-oxoglutarate only at a rate.3-1.% of the rate of 6-transamination of L-lysine. However, the alanine transamination was significantly stimulated by a high concentration of monovalent anions such as formate, acetate and halides. Table I shows the effect of potassium salts of these anions (.5 M) on the production of glutamate in the alanine transamination in potassium phosphate buffer (ph 8.). The order of effectiveness is HCOO- > Cl" > Br- > F- > CH 3 COO' > I~. Since sodium salts of the anions also show the rate-enhancing effect in the same order and magnitude, the stimulation observed is attributable to the action of the anion moiety of these salts. Longer carbon chain carboxylates such as propionate and butyrate, and aromatic carboxylates had no stimulating effect, but were rather inhibitory on the alanine transamination. Multivalent anions (e.g. sulfate and borate) were ineffective. The effect of concentration of anions on the rate of alanine transamination was examined with the most effective anions, formate and chloride (Fig. 1). The rate of pyruvate formation increased with an increase of concentrations of both anions. The saturation curves for the anions, however, were not hyperbolic but sigmoidal. A similar sigmoidality has been observed also for the formate-induced a,/?-elimination of -chloro-l-alanine catalyzed by aspartate aminotransferase (9, 1), although a mechanism for the anomalous saturation curves has not been elucidated. Figure 2 shows the apparent Michaelis constant for L-alanine in the alanine transamination by L-lysine 6-aminotransferase. The addition of.5 M formate or chloride did not change the K m for L-alanine (.34 M). Thus, the monovalent anions do not affect the affinity of the enzyme for L-alanine, but increase the maximum velocity of alanine transamination. Effect of Anions on Lysine Transamination In contrast to the activating effect on the alanine transamination, monovalent anions inhibit the transamination of L-lysine, the normal substrate /. Biochem.

3 EFFECT OF ANIONS ON LYSINE 6-AMINOTRANSFERASE 561 TABLE I. Effect of anions on L-lysine 6-aminotransferase-catalyzed reactions. The alanine transamination was carried out at 37 C for 8 h in the reaction mixture (.4 ml) containing 5 mm L-alanine, 5 mm potassium 2-oxoglutarate,.1 M potassium phosphate buffer (ph 8.),.5 M of an anion as indicated (as a potassium salt), and 1.8 //g of L-lysine 6-aminotransferase. After the reaction was terminated by the addition of.1 ml of 25% trichloroacetic acid, the amount of glutamate formed was determined with an amino aad analyzer. The lysine transamination was carried out at 37 C for 1 h in the reaction mixture (.4 ml) containing 8 mm L-lysine, 2 mm potassium 2-oxoglutarate,.1 M potassium phosphate buffer (ph 8.), 5 M of an anion as indicated, and 9 n% of L-lysine 6- aminotransferase. The amount of ^-piperideine 6-carboxylate (P-6-C) formed was determined as described previously (2). Anion None F~ ci- Bi- I- Formate Acetate Propionate Butyrate Benzoate Salicylate Alanine transamination Glutairuc aad formed (nmol/6 mm) FORMATE OR CHLORIDE (M) Fig. ]. Effect of formate and chloride anions on the alanine transamination catalyzed by L-lysine 6-aminotransferase. The reaction mixtures (.8 ml) contained 5 mm L-alanine, 5 mm potassium 2-oxoglutarate, 15 /*M NADH, 6 /(g of lactate dehydrogenase,.1 M potassium phosphate buffer (ph 8.), potassium formate (O) or potassium chloride ( ) as indicated, and 64 fig of L-lysine 6-aminotransferase. The reaction was started by the addition of L-lysine 6-aminotransferase, and the decrease in absorbance at 34 nm was followed at 25 C. Relative activity Lysine transamination P-6-C formed (//mol/6 min) Relative activity A 6 8 (L-ALAWNE, M)" 1 Fig. 2. Double reciprocal plots of the rate of the alanine transamination against concentrations of L-alanine. Reaction mixtures contained L-alanine as indicated, 5 mm potassium 2-oxoglutarate, 15 /JM NADH, 6 /ig of lactate dehydrogenase,.1 M potassium phosphate buffer (ph 8.),.5 M potassium formate ( ) or potassium chloride (A), and 98 //g of L-lysine 6-aminotransferase in a final volume of.8 ml. The reaction was initiated by the addition of L-lysine 6-aminotransferase and carried out at 25 C. The absorbance change at 34 nm was followed. Vol. 95, No. 2, 1984

4 562 T. YOSHIMURA, K. TAN1ZAWA, H. TANAKA, and K. SODA of L-lysine 6-aminotransferase (see Table I). The order of their effectiveness in inhibition is I~> CH,CH,COO- > CH,COO- > Br- > Cl- > HCOO" > F". The inhibitory effect of the anions increases linearly in proportion to the volume (cube of the radius) of anions (data not shown). Double reciprocal plots of the initial rate of lysine transamination against concentrations of L- lysine gave a family of straight lines when the reaction was carried out in the presence of various concentrations of formate or chloride (Fig. 3, A and B). The lines intersected at a common point on the ordinate, giving K t values of.16 M for formate and.18 M for chloride. This result indicates that the anions inhibit the lysine transamination by competition with L-lysine (probably with its a-carboxyl group) at the anion binding site -IX) 5 IX) (a-ketoglutarate, within or near the active region of enzyme. The other effective anions, e.g. acetate, propionate, fluoride, bromide, and iodide, also showed the competitive inhibition with L-lysine. In contrast, these anions inhibited the lysine transamination noncompetitively with regard to 2- oxo-glutarate (Fig. 3, C and D). This suggests that the anions do not affect the binding of 2-oxoglutarate to the enzyme. Effect of Phenylglyoxal Modification on Activity of L-Lysine 6-AminotransferaseThe findings described above support the suggestion that the enzyme has an anion binding site which normally binds the a-carboxyl group of L-lysine. To confirm the presence of such an anion binding site, we have modified the enzyme with an argininespecific reagent, phenylglyoxal (11, 12); arginyl B < (a- KETOGLUTARATE, mmt 1 Fig. 3. Inhibition of lysine transamination by formate (A, C) and chloride (B, D) ions. A, B: The reaction mixtures (.4 ml) contained various concentrations of L-lysine, 2 mm potassium 2-oxoglutarate, various concentrations of potassium formate (A) or potassium chloride (B),.1 M potassium phosphate buffer (ph 8.), and 9 ng of L-lysine 6-aminotransferase. J'-Piperideine 6-carboxylate formed was determined as described previously (2) by measuring the absorbance at 465 run. The anion concentrations were: O, OITIM;, 1HIM; A, 2 mm; A, 3 mm; D, 4mM. C, D; The reaction mixtures (.4 ml) contained various concentrations of potassium 2-oxoglutarate, 1 mm L-lysinc, various concentrations of potassium formate (C) or potassium chloride (D),.1 M potassium phosphate buffer (ph8.), and 9 fig of L-lysine 6-aminotransferase. The anion concentrations: were O, OITIM;, 2 mm; A,4mM; A,6mM; D,8mM. /. Biochem.

EFFECT OF ANIONS ON LYSINE 6-AMINOTRANSFERASE 563 residues in many proteins bind anionic ligands including substrates and cofactors, and are unusually reactive with phenylglyoxal and other similar

5 EFFECT OF ANIONS ON LYSINE 6-AMINOTRANSFERASE 563 residues in many proteins bind anionic ligands including substrates and cofactors, and are unusually reactive with phenylglyoxal and other similar reagents containing vicinal carbonyl groups (butanedione and cyclohexanedione) (13, 14). The time course of the inactivation of L-lysine 6- aminotransferase by 1 mm phenylglyoxal is shown in Fig. 4. The enzyme activity for the lysine transamination decreased to less than 35 % of the initial activity after 12 min incubation, whereas the activity for the alanine transamination was little affected by incubation with phenylglyoxal. The stimulatory effect of formate and chloride ions on the alanine transamination decreased in parallel with a decrease in the enzyme activity for the lysine transamination. Thus, the stimulatory effect of anions on the alanine transamination is diminished significantly by incubation of the enzyme with phenylglyoxal for 12 min. Ammo acid analysis of an acid hydrolysate of the phenylglyoxalmodified enzyme having 3% residual activity showed the disappearance of about 2% arginyl residues (17 residues out of 86 total arginyl resi TIME (min) Fig. 4. Inactivation of L-lysine 6-aminotransferase by incubation with phenylglyoxal. The reaction mixtures (.5 ml) contained 5 mm 4-{2-hydroxyethyl)-l-piperazineethanesulfonate buffer (ph 8.), 1 mm phenylglyoxal, and 5//g of L-lysine 6-aminotransferase. Incubation was at 37 C. Aliquots were removed at the indicated times for assay. The remaining activity for the lysine transamination (O) was determined by measuring J'-piperideine 6-carboxylate produced, as described before (1). The remaining activity for the alanine transamination was determined as described in Fig. 1 in the absence ( ) or presence of.5 M potassium formate (A) or potassium chloride (O). dues (5)); the other amino acid residues did not undergo a significant change. These results show that phenylglyoxal inactivates the enzyme through modification of an arginyl residue(s) which serves as a cationic site for binding the a-carboxyl group of L-lysine, and also for binding the effective monovalent anions in the alanine transamination. The enzyme inactivation by phenylglyoxal did not follow a simple pseudo-first-order kinetics (Fig. 4). This is probably due to the partial dissociation of the enzyme-bound pyridoxal-p from the enzyme to produce a half-resolved form, "semiapoenzyme" (6), which is very rapidly inactivated by phenylglyoxal (unpublished results). Since the enzyme-bound pyridoxamine 5'-phosphate (pyridoxamine-p) dissociates more easily from the enzyme than the bound pyridoxal-p (2), the incubation of the enzyme with phenylglyoxal in the presence of L-lysine, which reacts with the pyridoxal-p enzyme to produce the pyridoxamine- P form, results in more rapid inactivation of the enzyme. Therefore, it is not possible in practice to elucidate quantitatively the protective effect of L-lysine on the phenylglyoxal modification of arginyl residues, although we have observed strong protection of the enzyme against the phenylglyoxal inactivation by 2 mm L-lysine and 2 mm 2-oxoglutarate. Formate and chloride (.5-1 M) did not protect the enzyme from inactivation by phenylglyoxal, presumably because of the acceleration of pyridoxal-p dissociation from the enzyme by both anions and the consequent rapid inactivation. DISCUSSION The present results indicate that L-lysine 6-aminotransferase has an anion binding site which affects the activity of the enzyme by binding certain monovalent anions. The blocking of this site by anions such as formate and chloride results in the marked acceleration of the rate of L-alanine transamination without changing the affinity of the enzyme for L-alanine. The anion binding site probably binds the a-carboxyl group of L-lysine in the normal transamination (Scheme 1), because the anions inhibit the normal transamination competitively with L-lysine. We have found that cadaverine does not serve as a substrate, but 6-aminocaproate does, though poorly (i). This adds strong support for the suggestion that the a-car- Vol. 95, No. 2, 1984

6 564 T. YOSHIMURA, K. TANIZAWA, H. TANAKA, and K. SODA X e ^jxxfi B Scheme 1. A possible model for the substrate-binding site of L-lysine 6-aminotransferase. Binding of L-lysine (A) and of L-alanine in the presence of an effective anion (B). X~ is a monovalent anion which stimulates the alanine transarrunation (formate, acetate, F~, O~, Br~, or I~). boxyl group of L-lysine plays a significant role in binding to the enzyme, and that this binding stimulates the 6-transamination of L-lysine. When L-alanine is an amino donor, the anions added may bind to the same site as that of the a-carboxyl group of L-lysine to stimulate the alanine transamination (Scheme 1). The binding of halide or carboxylate anions may induce a preferred conformational change for the efficient catalysis, although no evidence has been obtained for such conformational change. Thus, the induced-fit effect caused by binding of a distal part of substrate probably leads to rigid regulation of the substrate specificity, and to enhancement of the catalytic efficiency of the enzyme in the L-lysine transamination. Similar enzyme activation by "fragmented substrates" (75) has been demonstrated for L- aspartate aminotransferase [EC ] (9, 1,16, 17) and L-lysine monooxygenase [EC ] (75). Monovalent carboxylates (9, 16) and halides (77) markedly activate aspartate aminotransferase in catalysis of the transamination of L-alanine, a very poor substrate, but competitively inhibit the normal L-aspartate transamination (18). The a,/jelimination of -chloro-l-alanine by L-aspartate aminotransferase as well as the concomitant inactivation of the enzyme also is greatly stimulated by formate anions (9, 1). This suggests that the enzyme has a discrete subsite in the active region which can bind formate anions, and normally binds the distal carboxyl group of the natural substrates L-aspartate and L-glutamate (9, 1, 16). L-Lysine monooxygenase does not catalyze the oxidation of L-alanine, L-norvaline, L-a-aminobutyrate, and some others under the assay conditions, but effectively does so in the presence of various alkylamines when the total number of carbon atoms of "fragmented substrates" is nearly identical to that of L-lysine (75). A high concentration of these effective compounds is required for not only both the enzymes but also lysine 6-aminotransferase. Studies of chemical modifications with the arginine-specific dicarbonyl reagents have shown that arginyl residues in many enzymes play a role for binding anionic group of substrates and cofactors, as reviewed by Riordan et al. (13) and Patthy and Thesz (14). It has been reported that a specific arginyl residue of 4-aminobutyrate aminotransferase [EC ] (79), L-glutamate decarboxylase [EC ] (2) and D-serine dehydrase [EC ] (21) binds the phosphate group of the bound coenzyme. The a- or distal carboxyl group of the substrate binds to a specific arginyl residue of L-aspartate aminotransferase (22-25), tryptophanase [EC ] (26), cystathionine y-iyase [EC ] (27), and tryptophan synthase [EC ] (28). Recent studies of the chemical modification of mitochondrial L-aspartate aminotransferase from chicken with arginine-specific reagents revealed that Arg-292 binds the distal carboxyl group of the substrate L-aspartate (25), in agreement with the proposal derived from X- ray crystallographic studies (29). The functional phenylglyoxal-modifiable arginyl residue (Arg-568) of glycogen phosphorylase binds the phosphate of the substrate, glucose-1-phosphate (3, 31). TJie results presented here show that L-lysine 6- aminotransferase also- contains a phenylglyoxal-./. Biochem

7 EFFECT OF AN1ONS ON LYSINE 6-AMINOTRANSFERASE 565 modifiable arginyl residues), which functions in the monovalent anion-enhanced alanine transamination and probably in the binding of the a-carboxyl group of L-lysine in the normal transamination (see Fig. 4). However, our preliminary observations that the semiapo form of the enzyme is much more rapidly inactivated by phenylglyoxal in the absence of pyridoxal-p also suggest the participation of an arginyl residue(s) in binding the phosphate group of pyridoxal-p. REFERENCES 1. Soda, K., Misono, H., & Yamamoto, T. (1968) Biochemistry 7, Soda, K. & Misono, H. (1968) Biochemistry 7, Misono, H., Yamamoto, T, & Soda, K. (1971) Bulletin of the Institute for Chemical Research, Kyoto University 49, Tanizawa, K., Yoshimura, T., Asada, Y., Sawada, S., Misono, H., & Soda, K. (1982) Biochemistry 21, Yagi, T., Misono, H., Kunhara, N., Yamamoto, T., Sawada, S., & Soda, K. (198) J. Biochem. 87, Misono, H. & Soda, K. (1977) /. Biochem. 82, Yagi, T., Yamamoto, T., & Soda, K. (198) Biochim. Biophys. Ada 614, Smith, L.T. & Opie, J.W. (1955) in Organic Syntheses (Homing, E.C., ed.) Collect. Vol. 3, pp , John Wiley & Sons, Inc., New York 9. Morino, Y. & Okamoto, M. (1972) Biochem. Biophys. Res. Commun. 47, Morino, Y, Osman, A.M., & Okamoto, M. (1974) /. Biol. Chem. 249, Takahashi, K. (1968) /. Biol. Chem. 243, Takahashi, K. (1977) J Biochem. 81, and Riordan, J.F., McElvany, K.D., & Borders, C.L., Jr. (1977) Science 195, Patthy, L. & Thesz, J. (198) Eur. J. Biochem. 15, Yamamoto, S., Yamauchi, T., & Hayaishi, O. (1972) Proc. Natl. Acad. Sci. U.S. 69, Saier, M.H. & Jenkins, W.T. (1967) /. Biol. Chem. 242, Harruff, H.C. & Jenkins, W.T. (1976) Arch. Biochem. Biophys. 177, Harruff, H.C. & Jenkins, W.T. (1978) Arch. Biochem. Biophys. 188, Tunnicliff, G. (198) Biochem. Biophys. Res. Commun. 97, Cheung, S.T. & Fonda, M.L. (1979) Arch Biochem. Biophys. 198, Kazarinoff, M.N. & Snell, E.E. (1976) /. Biol. Chem. 251, Riordan, J.F. & Scandurra, R. (1975) Biochem Biophys. Res. Commun. 66, Gilbert, H.F. & O'Leary, M.H. (1975) Biochem. Biophys. Res. Commun. 67, Miyawaki, M., Tanase, S, & Morino, Y. (1982) /. Biochem. 91, Sandmeier, E. & Christen, P. (1982) /. Biol. Chem. 257, Kazarinoff, M N. & Snell, E.E. (1977) /. Biol. Chem. 252, Chatagner, F. & Pierre, Y. (1977) FEBS Lett. 81, Tanizawa, K. & Miles, E.W. (1983) Biochemistry 22, Ford, G.C., Eichele, G., & Jansonius, J.N. (198) Proc. Natl. Acad. Sci. U.S. 77, Dreyfus, M., Vandenbunder, B., & Buc, H. (198) Biochemistry 19, Vandenbunder, B., Dreyfus, M., Bertrand, O., Dognin, M.J., Sibilli, L., & Buc, H. (1981) Biochemistry 2, Vol. 95, No. 2, 1984

UNIVERSITY OF GUELPH CHEM 4540 ENZYMOLOGY Winter 2005 Quiz #2: March 24, 2005, 11:30 12:50 Instructor: Prof R. Merrill ANSWERS

UNIVERSITY OF GUELPH CHEM 4540 ENZYMOLOGY Winter 2005 Quiz #2: March 24, 2005, 11:30 12:50 Instructor: Prof R. Merrill ANSWERS UNIVERSITY F GUELPH CHEM 4540 ENZYMLGY Winter 2005 Quiz #2: March 24, 2005, 11:30 12:50 Instructor: Prof R. Merrill ANSWERS Instructions: Time allowed = 80 minutes. Total marks = 30. This quiz represents