Reaction of liver pyruvate kinase with sulfhydryl reagents: effect on its activity

Biochimica et Biophysica Acta 913 (1987) 195-199

195

Elsevier BBA 32831

Reaction of liver pyruvate kinase with sulfhydryl reagents: effect on its activity Gale W. Rafter and James B. Blair Department of Biochemistry, West Virginia University School of Medicine, Morgantown, WV (U.S.A.) (Received 15 December 1986)

Key words: Pyruvate kinase; Chemical modification; Enzyme activity; Sulfhydryl group; (Rat liver)

Homogeneous liver pyruvate kinase was reacted with different sulfhydryl reagents, which included oiodosobenzoate, 5',5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide. Activity determinations of the treated enzyme made with and without Fru(l,6)P2 indicate that the protein contains two sulfhydryl groups per subunit important to its properties, one more accessible than the other. Fru(1,6)P2 added to mixtures prevented loss of activity obtained with o-iodosobenzoate and 5 ',5'-dithiobis(2-nitrobenzoic acid). It appears that Fru(1,6)P2 does not interfere with the reaction of the reagent with the sulfhydryl group, but prevents an ensuing conformationai change, which leads to changes in the enzyme's properties.

Introduction Pyruvate kinase (ATP-pyruvate phosphotransferase, EC 2.7.1.40) catalyzes the final step of glycolysis forming pyruvate from phosphoenolpyruvate. The liver enzyme had been purified to homogeneity. It is a tetramer of molecular weight about 240000 and it exhibits allosteric properties: its rate of reaction shows a sigmoidal dependence on phosphoenolpyruvate concentration, which is converted to a hyperbolic response by low concentrations of Fru(1,6)P2 [1]. Recently, the gene for the enzyme has been cloned and its amino acid

sequence has been deduced [2,3]. Six cysteinyl residues per subunit are found. Their role in the properties of the enzyme has not been much studied. Tanaka et al. [4] reported that after pmercuribenzoate treatment, activity is lost, while Van Berkel et al. [5] found that treatment with GSSG markedly decreased the enzyme's affinity for phosphoenolpyruvatewithout changing its activity when measured in the presence of Fru(1,6)PzIn the work described here, liver pyruvate kinase was treated with different sulfhydryl reagents and its effect on the enzyme's properties was examined. Materials and Methods

Abbreviations: Fru(1,6)P2, o-fructose 1,6-bis(phosphate); Ellman's reagent, 5'5'-dithiobis(2-nitrobenzoic acid); [Fru (1,6)P2h/2, the concentration of Fru(1,6)P 2 which gives a half-maximal activity with 1.5 mM phosphoenolpyruvate in the standard assay mixture. Maximal activity is the activity obtained with 160/xM Fru(1,6)P 2 and 1.5 mM phosphoenolpyruvate in the standard assay mixture. Correspondence: G.W. Rafter, Department of Biochemistry, West Virginia University School of Medicine, Morgantown, WV 26506, U.S.A.

Pyruvate kinase was prepared from livers of fed rats as previously described [6]. The crystalline enzyme prepared in this manner is homogeneous as judged by SDS-polyacrylamide gel electrophoresis. Enzyme assays were conducted with a thermostated Gilford recording spectrophotometer by coupling the enzyme activity to lactate dehydrogenase [7]. The assay buffer (pH 7.5) contained 136 mM Tris-C1, 66 mM KC1, 10 mM MgSO4, 2.5 mM ADP, 0.16 mM NADH and 3.2

0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

196 units per ml of lactate dehydrogenase (Boehringer Mannheim). The concentration of phosphoenolpyruvate and Fru(1,6)P 2 in mixtures, as well as the incubation conditions for reacting the sulfhydryl reagents with the enzyme are described with the individual experiments. The final assay vol. was 630 /d, unless otherwise noted, and the assay temperature was 25 °C. The reactions was initiated by addition of phosphoenolpyruvate. The same preparation of pyruvate kinase with a spec. act. of 160 units per mg of protein was used in all experiments. 1 unit of enzyme activity is defined as that amount catalyzing the formation of I #mol of pyruvate per min at 25 °C. Before use, it was freed of mercaptoethanol by precipitating it with ammonium sulfate, washing the protein pellet several times with saturated ammonium sulfate, and redissolving it in 0.1 M potassium phosphate (pH 6.7) buffer which contained 10 mM EDTA and 5 mM MgSO 4. The enzyme is stable in this buffer for several weeks at 4°C. For sulfhydryl group determination, pyruvate kinase was incubated with 200 /tM Ellman's reagent in 0.2 M Tris-C1 buffer (pH 8.1). The increase in absorbance at 412 nm at 1 rain, corrected for reagent alone, was used to estimate protein sulfhydryl content utilizing an extinction coefficient for the released thionitrobenzoate anion of 13600 M - 1 . cm 1 [8] and Al~0 for pyruvate kinase of 0.500 [6]. To remove the last traces of mercaptoethanol, the enzyme was exhaustively dialyzed against 0.1 M potassium phosphate buffer (pH 6.7) which contained 10 mM EDTA, or was subjected to gel filtration.

Fru(1,6)P 2 (Fig. 1). It should be noted that after 30 min incubation, only about 10% of the activity was obtained with 7.5 mM phosphoenolpyruvate in the absence of Fru(1,6)P 2. The [Fru(1,6)P2]l/2 of the treated enzyme was also altered; it was about 10 ~M compared to less than 1.0 ttM for the untreated enzyme (data not shown). Addition of Fru(1,6)P 2 to enzyme mixtures before adding the o-iodosobenzoate protected the enzyme against the inactivation seen when the assay was made with phosphoenolpyruvate alone (Table I). As can be seen in Table I, inactivation was obtained, comparable to that shown in Fig. 1, when Fru(1,6)P 2 was added after the o-iodosobenzoate treatment period. The final concentration of Fru(1,6)P 2 in the assay mixture of 0.8 #M has a negligible activating effect on the o-iodosobenzoate-treated enzyme. If o-iodosobenzoate-treated enzyme (15 min) was further incubated with 2 mM N-ethylmalei-

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Results As inferred from its sensitivity to treatment with p-mercuribenzoate and GSSG liver pyruvate kinase contains reduced sulfhydryl groups. Estimation of its sulfhydryl content with Ellman's reagent showed 1.3 + 0.2 (S.E., n = 4) groups per subunit. To examine the role of the sulfhydryl groups, the enzyme was incubated with several different sulfhydryl reagents and assayed under various conditions. After incubation with oiodosobenzoate, activity was lost when assayed with phosphoenolpyruvate alone, but retained when assayed with phosphoenolpyruvate plus

I

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5

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15

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MINUTES

Fig. l. o-lodosobenzoatetreatment of pyruvate kinase. Incubation was performed at 25°C in 100 /tl of 0.1 M potassium phosphate buffer (pH 7.5) which contained 10 mM EDTA and 370 /~M o-iodosobenzoate. For enzyme determination, mixtures were diluted with 500 txl of assay buffer which gave either 7.5 mM (e e) or 1.5 mM phosphoenolpyruvate+160 #M Fru(1,6)P2 (O O) final concentrations in assay mixtures.

197 TABLE I

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EFFECT OF Fru(1,6)P 2 ON LOSS OF ACTIVITY UPON REACTING PYRUVATE KINASE WITH o-IODOSOBENZOATE OR ELLMAN'S REAGENT OR BY INCUBATION Enzyme in 100 ttl of 0.1 M potassium phosphate buffer (pH 7.5) was incubated with 370/~M o-iodosobenzoate for 30 min at 25 o C, or with no reagent for 30 min at 25 ° C. Enzyme was also incubated with 250 ~tM Ellman's reagent for 1 min at 25 o C, which was followed by a 12 molar excess of GSH. The Fru(1,6)P 2 concentration in the o-iodosobenzoate experiment was 5/~M and in the Ellman and incubation experiment it was 10 /~M. For enzyme determination mixtures were diluted with 500 /~1 of assay buffer. The final concentration of phosphoenolpyruvate in the assay mixture was 1.6 mM, and of Fru(1,6)P 2 was 0.8 /tM in the o-iodosobenzoate experiment and 1.6 /tM in the Ellman's reagent and incubation experiment. Reagent or Treatment

o-lodosobenzoate Incubation alone Ellman's reagent

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0.5

1

Percent of control activity Fru(1,6) P 2 added before reagent a n d / o r incubation

Fru(1,6) P 2 added after reagent a n d / o r incubation

100 100 100

15 68 35

3

6

MINUTES Fig. 2. N-Ethylmaleimide treatment of pyruvate kinase. Incubation was performed at 25°C in 500 #1 of assay buffer which contained 2 mM N-ethylmaleimide. Enzyme measurements were made with either 1.8 mM (O O) or 1.8 mM phosphoenolpyruvate+190 ttM Fru(1,6)P2 (e O). The final volume of the assay mixtures was 530/tl.

1.0 m i d e f o r 10 m i n a t 25 ° C, it l o s t all its a c t i v i t y , e v e n w h e n a s s a y e d w i t h phosphoenolpyruvate p l u s F r u ( 1 , 6 ) P 2 as i n Fig. 1. L i k e w i s e , u n t r e a t e d e n zyme incubated with N-ethylmaleimide also lost a c t i v i t y . A s c a n b e s e e n i n Fig. 2, t h e loss o f activity was complete after 1 min incubation when t h e a s s a y w a s p e r f o r m e d w i t h phosphoenolpyruv a t e a l o n e , w h i l e m o r e t h a n 60% o f t h e a c t i v i t y w a s r e t a i n e d , if t h e a s s a y w a s p e r f o r m e d w i t h phosphoenolpyruvate p l u s F r u ( 1 , 6 ) P 2. I n t h i s l a t t e r e x p e r i m e n t , if t h e F r u ( 1 , 6 ) P 2 i n t h e e n z y m e assay was added before treatment of the enzyme w i t h N - e t h y l m a l e i m i d e , t h e r a t e of i n a c t i v a t i o n w a s r e d u c e d b y a b o u t 50%. P y r u v a t e k i n a s e i n c u b a t e d f o r 30 m i n a t 2 5 ° C l o s t a c t i v i t y i n a p H - d e p e n d e n t m a n n e r w h e n ass a y e d w i t h 1.5 m M phosphoenolpyruvate ( F i g . 3). Including 5 mM GSH in the incubation mixture d i d n o t p r e v e n t t h e loss o f a c t i v i t y n o r d i d t r e a t m e n t of t h e i n c u b a t e d e n z y m e b e f o r e its a s s a y w i t h 10 m M d i t h i o t h r e i t o l f o r 30 m i n a t 2 5 ° C r e s t o r e its l o s t a c t i v i t y . T h e loss o f a c t i v i t y w a s

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6.8

7.2

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pH Fig. 3. Effect of pH on loss of pyruvate kinase activity upon incubation. Incubation was performed at 25 ° C for 30 rain in 100/~l of 0.1 M potassium phosphate buffers which contained 10 mM EDTA. For enzyme determination, mixtures were diluted with 500 /tl of assay buffer which gave 1.5 mM phosphoenolpyruvate final concentration in assay mixtures. No incubation (O o); incubated (O O).

198

prevented by including Fru(1,6)P 2 in the incubation mixture (Table I). If incubated enzyme was assayed with phosphoenolpyruvate plus Fru(1,6)P 2 (160 #M), control activity was obtained. Unlike the o-iodosobenzoate-treated enzyme, 65-90% of the activity was obtained when the incubated enzyme was assayed with 7.5 mM phosphoenolpyruvate in the absence of Fru(1,6)P 2. The protection afforded by Fru(1,6)P 2 against inactivation of pyruvate kinase by o-iodosobenzoate treatment or by incubation (Table I) can be explained in at least two different ways: it prevents modification of a sulfhydryl group or prevents an ensuing reaction promoted by modification of a sulfhydryl group. The second explanation seems the most likely as Fru(1,6)P 2 does not interfere with the reaction of o-iodosobenzoate with the protein, as measured by the enzyme's loss of reactivity with Ellman's reagent, nor does it affect the reactivity of the untreated enzyme with Ellman's reagent. The result obtained by treating the enzyme with Ellman's reagent also supports the second explanation. Pyruvate kinase incubated with this reagent loss activity which was not reversed by adding GSH to remove the reagent as the mixed disulfide (Table I). The inactivation was prevented by including Fru(1,6)P 2 in mixtures during the treatment period (Table I).

Discussion How modification of one protein functional group affects the overall function of a protein affords a useful approach to understanding protein structure function relationships. Previously, phosphorylation of a seryl residue near the Nterminus of pyruvate kinase has been shown to decrease its apparent affinity for its substrate, phosphoenolpyruvate, and its allosteric effector, Fru(1,6)P 2 [9]. As reported here, modification of a protein sulfhydryl group brings about the same qualitative effect. It appears that two sulfhydryl groups per subunit of differing reactivity are important to liver pyruvate kinase's properties. One group available to N-ethylmaleimide, when modified, results in irreversible loss of activity. A second more available group, when modified, leads to alteration of the enzyme's kinetic properties, which is the primary concern of this paper. Ellman's reagent or o-iodosobenzoate treatment gave

an enzyme with a markedly reduced affinity for its substrate in the absence of Fru(1,6)P 2. A less drastic change in substrate affinity was obtained upon incubating the enzyme at alkaline pH values. In this case, the modification of the sulfhydryl group is attributed to its ionization to form a thiolate ion, which is probably the least intrusive of the procedures used. Likewise, a charged sulfur is suggested as the product of the reaction of o-iodosobenzoate with the protein. The product of o-iodosobenzoate reaction with GSH is GSSG [10]; the product of its reaction with protein sulfhydryl groups is not clear. Presumably, the proper juxtaposition of two sulfhydryl groups would form a disulfide bond, but lacking this arrangement, a sulfur of a higher oxidation state, not involving a second group, will form. In this connection reaction of the reagent with either muscle or yeast glyceraldehye-3-PO4 dehydrogenase showed a mole for mole relationship [11]. Unlike phosphorylation, the kinetic changes obtained upon modification of sulfhydryl group in pyruvate kinase were not reversible in the two cases in which it was expected: Ellman's reagent incubation followed by GSH treatment, and incubation in alkaline pH buffers. Fru(1,6)P 2 exerts two different effects on this process; it restores the activity of the treated enzyme, and if present during the treatment period, prevents the changes. It appears that Fru(1,6)P 2 does not interfere with the modification of the sulfhydryl group, but prevents an ensuing reaction, presumably a conformational change, which is expressed in the altered kinetic properties of the enzyme. A possible structural change to account for the kinetic changes is perturbation of the arrangement of the four subunits in the enzyme, which is suggested by the previously described effects of sulfhydryl reagents on the properties of hemoglobin [12].

Acknowledgement This work was supported by an N I H research grant, AM 17664, awarded to J.B.

References 1 Blair, J.B. (1980) in The Regulation of Carbohydrate Formation and Utilization in Mammals (Veneziale, C.M., ed.), pp. 121-151, University Park Press, Baltimore

199 2 Lone, Y.-C., Simon, M.-P., Kahn, A. and Marie, J. (1986) FEBS Lett. 195, 97-100 3 Inoue, H., Noguci, T. and Tanaka, T. (1986) Eur. J. Biochem. 154, 465-469 4 Tanaka, T., Harano, Y., Sue, F. and Morimura, H. (1967) J. Biochem. (Tokyo) 62, 71-91 5 Van Berkel, J.C., Koster, J.F. and Hulsman, W.C. (1973i Biochim. Biophys. Acta 293, 118-124 6 Blair, J.B., Cimbala, M.A. and James, M.E. (1982) J. Biol. Chem. 157, 7595-7602

7 Blair, J.B., Cimbala, M.A., Foster, J.L. and Morgan, R.A. (1976) J. Biol. Chem. 251, 3756-3762 8 Ellman, G.L. (1959) Arch. Biochem. Biophys. 82, 70-77 9 Ljungstrom, O., Berglund, L. and Engstrom, L. (1976) Eur. J. Biochem. 68, 497-506 10 Chinard, F.P. and Hellerman, L. (1954) Methods Biochem. Anal. 1, 1-26 11 Rafter, G.W. (1957) Arch. Biochem. Biophys. 67, 269-271 12 Jocelyn, P.C. (1972) Biochemistry of the SH Group, p. 243, Academic press, New York