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2,3-bisphosphoglycerate synthase-phosphatase from pig brain
Comp. Biochem. Physiol. Vol. 87B, No. i, pp. 11%124, 1987
0305-0491/87 $3.00+0.00 © 1987 Pergamon Journals Ltd
Printed in Great Britain
METABOLISM OF GLYCERATE 2,3-P2--XII. CHARACTERIZATION OF THE 2,3-BISPHOSPHOGLYCERATE SYNTHASE-PHOSPHATASE A N D OF THE HYBRID PHOSPHOGLYCERATE MUTASE/2,3-BISPHOSPHOGLYCERATE SYNTHASE-PHOSPHATASE FROM PIG BRAIN ALBERT TAULER and Jo$1~ CARRERAS* Departamento de Bioquimica, Facultad de Medicina, Universidad de Barcelona, Casanova 143, 08036 Barcelona, Spain (Received 23 June 1986)
Abstract--1.2,3-Bisphosphoglycerate synthase-phosphatase and the hybrid phosphoglycerate mutase/2,3bisphosphoglycerate synthase-phosphatase have been partially purified from pig brain. 2. Their 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities are concurrently lost upon heating and treatment with reagents specific for histidyl, arginyl and lysyl residues. 3. The two enzymes differ in their thermal stability and sensitivity to tetrathionate. 4. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. 5. The synthase activity of both enzymes shows a nonhypcrbolic pattern which fits to a second degree polynomial. 6. The KIn,/~ and optimum pH values are similar to those of the 2,3-bisphosphoglycerate synthasephosphatase from erythrocytes and the hybrid enzyme from skeletal muscle. 7. The synthase activity is inhibited by inorganic phosphate and it is stimulated by glycolyate 2-P.
INTRODUCTION From pig and cat tissues six multifunctional enzymes (forms I-A to I-F) have been isolated which possess 2,3-bisphosphoglycerate synthase (glycerate 3-P + glycerate 1,3-P2--,glycerate 2,3-P2 + glycerate 3-P), 2,3-bisphosphoglycerate phosphatase (glycerate 2,3-P2--,glycerate 3 - P + P i ) and phosphogiycerate mutase (glycerate 3-P~-~glycerate 2-P) activities in different proportions (Carreras et al., 1981, 1983; Pons et al., 1985a). Since the distribution of these enzymes varies in mammalian tissues, their relative contribution to giycerate 2,3-P2 metabolism differs with the tissue. It can contribute to explain the different levels of glycerate 2,3-P 2 in erytbrocytes and in the other tissues. It has been recently proposed that the six enzyme forms result from the homodimeric and heterodimeric combinations of three distinct subunits: M (muscle type), B (brain type) and E (erythrocyte type) (Ports et al., 1985b). Forms I-A, I-B and I-D, which correspond to the phosphoglycerate mutase isozymes, result from combination of types M and B subunits (Bartrons and Carreras, 1982). Form I-F, which corresponds to the 2,3-bisphosphoglycerate synthase-phosphatase originally characterized from erytbrocytes, results from combination of two subunits type E (Sasaki et al., 1975, 1976; Rose and Dube, 1976a; Kappel and Hass, 1976; Hass et al., *Author to whom correspondence should be addressed. 117
1978). Forms I-C and I-E would constitute the combination of a subunit type E with a subunit type M and B, respectively (Rosa et al., 1984; Pons et al., 1985b). Forms I-A, I-B, I-C and I-D have been purified and characterized from pig heart and skeletal muscle (Bartrons and Carreras, 1982; Pons and Carreras, 1985; Berrocal and Carreras, 1987; Tauler et al., 1986). This paper reports the partial purification and characterization of forms I-E and I-F from pig brain. MATERIALSAND
METHODS
Chemicals
Enzymes, substrates, cofactors, reagents and other products were obtained from the sources previously reported (Berrocal and Carreras, 1987). Enzyme and protein assay
2,3-Bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities were assayed, and protein was estimated as previously described (Bartrons and Carreras, 1982;Tauler et al., 1986). Determination of the molecular weight
The molecular weight was estimated by gel-filtration on Ultrogel AcA-44 as previously reported (Bartrons and Carreras, 1982). Modification of specific amino acid residues
Histidyl, arginyl and lysyl residues were modified as previously described (Tauler et al., 1986).
118
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Purification of form I-C Form I-C was obtained as already reported (Pons and Carreras, 1985) from pig skeletal muscle.
RESULTS
Enzyme purification Forms I-E and I-F were obtained from pig brain by modifying the method used to prepare form I-C (Pons and Carreras, 1985) as follows. All steps were carried out at 0-3°C. Pig brains were obtained from a local slaughter house. Nervous tissue was cleaned, minced and homogenized in 3 vol. (v/w) of 20 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA, 1 mM fl-mercaptoethanol in a Sorvall-Omnimixer homogenizer (5000rpm, 20see). After centrifugation at 8000g for 30 min, the supernatant (fraction I) was precipitated with ammonium sulfate (42-75%, pH 7.0 + 0.1), and resuspended in 10 mM phosphate buffer (pH 6.9) containing 1 mM fl-mercaptoethanol. After centrifugation to discard insoluble protein, the solution (fraction II) was applied ( ~ 175 ml; ~ 6.0 mg protein/ml) to a column (3 x 15 cm) of hydroxyapatite equilibrated with the same buffer. The column was eluted first with buffer until the A280nm returned to the original value, and then with a 500 ml linear gradient of phosphate buffer (pH 6.9) ranging from 10 to 200raM (Fig. 1). Two peaks of 2,3-bisphosphoglycerate synthase activity were eluted at 80 and 120 mM phosphate, respectively. The first peak (fraction III-A), possessing most of the 2,3-bisphosphoglycerate phosphatase and of the phosphoglycerate mutase activities eluted, contained forms I-D and I-E. The second peak (fraction III-B) corresponded to form I-F with some contaminant form I-D. The two peaks from the hydroxyapatite column were precipitated by addition of 3 vol. of saturated ammonium sulfate (pH 7.0 + 0.1) and resuspended in
10mM Tris-HC1 buffer (pH 7.4) containing I mM fl-mercaptoethanol. After dialysis, the solutions (100ml; l-2.Smg protein/ml) were applied to columns (3 x 15 cm) of Cibacron Blue Sepharose CL-6B equilibrated with the same buffer. The columns were washed first with 150 ml of equilibrating buffer, and then with 50 ml of 3 mM sodium pyrophosphate to elute form I-D, and with 100 ml of l0 mM sodium pyrophosphate to elute forms I-E and I-F (Fig. 2). Glycerate 2-P, glycerate 3-P and glycolate 2-P were also able to differentially elute forms I-E, I-F and I-D, but the degree of separation was lower than that achieved with pyrophosphate. A summary of the purification procedure is given in Table I. Disc gel electrophoresis on polyacrylamide containing SDS revealed the presence of contaminant proteins in the preparations of forms I-E and I-F. All attempts of further purification failed because of the high unstability of the enzymes and the very low activity of the original extract. However, the preparations obtained were free of other enzymes with 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities present in the original extract. The final preparations were reasonably stable. They may be stored for several weeks at 0°C in ammonium sulfate without loss of activity. Molecular weight The molecular weight of forms I-E and I-F was estimated by gel filtration. Both enzymes emerged between bovine serum albumin and ovalbumin (not shown). The apparent molecular weight was found to be 56,000 _ 1000 for form I-E, and 59,000 + 1000 for form I-F. Intrinsic activities. Critical amino acids As form I-C (Pons and Carreras, 1985), forms I-E and I-F possess 2,3-bisphosphoglyccrate synthase, glycolate 2-P-stimulated 2,3-bisphosphoglyccrate phosphatase and phosphoglycerate mutase activities
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Fig. 2. Elution profiles from CL-6B Cibacron Blue Sepharose. Fraction III-A (175 m g total protein) and fraction III-B (132rag total protein) were applied to the columns (3 x 15 cm) and eluted as described in the text. At a flow rate of 16ml/hr, ~ 3 ml fractions were collected. Panel A: fraction Ill-A; panel B: fraction III-B. ( - - - - ) A~0 nm; ( 0 ) 2,3-bisphosphoglycerate synthase; ( A ) phosphoglycerate mutase. The two-headed arrows indicate fractions pooled to give fractions IV-A 1, IV-A2 and IV-B.
(Table 1). Form I-F shows the synthase and the phosphatase activities in higher proportion than form I-E. Evidence that the three enzymatic activities are displayed by the same proteins was obtained from experiments of inactivation by heating and by treat-
ment with reagents specific for histidine, arginine, lysine and cysteine residues. The three enzyme forms differ in their thermal lability, form I-F being the less stable (Fig. 3). All enzymes lose concurrently their synthase, phos-
Table 1. Summary of enzyme purification Specific activity (U/mg protein) Yield (%) Protein Fraction (mg) BPGS BPGP PGM BPGS BPGP PGM I Extract 4960 0.0013 0.009 4.5 100 100 100 II (NH4)2 SO4 fractionation 1050 0.005 0.031 17.7 80 73 83 III-A Hydroxyapatite peak I 312 0.007 0.052 39.2 32 37 54 III-B Hydroxyapatite peak II 123 0.015 0.007 0.18 29 1 0.I IV-A I Cibacron Blue Svpharose Peak I 57 0.004 0.175 200 2 14 31 tV-A2 Cibacron Blue Sepharose Peak II 8.2 0.140 0.02 4.0 18 0.7 0.1 IV-B Cibacron Blue Sepharose 4 0;300 0.02 0.6 18 0.5 0.1 Enzymes were purified from 300 g of brain. BPGS, 2,3-bisphosphoglycerate synthase activity; BPGP, glycolate 2-P-stimulated 2,3-bisphosphoglycerate phosphatase activity; PGM, phosphoglycerate mutas¢ activity.
120
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TIME (mln) Fig. 3. Rates of thermal inactivation of the activities associated with forms I-E, I-F and I-C. The enzymes were heated at 60°C in 20mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA and 1 mM fl-mercaptoethanol. Protein was adjusted to 2 mg/ml with bovine serum albumin. Aliquots were removed at the indicated intervals and were quickly cooled in ice prior to assay. Open symbols, form I-C; closed symbols, form I-E; half-closed symbols, form I-F. Circles, 2,3-bisphosphoglycerate synthase activity; squares, glycolate 2-P-stimulated 2,3-bisphosphoglycerate phosphatase activity; triangles, phosphoglycerate mutase activity. The broken line represents experiments conducted in the presence of 1 mM glycerate 2,3-P2.
JOSl~ C A R R E R A S
phatase and mutase activities upon heating. Glycerate 2,3-P2 protects against inactivation. Incubation of form I-F with diethylpyrocarbonate (Fig. 4A), Rose Bengal (Fig. 4B), 2,3-butanedione (Fig. 5A) and trinitrobenzenesulfonate (Fig. 5B) produced a progressive and concurrent loss of its 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities. Glycerate 2,3-P2, glycerate 1,3-P2, glycerate 3-P and glycolate 2-P partially protected the enzyme against diethylpyrocarbonate and trinitrobenzenesulfonate. All those compounds similarly decreased the rate of inactivation of the synthase, phosphatase and mutase activities. In contrast, the enzyme was not protected by the metabolites against photo-oxidation with Rose Bengal and against inactivation with 2,3-butanedione. In the presence of substrates and cofactor the three enzymatic activities were lost a rate similar to that of the controls. Similar results were obtained with form I-E, although they are not shown for compactness. No significant differences were observed between the two enzyme forms. As summarized in Table 2, incubation with tetrathionate affected differently forms I-E and I-F. As form I-C, form I-F lost the synthase, the phosphatase
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Fig. 4. Time course of the inactivation of form I-F by diethylpyrocarbonate and by photooxidation with Rose Bengal. Form I-F (1 rag/protein ml) in 25 mM acetate buffer (pH 6.0) was incubated at 25°C with 10 mM diethylpyrocarbonate (panel A) and was photo-oxidized with Rose Bengal (100 #g/ml) at 4°C (panel B) in the absence of any addition (closed symbols) and in the presence of 0.5 mM glyceratc 1,3-P2 (©), I mM glycerate 2,3-P2 ([-]), 1 mM glycerate 3-P (A), and 1 mM glycolate 2-P (W). At intervals aliquots were removed and assayed for 2,3-bisphosphoglycerate synthase (BPGS), glycolate 2-P-stimulated 2,3-bisphosphoglycerate phosphatas¢ (BPGP) and phosphoglycerate mutase (PGM) activities. ('A') control without addition of modifying reagent.
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Fig. 5. Time course of the inactivation of form I-F by 2,3-butanedione and by trinitrobcnzenesulfonate. Form I-F (1 mg protein/ml) in 25mM borate buffer (pH 8.2) was incubated at 25°C with 1.5 mM 2,3-butanedione (panel A) and with 0.5 trinitrobcnzcnesulfonate (panel B), as indicated in the legend to Fig. 4. and the mutase activities in similar proportion. In contrast, form I-E retained most of the mutase activity after tetrathionate treatment.
Kinetic parameters The effects o f the concentration of the substrates on the 2,3-bisphosphoglycerate synthase activity o f forms I-E and I-F was determined over a range of 0.5-50 # M glycerate 1,3-P2, and of 0.5-500 # M glycerate 3-P. The kinetic patterns were similar for the two enzymes, although for compactness only data for form I-F are presented. The Lineweaver-Burk plots of 1/V vs 1/glycerate 3-P at different glycerate 1,3-P2 concentrations (Fig. 6), and of l/V vs 1/glycerate 1,3-P2 at different glycerate 3-P concentrations (not shown) provided a series of nonparallel lines and
Table 2. Inactivation of forms I-F, I-E and I-C by tetrathionat¢ Residual activity (%) Enzyme BPGS BPGP PGM Form I-F 11 12 14 Form I-E 85 20 16 Form I-C 8 4 8 The enzyme (2.25 mg protein/ml) in 20 mM Tris-HCl buffer (pH 7.4) was incubated at 15°Cin the absence of any addition and in the presence of I mM potassium tctrathionat©. After 15 rain the incubation mixture was cooled at 0°C and assayed for activity. BPGS, 2,3-bisphosphoglycerate synthas¢ activity; BPGP, glycolate 2-P-stimulated 2,3-bisphosphoglyccrat© phosphatas¢ activity; PGM, phosphoglycerate mutas¢ activity.
competitive inhibition at high substrate concentrations. The reciprocal plots were nonlinear and curved downward. The statistical analysis o f the
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122
ALBERTTAULERand Jos~ CARRERAS Table 3. Kinetic parameters of forms I-F, I-E and I-C* BPGS/BPGP/PGM'[" • Km(~M) Enzymeform activity ratio 1,3-BPG 3-PG I-F 500/30/1000 0.9 0.5 I-E
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I-C:~ 37/7/1000 0.75 1.0 250 *Calculated from the initial rate values correspondingto the 0.5-5#M concentration of glycerate 3-P (3-PG) and glycerate 1,3-P2 (I,3-BPG). tBPGS, 2,3-bispbosphoglyccratesynthase; BPGP, glycolate 2-P-stimulated 2,3-bisphosphoglyceratephospbatase; PGM, phosphoglyceratemutase. :~Datafrom Tauler et al. (1986). experimental data (Tauler et al., 1986) showed that they fit to a second degree polynomial. As summarized in Table 3, no significant differences were observed between the Km and the K~ values of forms I-E and I-F, and those previously reported for form I-C (Tauler et al., 1986). The three enzyme forms also possess similar pH optimum. Their initial rates show a maximum value at pH 7.5, and fall to one half about pH 6.0 and 8.5. Effects o f modifiers It has been shown that 2,3-bisphosphoglycerate synthase-phosphatase from erythrocytes (form I-F) is inhibited by inorganic phosphate and is stimulated by glycolate 2-P (Rose, 1980). Form I-E from pig brain is also competitively inhibited by Pi (K~ = 8 mM) (Fig. 7). Glycolate 2-P possesses some activatory effect at low concentration, and shows some inhibitory effect at mM concentration (Fig. 8). Bisphosphorylated sugars, ~- and fl-glycerophosphate, phosphoenolpyruvate and adenilic nucleotides (1 mM) did not produce any effect (not shown). DISCUSSION
It is known that erythrocyte 2,3-bisphosphoglycerate synthase-phosphatase (form I-F), type M phosphoglycerate mutase (form I-A) and type B phosphoglycerate mutase from kidney (form I-D) possess a similar ping-pong mechanism which involves the formation of a phosphoenzyme as an intermediate (Rose, 1970, 1980; Rose and Whalen, 1973; Rose et al., 1975; Rose and Dube, 1976b; Han
and Rose, 1979; Britton and Clarke, 1972; Britton et al., 1972b, 1973; Chiba and Sasaki, 1978; Hass et al., 1980; Haggarty and Fothergill, 1980a,b). However they differ in two catalytic properties: the substrate used as donor of the phosphoryl group and the product released in the catalytic cycle. Whereas phosphoglycerate mutase prefers glycerate 2,3-P2 as donor of the phosphoryl group, 2,3-bisphosphoglycerate synthase-phosphatase preferentially uses glycerate 1,3-P2. In the catalytic cycle, phosphoglycerate mutase generally releases monophosphoglycerate and regenerates the phosphoenzyme. In contrast, 2,3-bisphosphoglycerate synthase-phosphatase usually releases glycerate 1,3-P2 and regenerates the free enzyme (Rose, 1980). The nature of the structural features of 2,3-bisphosphoglycerate synthase-phosphatase subunit (type E subunit) and phosphoglycerate mutase (types M and B) subunits responsible for the different functional properties of both enzymes remain unknown. Extensive structural homology between human red cell phosphoglycerate mutase and 2,3-bisphosphoglycerate synthasephosphatase has been reported (Hass et aL, 1976, 1978) but no comparative data exists about their active sites. The inactivation experiments of forms I-E and I-F herein reported, the experiments previously published about inactivation of forms I-A, I-B and I-D (Berrocal and Carreras, 1987) and I-C (Tauler et al., 1986), and the inactivation of erythrocyte 2,3-bisphosphoglycerate synthase-phosphatase upon trinitrophenylation (Ikura et al., 1976) show that histidyi, lysyl and arginyl residues are critical for
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Fig. 8. Effect of glycolate 2-P on the 2,3-bisphosphoglycerate synthase activity of form I-E. The assay mixture contained 50 mM glycyl-glycine buffer (pH 7.2), 1 #M glyeerate 1,3-P2, 1 #M glycerate 3-P and glycolate 2-P at the indicated concentrations.
Glycerate 2,3-P2 metabolism subunits type M, B and E. The concurrent loss of the 2,3-bisphosphogiycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities upon treatment with the amino acid specific reagents may result from the modification of a residue(s) at the active site directly involved in the enzymatic mechanism. However, inactivation could also be brought about by steric hindrance or by a conformational change induced by the bulking groups introduced in the modified residues. The protective effects of substrates, products and cofactors on inactivation suggest that the modified residues are located at or near the active site, although binding could also develop the protective effect at a distance through conformational changes. The different effectiveness of substrates and cofactors to protect against inactivation support the existence of separate binding sites at the active center, as postulated by others (Chiba and Sasaki, 1978; Rose, 1980). The differences observed between the six enzymatic forms in the protection by substrates and cofactors against inactivation with histidine and lysine specific reagents, and the lack of protection of forms I-C, I-E and I-F against modification of arginyl residues suggest structural differences between subunits type E and types M and B, but further studies are needed. The small differences observed in the rate of inactivation of the synthase, phosphatase and mutase activities upon modification of the amino acid residues could result from different sensitivity of the subunits to the modifying reagent, and from the different contribution of each subunit to the total activities of the dimeric molecules. It has been found that 2,3-bisphosphoglycerate synthase-phosphatase and phosphoglycerate mutase isozymes differ in their sensitivity to the sulfhydryl group reagents and in their thermal lability (Sasaki et al., 1975; Mezquita et al., 1981; Bartrons and Carreras, 1982; Carreras et al., 1982; Prehu et al., 1984). The data previously reported and the data now presented can be explained on basis of the subunit structure of the different enzyme forms, if it is assumed that types M and E subunits are sensitive to the - S H group reagents and possess high thermal stability, whereas type M subunit is resistent to the - S H group modifiers and has low thermal stability. Previous results (Tauler et al., 1986) and the data now reported show that the 2,3-bisphosphoglycerate synthase activity of forms I-C, I-E and I-F possesses nonhyperbolic kinetics. This type of pattern could result from either a random Bi Bi or a hybrid pingpong sequential mechanism. However, it could also reflect the existence of two enzyme conformations with different substrate affinities induced by the substrate, or the presence in the enzyme molecule of several substrate binding sites with either different affinities or negative cooperativity (Segel, 1975; Plowman, 1973). The kinetic p a t t e r n s of 2,3-bisphosphoglycerate synthase-phosphatase from erythrocytes have been conflicting. Lineweaver-Burk plots of the enzyme from horse erythrocytes showed a parallel line relationship as a function of second substrate concentration (Rose and Dube, 1976a). With the enzyme from human erythrocytes, patterns with intersecting lines were originally obtained (Rose, 1968), although it was later reported to be an error
123
(Rose, 1980). The ping-pong mechanism postulated for erythrocyte 2,3-bisphosphoglycerate synthasephosphatase (Rose, 1980) might give either a parallel or a converging line pattern (Britton et al., 1972). Lineweaver-Burk plots of the kinetic data corresponding to forms I-C, I-E and I-F from pig tissues show intersecting patterns, although due to the nonlinear character of the reciprocal plots parallel lines can be traced at some concentration ranges. Forms I-E and I-F possess kinetic constants and optimum pH similar to those reported for form I-C from pig muscle (Tauler et al., 1986) and for 2,3-bisphosphoglycerate synthase-phosphatase from human and horse erythrocytes (Rose, 1980). Like this enzyme (Rose, 1980), form I-F is competitively inhibited by inorganic phosphate and stimulated by glycolate 2-P. It is concluded that no significant kinetic differences exist between the homodimeric 2,3-bisphosphoglycerate synthase-phosphatase and the hybrids with types M and B phosphoglycerate mutase subunit. Acknowledgements--This work has been supported by the Spanish Comisitn Asesora de Investigacitn Cientifica y T~cnica.
REFERENCES
Bartrons R. and Carreras J. (1982) Purification and characterization of phospheglycerate mutase isezymes from pig heart. Biochim. biophys. Acta 708, 167-177. Berrocal F. and Carreras J. (1987) Metabolism of glycerate 2,3-P2-XI. Essential amino acids of pig phosphoglycerate mutase isozymes. Comp. Biochem. Physiol. in press. Britton H. G. and Clarke J. B. (1972) Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle. Biochem. J. 130, 397-410. Britton H. G., Carreras J. and Grisolia S. (1972a) Mechanism of yeast phosphoglycerate mutase. Biochemistry 11, 3008-3014. Britton H. G., Carreras J. and Grisolia S. (1972b) Formation of an active phosphoenzyme by diphosphoglycerate-dependent phosphoglycerate mutases from muscle, kidney and yeast. Biochim. biophys. Acta 289, 311-322. Britton H. G., Carreras J. and Grisolia S. (1973) Application of the induced-transport test to the mechanism of pig-kidney phosphoglycerate mutase. Eur. J. Biochem. 36, 495-503. Carreras J., Bartrons R., Bosch J. and Pons G. (1981) Metabolism of glycerate-2,3-P2-I. Distribution of the enzymes involved in the glycerate-2,3-P2 metabolism in pig tissues. Comp. Biochem. Physiol. 70B, 477-485. Carreras J., Bosch J. and Mezquita J. (1982) Phylogeny and ontogeny of phosphoglycerate mutases--III. Inactivation of rabbit muscle phosphoglycerate mutase (type M isozyme) by the sulfhydryl group reagents. Comp. Biochem. Physiol. 71B, 5741. Carreras J., Bartrons R., Berrocal F., Pons G. and Tauler A. (1983) Comparative studies of the enzymes involved in the metabolism of 2,3-bisphosphoglycerate in pig and cat tissues. Biomed. biochim. Acta 42, 306-310. Chiba H. and Sasaki R. (1978) Functions of 2,3-bisphosphoglycerate and its metabolism. In Current Topics in Cellular Regulation (Edited by Horecker B. L. and Stadmann E. R.), Vol. 14, pp. 75-116. Academic Press, New York. Haggarty N. W. and Fothergill L. A. (1980a) Amino acid sequences of active-site histidine peptides from rabbit muscle phosphoglycerate mutase. FEBS Lett. 109, 18-20.
124
ALBERT TAULr,R and JOS~ CAR~RAS
Haggarty N. W. and Fothergill L. A. (1980b) Amino acid sequences of an active site fragment from human diphosphogiycerate mutase. FEBS Left. 114, 124-126. Han Ch. H. and Rose Z. B. (1979) Active site phosphohistidine peptides from red cell diphosphoglycerate synthase and yeast phosphoglycerate mutase. J. biol. Chem. 254, 8836-8840. Hass L. F., Sheibley R. H., Kappel W. K. and Miller K. B. (1976) A comparison of some of the physical and chemical properties of the phosphoglycerate mutases from human erythrocytes. Biochem. biophys. Res. Commun. 72, 976-983. Hass L. F., Kappel W. K., Miller K. B. and Engle R. L. (1978) Evidence for structural homology between human red cell phosphoglycerate mutase and 2,3-bisphosphoglycerate synthase. J. biol. Chem. 253, 77-81. Hass L. F., Place A. R., Miller K. B. and Powers D. A. (I 980) The isolation and characterization of an active site phosphohistidine peptide from human erythrocytes bisphosphoglycerate synthase. Biochem. biophys. Res. Commun. 95, 1570-1576. Ikura K., Sasaki R., Narita H., Sugimoto E. and Chiba H. (1976) Multifunctional enzyme, bisphosphoglyceromutase/2,3-bisphosphoglycerate phosphatase/phosphoglyceromutase from human erythrocytes. Evidence for a common active site. Eur. J. Biochem. 66, 515-523. Kappel W. K. and Hass L. F. (1976) The isolation and partial characterization of diphosphoglycerate mutase from human erythrocytes. Biochemistry 15, 290-295. Mezquita J., Bartrons R., Pons G. and Carreras J. (1981) Phylogeny and ontogeny of the phosphogiycerate mutases. II. Characterization of phosphoglycerate mutase isozymes from vertebrates by their thermal lability and sensitivity to the sulfhydryl group reagents. Comp. Biochem. Physiol. 70B, 247-255. Plowman K. M. (1973) Enzymatic Kinetics McGraw Hill, New York. Pons G. and Carreras J. (1985) Purification of 2,3-bisphosphoglycerate synthase-phosphatase from pig skeletal muscle. Biochim. biophys. Acta 842, 56-61. Pons G., Berrocal F., Tauler A. and Carreras J. (1985a) Metabolism of glycerate-2,3-P2--VII. Enzymes involved in the metabolism of glycerate 2,3-P2 in cat tissues. Comp. Biochem. Physiol. 80B, 551-556. Pons G., Bartrons R. and Carreras J. (1985b) Hybrid forms of phosphoglycerate mutase and 2,3-bisphosphoglycerate
synthase-phosphatase. Biochem. biophys. Res. Commun. 129, 658-663. Prehu M. O., Calvin M. C., Prehu C. and Rosa R. (1984) Biochemical and immunological arguments for homology between red cell and liver phosphoglyceratemutase isozymes. Biochim. biophys. Acta 787, 270-274. Rosa R., Calvin M.-C., Prehu M.-O., Lewis-Strauss M. and Rosa J. (1984) Evidence for the presence of a hybrid of phosphoglyceromutase/bisphosphoglyceromutase in the red cell: partial characterization of the hybrid. Biochem. biophys. Res. Commun. 120, 715-720. Rose Z. B. (1968) The purification and properties of diphosphoglycerate mutase from human erythrocytes. J. biol. Chem. 243, 4810-4820. Rose Z. B. (1970) Evidence for a phosphohistidine protein intermediate in the phosphoglycerate mutase reaction. Archs Biochem. Biophys. 140, 508-513. Rose Z. B. and Dube S. (1976a) The purification and kinetic properties of bisphosphoglycerate synthase from horse red blood cells. Archs Biochem. Biophys. 177, 284-292. Rose Z. B. and Dube S. (1976b) Rates of phosphorylation and dephosphorylation of phosphogiyeerate mutase and bisphosphoglyeerate synthase. J. biol. Chem. 251, 4817-4822. Rose Z. B., Hamasaki N. and Dube S. (1975) The sequence of a peptide containing the active site phosphohistidine residue of phosphoglycerate mutase from chicken breast muscle. J. biol. Chem. 250, 7939-7942. Rose Z. B. (1980) The enzymology of 2,3-bisphosphoglycerate. Adv. Enzym. 51, 211-253. Rose Z. B. and Whalen R. G. (1973) The phosphorylation of diphosphogiycerate mutase. J. biol. Chem. 248, 1513-1519. Sasaki R., Ikura K., Sugimoto E. and Chiba H. (1975) Purification of bisphosphoglyceromutase 2,3-bisphosphoglycerate phosphatase and phosphoglyceromutase from human erythrocytes. Eur. J. Biochem. 50, 581-593. Sasaki R., Ikura K., Narita H. and Chiba H. (1976) Multifunctionality of the enzyme involved in the 2,3-diphosphogiycerate metabolism of pig erythrocyte. Agr. bioL Chem. 40, 2213-2224. Segel I. H. (1975) Enzyme Kinetics. John Wiley, New York. Tauler A., Pons G. and Carreras J. (1986) Kinetic properties and essential amino acids of the 2,3-bisphosphogiycerate synthase-phosphatase from pig skeletal muscle. Biochim. biophys. Acta 872, 201-207.
0305-0491/87 $3.00+0.00 © 1987 Pergamon Journals Ltd
Printed in Great Britain
METABOLISM OF GLYCERATE 2,3-P2--XII. CHARACTERIZATION OF THE 2,3-BISPHOSPHOGLYCERATE SYNTHASE-PHOSPHATASE A N D OF THE HYBRID PHOSPHOGLYCERATE MUTASE/2,3-BISPHOSPHOGLYCERATE SYNTHASE-PHOSPHATASE FROM PIG BRAIN ALBERT TAULER and Jo$1~ CARRERAS* Departamento de Bioquimica, Facultad de Medicina, Universidad de Barcelona, Casanova 143, 08036 Barcelona, Spain (Received 23 June 1986)
Abstract--1.2,3-Bisphosphoglycerate synthase-phosphatase and the hybrid phosphoglycerate mutase/2,3bisphosphoglycerate synthase-phosphatase have been partially purified from pig brain. 2. Their 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities are concurrently lost upon heating and treatment with reagents specific for histidyl, arginyl and lysyl residues. 3. The two enzymes differ in their thermal stability and sensitivity to tetrathionate. 4. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. 5. The synthase activity of both enzymes shows a nonhypcrbolic pattern which fits to a second degree polynomial. 6. The KIn,/~ and optimum pH values are similar to those of the 2,3-bisphosphoglycerate synthasephosphatase from erythrocytes and the hybrid enzyme from skeletal muscle. 7. The synthase activity is inhibited by inorganic phosphate and it is stimulated by glycolyate 2-P.
INTRODUCTION From pig and cat tissues six multifunctional enzymes (forms I-A to I-F) have been isolated which possess 2,3-bisphosphoglycerate synthase (glycerate 3-P + glycerate 1,3-P2--,glycerate 2,3-P2 + glycerate 3-P), 2,3-bisphosphoglycerate phosphatase (glycerate 2,3-P2--,glycerate 3 - P + P i ) and phosphogiycerate mutase (glycerate 3-P~-~glycerate 2-P) activities in different proportions (Carreras et al., 1981, 1983; Pons et al., 1985a). Since the distribution of these enzymes varies in mammalian tissues, their relative contribution to giycerate 2,3-P2 metabolism differs with the tissue. It can contribute to explain the different levels of glycerate 2,3-P 2 in erytbrocytes and in the other tissues. It has been recently proposed that the six enzyme forms result from the homodimeric and heterodimeric combinations of three distinct subunits: M (muscle type), B (brain type) and E (erythrocyte type) (Ports et al., 1985b). Forms I-A, I-B and I-D, which correspond to the phosphoglycerate mutase isozymes, result from combination of types M and B subunits (Bartrons and Carreras, 1982). Form I-F, which corresponds to the 2,3-bisphosphoglycerate synthase-phosphatase originally characterized from erytbrocytes, results from combination of two subunits type E (Sasaki et al., 1975, 1976; Rose and Dube, 1976a; Kappel and Hass, 1976; Hass et al., *Author to whom correspondence should be addressed. 117
1978). Forms I-C and I-E would constitute the combination of a subunit type E with a subunit type M and B, respectively (Rosa et al., 1984; Pons et al., 1985b). Forms I-A, I-B, I-C and I-D have been purified and characterized from pig heart and skeletal muscle (Bartrons and Carreras, 1982; Pons and Carreras, 1985; Berrocal and Carreras, 1987; Tauler et al., 1986). This paper reports the partial purification and characterization of forms I-E and I-F from pig brain. MATERIALSAND
METHODS
Chemicals
Enzymes, substrates, cofactors, reagents and other products were obtained from the sources previously reported (Berrocal and Carreras, 1987). Enzyme and protein assay
2,3-Bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities were assayed, and protein was estimated as previously described (Bartrons and Carreras, 1982;Tauler et al., 1986). Determination of the molecular weight
The molecular weight was estimated by gel-filtration on Ultrogel AcA-44 as previously reported (Bartrons and Carreras, 1982). Modification of specific amino acid residues
Histidyl, arginyl and lysyl residues were modified as previously described (Tauler et al., 1986).
118
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Fig. 1. Elution profile from hydroxyapatite. Fraction II (1 mg total protein) was applied to a column (3 x 15 cm) of hydroxyapatite and ©luted as described in the text. At a flow rate of 15 ml/hr, 3.5 ml fractions were collected. (---) A2~nm; (0) 2,3-bisphosphoglyeerate synthase; (A) phosphogiycerate mutase. The two-heated arrows indicated fractions pooled to give fractions III-A and III-B.
Purification of form I-C Form I-C was obtained as already reported (Pons and Carreras, 1985) from pig skeletal muscle.
RESULTS
Enzyme purification Forms I-E and I-F were obtained from pig brain by modifying the method used to prepare form I-C (Pons and Carreras, 1985) as follows. All steps were carried out at 0-3°C. Pig brains were obtained from a local slaughter house. Nervous tissue was cleaned, minced and homogenized in 3 vol. (v/w) of 20 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA, 1 mM fl-mercaptoethanol in a Sorvall-Omnimixer homogenizer (5000rpm, 20see). After centrifugation at 8000g for 30 min, the supernatant (fraction I) was precipitated with ammonium sulfate (42-75%, pH 7.0 + 0.1), and resuspended in 10 mM phosphate buffer (pH 6.9) containing 1 mM fl-mercaptoethanol. After centrifugation to discard insoluble protein, the solution (fraction II) was applied ( ~ 175 ml; ~ 6.0 mg protein/ml) to a column (3 x 15 cm) of hydroxyapatite equilibrated with the same buffer. The column was eluted first with buffer until the A280nm returned to the original value, and then with a 500 ml linear gradient of phosphate buffer (pH 6.9) ranging from 10 to 200raM (Fig. 1). Two peaks of 2,3-bisphosphoglycerate synthase activity were eluted at 80 and 120 mM phosphate, respectively. The first peak (fraction III-A), possessing most of the 2,3-bisphosphoglycerate phosphatase and of the phosphoglycerate mutase activities eluted, contained forms I-D and I-E. The second peak (fraction III-B) corresponded to form I-F with some contaminant form I-D. The two peaks from the hydroxyapatite column were precipitated by addition of 3 vol. of saturated ammonium sulfate (pH 7.0 + 0.1) and resuspended in
10mM Tris-HC1 buffer (pH 7.4) containing I mM fl-mercaptoethanol. After dialysis, the solutions (100ml; l-2.Smg protein/ml) were applied to columns (3 x 15 cm) of Cibacron Blue Sepharose CL-6B equilibrated with the same buffer. The columns were washed first with 150 ml of equilibrating buffer, and then with 50 ml of 3 mM sodium pyrophosphate to elute form I-D, and with 100 ml of l0 mM sodium pyrophosphate to elute forms I-E and I-F (Fig. 2). Glycerate 2-P, glycerate 3-P and glycolate 2-P were also able to differentially elute forms I-E, I-F and I-D, but the degree of separation was lower than that achieved with pyrophosphate. A summary of the purification procedure is given in Table I. Disc gel electrophoresis on polyacrylamide containing SDS revealed the presence of contaminant proteins in the preparations of forms I-E and I-F. All attempts of further purification failed because of the high unstability of the enzymes and the very low activity of the original extract. However, the preparations obtained were free of other enzymes with 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities present in the original extract. The final preparations were reasonably stable. They may be stored for several weeks at 0°C in ammonium sulfate without loss of activity. Molecular weight The molecular weight of forms I-E and I-F was estimated by gel filtration. Both enzymes emerged between bovine serum albumin and ovalbumin (not shown). The apparent molecular weight was found to be 56,000 _ 1000 for form I-E, and 59,000 + 1000 for form I-F. Intrinsic activities. Critical amino acids As form I-C (Pons and Carreras, 1985), forms I-E and I-F possess 2,3-bisphosphoglyccrate synthase, glycolate 2-P-stimulated 2,3-bisphosphoglyccrate phosphatase and phosphoglycerate mutase activities
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Fig. 2. Elution profiles from CL-6B Cibacron Blue Sepharose. Fraction III-A (175 m g total protein) and fraction III-B (132rag total protein) were applied to the columns (3 x 15 cm) and eluted as described in the text. At a flow rate of 16ml/hr, ~ 3 ml fractions were collected. Panel A: fraction Ill-A; panel B: fraction III-B. ( - - - - ) A~0 nm; ( 0 ) 2,3-bisphosphoglycerate synthase; ( A ) phosphoglycerate mutase. The two-headed arrows indicate fractions pooled to give fractions IV-A 1, IV-A2 and IV-B.
(Table 1). Form I-F shows the synthase and the phosphatase activities in higher proportion than form I-E. Evidence that the three enzymatic activities are displayed by the same proteins was obtained from experiments of inactivation by heating and by treat-
ment with reagents specific for histidine, arginine, lysine and cysteine residues. The three enzyme forms differ in their thermal lability, form I-F being the less stable (Fig. 3). All enzymes lose concurrently their synthase, phos-
Table 1. Summary of enzyme purification Specific activity (U/mg protein) Yield (%) Protein Fraction (mg) BPGS BPGP PGM BPGS BPGP PGM I Extract 4960 0.0013 0.009 4.5 100 100 100 II (NH4)2 SO4 fractionation 1050 0.005 0.031 17.7 80 73 83 III-A Hydroxyapatite peak I 312 0.007 0.052 39.2 32 37 54 III-B Hydroxyapatite peak II 123 0.015 0.007 0.18 29 1 0.I IV-A I Cibacron Blue Svpharose Peak I 57 0.004 0.175 200 2 14 31 tV-A2 Cibacron Blue Sepharose Peak II 8.2 0.140 0.02 4.0 18 0.7 0.1 IV-B Cibacron Blue Sepharose 4 0;300 0.02 0.6 18 0.5 0.1 Enzymes were purified from 300 g of brain. BPGS, 2,3-bisphosphoglycerate synthase activity; BPGP, glycolate 2-P-stimulated 2,3-bisphosphoglycerate phosphatase activity; PGM, phosphoglycerate mutas¢ activity.
120
ALBERT TAULER and
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TIME (mln) Fig. 3. Rates of thermal inactivation of the activities associated with forms I-E, I-F and I-C. The enzymes were heated at 60°C in 20mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA and 1 mM fl-mercaptoethanol. Protein was adjusted to 2 mg/ml with bovine serum albumin. Aliquots were removed at the indicated intervals and were quickly cooled in ice prior to assay. Open symbols, form I-C; closed symbols, form I-E; half-closed symbols, form I-F. Circles, 2,3-bisphosphoglycerate synthase activity; squares, glycolate 2-P-stimulated 2,3-bisphosphoglycerate phosphatase activity; triangles, phosphoglycerate mutase activity. The broken line represents experiments conducted in the presence of 1 mM glycerate 2,3-P2.
JOSl~ C A R R E R A S
phatase and mutase activities upon heating. Glycerate 2,3-P2 protects against inactivation. Incubation of form I-F with diethylpyrocarbonate (Fig. 4A), Rose Bengal (Fig. 4B), 2,3-butanedione (Fig. 5A) and trinitrobenzenesulfonate (Fig. 5B) produced a progressive and concurrent loss of its 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities. Glycerate 2,3-P2, glycerate 1,3-P2, glycerate 3-P and glycolate 2-P partially protected the enzyme against diethylpyrocarbonate and trinitrobenzenesulfonate. All those compounds similarly decreased the rate of inactivation of the synthase, phosphatase and mutase activities. In contrast, the enzyme was not protected by the metabolites against photo-oxidation with Rose Bengal and against inactivation with 2,3-butanedione. In the presence of substrates and cofactor the three enzymatic activities were lost a rate similar to that of the controls. Similar results were obtained with form I-E, although they are not shown for compactness. No significant differences were observed between the two enzyme forms. As summarized in Table 2, incubation with tetrathionate affected differently forms I-E and I-F. As form I-C, form I-F lost the synthase, the phosphatase
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Fig. 4. Time course of the inactivation of form I-F by diethylpyrocarbonate and by photooxidation with Rose Bengal. Form I-F (1 rag/protein ml) in 25 mM acetate buffer (pH 6.0) was incubated at 25°C with 10 mM diethylpyrocarbonate (panel A) and was photo-oxidized with Rose Bengal (100 #g/ml) at 4°C (panel B) in the absence of any addition (closed symbols) and in the presence of 0.5 mM glyceratc 1,3-P2 (©), I mM glycerate 2,3-P2 ([-]), 1 mM glycerate 3-P (A), and 1 mM glycolate 2-P (W). At intervals aliquots were removed and assayed for 2,3-bisphosphoglycerate synthase (BPGS), glycolate 2-P-stimulated 2,3-bisphosphoglycerate phosphatas¢ (BPGP) and phosphoglycerate mutase (PGM) activities. ('A') control without addition of modifying reagent.
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Fig. 5. Time course of the inactivation of form I-F by 2,3-butanedione and by trinitrobcnzenesulfonate. Form I-F (1 mg protein/ml) in 25mM borate buffer (pH 8.2) was incubated at 25°C with 1.5 mM 2,3-butanedione (panel A) and with 0.5 trinitrobcnzcnesulfonate (panel B), as indicated in the legend to Fig. 4. and the mutase activities in similar proportion. In contrast, form I-E retained most of the mutase activity after tetrathionate treatment.
Kinetic parameters The effects o f the concentration of the substrates on the 2,3-bisphosphoglycerate synthase activity o f forms I-E and I-F was determined over a range of 0.5-50 # M glycerate 1,3-P2, and of 0.5-500 # M glycerate 3-P. The kinetic patterns were similar for the two enzymes, although for compactness only data for form I-F are presented. The Lineweaver-Burk plots of 1/V vs 1/glycerate 3-P at different glycerate 1,3-P2 concentrations (Fig. 6), and of l/V vs 1/glycerate 1,3-P2 at different glycerate 3-P concentrations (not shown) provided a series of nonparallel lines and
Table 2. Inactivation of forms I-F, I-E and I-C by tetrathionat¢ Residual activity (%) Enzyme BPGS BPGP PGM Form I-F 11 12 14 Form I-E 85 20 16 Form I-C 8 4 8 The enzyme (2.25 mg protein/ml) in 20 mM Tris-HCl buffer (pH 7.4) was incubated at 15°Cin the absence of any addition and in the presence of I mM potassium tctrathionat©. After 15 rain the incubation mixture was cooled at 0°C and assayed for activity. BPGS, 2,3-bisphosphoglycerate synthas¢ activity; BPGP, glycolate 2-P-stimulated 2,3-bisphosphoglyccrat© phosphatas¢ activity; PGM, phosphoglycerate mutas¢ activity.
competitive inhibition at high substrate concentrations. The reciprocal plots were nonlinear and curved downward. The statistical analysis o f the
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122
ALBERTTAULERand Jos~ CARRERAS Table 3. Kinetic parameters of forms I-F, I-E and I-C* BPGS/BPGP/PGM'[" • Km(~M) Enzymeform activity ratio 1,3-BPG 3-PG I-F 500/30/1000 0.9 0.5 I-E
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I-C:~ 37/7/1000 0.75 1.0 250 *Calculated from the initial rate values correspondingto the 0.5-5#M concentration of glycerate 3-P (3-PG) and glycerate 1,3-P2 (I,3-BPG). tBPGS, 2,3-bispbosphoglyccratesynthase; BPGP, glycolate 2-P-stimulated 2,3-bisphosphoglyceratephospbatase; PGM, phosphoglyceratemutase. :~Datafrom Tauler et al. (1986). experimental data (Tauler et al., 1986) showed that they fit to a second degree polynomial. As summarized in Table 3, no significant differences were observed between the Km and the K~ values of forms I-E and I-F, and those previously reported for form I-C (Tauler et al., 1986). The three enzyme forms also possess similar pH optimum. Their initial rates show a maximum value at pH 7.5, and fall to one half about pH 6.0 and 8.5. Effects o f modifiers It has been shown that 2,3-bisphosphoglycerate synthase-phosphatase from erythrocytes (form I-F) is inhibited by inorganic phosphate and is stimulated by glycolate 2-P (Rose, 1980). Form I-E from pig brain is also competitively inhibited by Pi (K~ = 8 mM) (Fig. 7). Glycolate 2-P possesses some activatory effect at low concentration, and shows some inhibitory effect at mM concentration (Fig. 8). Bisphosphorylated sugars, ~- and fl-glycerophosphate, phosphoenolpyruvate and adenilic nucleotides (1 mM) did not produce any effect (not shown). DISCUSSION
It is known that erythrocyte 2,3-bisphosphoglycerate synthase-phosphatase (form I-F), type M phosphoglycerate mutase (form I-A) and type B phosphoglycerate mutase from kidney (form I-D) possess a similar ping-pong mechanism which involves the formation of a phosphoenzyme as an intermediate (Rose, 1970, 1980; Rose and Whalen, 1973; Rose et al., 1975; Rose and Dube, 1976b; Han
and Rose, 1979; Britton and Clarke, 1972; Britton et al., 1972b, 1973; Chiba and Sasaki, 1978; Hass et al., 1980; Haggarty and Fothergill, 1980a,b). However they differ in two catalytic properties: the substrate used as donor of the phosphoryl group and the product released in the catalytic cycle. Whereas phosphoglycerate mutase prefers glycerate 2,3-P2 as donor of the phosphoryl group, 2,3-bisphosphoglycerate synthase-phosphatase preferentially uses glycerate 1,3-P2. In the catalytic cycle, phosphoglycerate mutase generally releases monophosphoglycerate and regenerates the phosphoenzyme. In contrast, 2,3-bisphosphoglycerate synthase-phosphatase usually releases glycerate 1,3-P2 and regenerates the free enzyme (Rose, 1980). The nature of the structural features of 2,3-bisphosphoglycerate synthase-phosphatase subunit (type E subunit) and phosphoglycerate mutase (types M and B) subunits responsible for the different functional properties of both enzymes remain unknown. Extensive structural homology between human red cell phosphoglycerate mutase and 2,3-bisphosphoglycerate synthasephosphatase has been reported (Hass et aL, 1976, 1978) but no comparative data exists about their active sites. The inactivation experiments of forms I-E and I-F herein reported, the experiments previously published about inactivation of forms I-A, I-B and I-D (Berrocal and Carreras, 1987) and I-C (Tauler et al., 1986), and the inactivation of erythrocyte 2,3-bisphosphoglycerate synthase-phosphatase upon trinitrophenylation (Ikura et al., 1976) show that histidyi, lysyl and arginyl residues are critical for
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(mM)
Fig. 8. Effect of glycolate 2-P on the 2,3-bisphosphoglycerate synthase activity of form I-E. The assay mixture contained 50 mM glycyl-glycine buffer (pH 7.2), 1 #M glyeerate 1,3-P2, 1 #M glycerate 3-P and glycolate 2-P at the indicated concentrations.
Glycerate 2,3-P2 metabolism subunits type M, B and E. The concurrent loss of the 2,3-bisphosphogiycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities upon treatment with the amino acid specific reagents may result from the modification of a residue(s) at the active site directly involved in the enzymatic mechanism. However, inactivation could also be brought about by steric hindrance or by a conformational change induced by the bulking groups introduced in the modified residues. The protective effects of substrates, products and cofactors on inactivation suggest that the modified residues are located at or near the active site, although binding could also develop the protective effect at a distance through conformational changes. The different effectiveness of substrates and cofactors to protect against inactivation support the existence of separate binding sites at the active center, as postulated by others (Chiba and Sasaki, 1978; Rose, 1980). The differences observed between the six enzymatic forms in the protection by substrates and cofactors against inactivation with histidine and lysine specific reagents, and the lack of protection of forms I-C, I-E and I-F against modification of arginyl residues suggest structural differences between subunits type E and types M and B, but further studies are needed. The small differences observed in the rate of inactivation of the synthase, phosphatase and mutase activities upon modification of the amino acid residues could result from different sensitivity of the subunits to the modifying reagent, and from the different contribution of each subunit to the total activities of the dimeric molecules. It has been found that 2,3-bisphosphoglycerate synthase-phosphatase and phosphoglycerate mutase isozymes differ in their sensitivity to the sulfhydryl group reagents and in their thermal lability (Sasaki et al., 1975; Mezquita et al., 1981; Bartrons and Carreras, 1982; Carreras et al., 1982; Prehu et al., 1984). The data previously reported and the data now presented can be explained on basis of the subunit structure of the different enzyme forms, if it is assumed that types M and E subunits are sensitive to the - S H group reagents and possess high thermal stability, whereas type M subunit is resistent to the - S H group modifiers and has low thermal stability. Previous results (Tauler et al., 1986) and the data now reported show that the 2,3-bisphosphoglycerate synthase activity of forms I-C, I-E and I-F possesses nonhyperbolic kinetics. This type of pattern could result from either a random Bi Bi or a hybrid pingpong sequential mechanism. However, it could also reflect the existence of two enzyme conformations with different substrate affinities induced by the substrate, or the presence in the enzyme molecule of several substrate binding sites with either different affinities or negative cooperativity (Segel, 1975; Plowman, 1973). The kinetic p a t t e r n s of 2,3-bisphosphoglycerate synthase-phosphatase from erythrocytes have been conflicting. Lineweaver-Burk plots of the enzyme from horse erythrocytes showed a parallel line relationship as a function of second substrate concentration (Rose and Dube, 1976a). With the enzyme from human erythrocytes, patterns with intersecting lines were originally obtained (Rose, 1968), although it was later reported to be an error
123
(Rose, 1980). The ping-pong mechanism postulated for erythrocyte 2,3-bisphosphoglycerate synthasephosphatase (Rose, 1980) might give either a parallel or a converging line pattern (Britton et al., 1972). Lineweaver-Burk plots of the kinetic data corresponding to forms I-C, I-E and I-F from pig tissues show intersecting patterns, although due to the nonlinear character of the reciprocal plots parallel lines can be traced at some concentration ranges. Forms I-E and I-F possess kinetic constants and optimum pH similar to those reported for form I-C from pig muscle (Tauler et al., 1986) and for 2,3-bisphosphoglycerate synthase-phosphatase from human and horse erythrocytes (Rose, 1980). Like this enzyme (Rose, 1980), form I-F is competitively inhibited by inorganic phosphate and stimulated by glycolate 2-P. It is concluded that no significant kinetic differences exist between the homodimeric 2,3-bisphosphoglycerate synthase-phosphatase and the hybrids with types M and B phosphoglycerate mutase subunit. Acknowledgements--This work has been supported by the Spanish Comisitn Asesora de Investigacitn Cientifica y T~cnica.
REFERENCES
Bartrons R. and Carreras J. (1982) Purification and characterization of phospheglycerate mutase isezymes from pig heart. Biochim. biophys. Acta 708, 167-177. Berrocal F. and Carreras J. (1987) Metabolism of glycerate 2,3-P2-XI. Essential amino acids of pig phosphoglycerate mutase isozymes. Comp. Biochem. Physiol. in press. Britton H. G. and Clarke J. B. (1972) Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle. Biochem. J. 130, 397-410. Britton H. G., Carreras J. and Grisolia S. (1972a) Mechanism of yeast phosphoglycerate mutase. Biochemistry 11, 3008-3014. Britton H. G., Carreras J. and Grisolia S. (1972b) Formation of an active phosphoenzyme by diphosphoglycerate-dependent phosphoglycerate mutases from muscle, kidney and yeast. Biochim. biophys. Acta 289, 311-322. Britton H. G., Carreras J. and Grisolia S. (1973) Application of the induced-transport test to the mechanism of pig-kidney phosphoglycerate mutase. Eur. J. Biochem. 36, 495-503. Carreras J., Bartrons R., Bosch J. and Pons G. (1981) Metabolism of glycerate-2,3-P2-I. Distribution of the enzymes involved in the glycerate-2,3-P2 metabolism in pig tissues. Comp. Biochem. Physiol. 70B, 477-485. Carreras J., Bosch J. and Mezquita J. (1982) Phylogeny and ontogeny of phosphoglycerate mutases--III. Inactivation of rabbit muscle phosphoglycerate mutase (type M isozyme) by the sulfhydryl group reagents. Comp. Biochem. Physiol. 71B, 5741. Carreras J., Bartrons R., Berrocal F., Pons G. and Tauler A. (1983) Comparative studies of the enzymes involved in the metabolism of 2,3-bisphosphoglycerate in pig and cat tissues. Biomed. biochim. Acta 42, 306-310. Chiba H. and Sasaki R. (1978) Functions of 2,3-bisphosphoglycerate and its metabolism. In Current Topics in Cellular Regulation (Edited by Horecker B. L. and Stadmann E. R.), Vol. 14, pp. 75-116. Academic Press, New York. Haggarty N. W. and Fothergill L. A. (1980a) Amino acid sequences of active-site histidine peptides from rabbit muscle phosphoglycerate mutase. FEBS Lett. 109, 18-20.
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Haggarty N. W. and Fothergill L. A. (1980b) Amino acid sequences of an active site fragment from human diphosphogiycerate mutase. FEBS Left. 114, 124-126. Han Ch. H. and Rose Z. B. (1979) Active site phosphohistidine peptides from red cell diphosphoglycerate synthase and yeast phosphoglycerate mutase. J. biol. Chem. 254, 8836-8840. Hass L. F., Sheibley R. H., Kappel W. K. and Miller K. B. (1976) A comparison of some of the physical and chemical properties of the phosphoglycerate mutases from human erythrocytes. Biochem. biophys. Res. Commun. 72, 976-983. Hass L. F., Kappel W. K., Miller K. B. and Engle R. L. (1978) Evidence for structural homology between human red cell phosphoglycerate mutase and 2,3-bisphosphoglycerate synthase. J. biol. Chem. 253, 77-81. Hass L. F., Place A. R., Miller K. B. and Powers D. A. (I 980) The isolation and characterization of an active site phosphohistidine peptide from human erythrocytes bisphosphoglycerate synthase. Biochem. biophys. Res. Commun. 95, 1570-1576. Ikura K., Sasaki R., Narita H., Sugimoto E. and Chiba H. (1976) Multifunctional enzyme, bisphosphoglyceromutase/2,3-bisphosphoglycerate phosphatase/phosphoglyceromutase from human erythrocytes. Evidence for a common active site. Eur. J. Biochem. 66, 515-523. Kappel W. K. and Hass L. F. (1976) The isolation and partial characterization of diphosphoglycerate mutase from human erythrocytes. Biochemistry 15, 290-295. Mezquita J., Bartrons R., Pons G. and Carreras J. (1981) Phylogeny and ontogeny of the phosphogiycerate mutases. II. Characterization of phosphoglycerate mutase isozymes from vertebrates by their thermal lability and sensitivity to the sulfhydryl group reagents. Comp. Biochem. Physiol. 70B, 247-255. Plowman K. M. (1973) Enzymatic Kinetics McGraw Hill, New York. Pons G. and Carreras J. (1985) Purification of 2,3-bisphosphoglycerate synthase-phosphatase from pig skeletal muscle. Biochim. biophys. Acta 842, 56-61. Pons G., Berrocal F., Tauler A. and Carreras J. (1985a) Metabolism of glycerate-2,3-P2--VII. Enzymes involved in the metabolism of glycerate 2,3-P2 in cat tissues. Comp. Biochem. Physiol. 80B, 551-556. Pons G., Bartrons R. and Carreras J. (1985b) Hybrid forms of phosphoglycerate mutase and 2,3-bisphosphoglycerate
synthase-phosphatase. Biochem. biophys. Res. Commun. 129, 658-663. Prehu M. O., Calvin M. C., Prehu C. and Rosa R. (1984) Biochemical and immunological arguments for homology between red cell and liver phosphoglyceratemutase isozymes. Biochim. biophys. Acta 787, 270-274. Rosa R., Calvin M.-C., Prehu M.-O., Lewis-Strauss M. and Rosa J. (1984) Evidence for the presence of a hybrid of phosphoglyceromutase/bisphosphoglyceromutase in the red cell: partial characterization of the hybrid. Biochem. biophys. Res. Commun. 120, 715-720. Rose Z. B. (1968) The purification and properties of diphosphoglycerate mutase from human erythrocytes. J. biol. Chem. 243, 4810-4820. Rose Z. B. (1970) Evidence for a phosphohistidine protein intermediate in the phosphoglycerate mutase reaction. Archs Biochem. Biophys. 140, 508-513. Rose Z. B. and Dube S. (1976a) The purification and kinetic properties of bisphosphoglycerate synthase from horse red blood cells. Archs Biochem. Biophys. 177, 284-292. Rose Z. B. and Dube S. (1976b) Rates of phosphorylation and dephosphorylation of phosphogiyeerate mutase and bisphosphoglyeerate synthase. J. biol. Chem. 251, 4817-4822. Rose Z. B., Hamasaki N. and Dube S. (1975) The sequence of a peptide containing the active site phosphohistidine residue of phosphoglycerate mutase from chicken breast muscle. J. biol. Chem. 250, 7939-7942. Rose Z. B. (1980) The enzymology of 2,3-bisphosphoglycerate. Adv. Enzym. 51, 211-253. Rose Z. B. and Whalen R. G. (1973) The phosphorylation of diphosphogiycerate mutase. J. biol. Chem. 248, 1513-1519. Sasaki R., Ikura K., Sugimoto E. and Chiba H. (1975) Purification of bisphosphoglyceromutase 2,3-bisphosphoglycerate phosphatase and phosphoglyceromutase from human erythrocytes. Eur. J. Biochem. 50, 581-593. Sasaki R., Ikura K., Narita H. and Chiba H. (1976) Multifunctionality of the enzyme involved in the 2,3-diphosphogiycerate metabolism of pig erythrocyte. Agr. bioL Chem. 40, 2213-2224. Segel I. H. (1975) Enzyme Kinetics. John Wiley, New York. Tauler A., Pons G. and Carreras J. (1986) Kinetic properties and essential amino acids of the 2,3-bisphosphogiycerate synthase-phosphatase from pig skeletal muscle. Biochim. biophys. Acta 872, 201-207.