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Metabolism of glycerate-2,3-P2—VI. Lysyl-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P2 synthase and glycerate-2,3-P2 phosphatase activities of rabbit muscle phosphoglycerate mutase
Comp. Biochem. Physiol. Vol. 77B, No. 3, pp. 475 481, 1984 Printed in Great Britain
0305-0491/84 $3.00+0.00 ~2 1984PergamonPress Ltd
METABOLISM OF GLYCERATE-2,3-P2--VI. LYSYL-SPECIFIC REAGENTS INACTIVATE THE PHOSPHOGLYCERATE MUTASE, GLYCERATE-2,3-P2 SYNTHASE AND GLYCERATE-2,3-P2 PHOSPHATASE ACTIVITIES OF RABBIT MUSCLE PHOSPHOGLYCERATE MUTASE FI~LIX BERROCAL and Jos~ CARRERAS* Departamento de Bioquimica, Facultad de Medicina, Universidad de Barcelona, Casanova 143, Barcelona (36), Spain (Received 25 July 1983) A b s t r a c t - - 1 . Treatment of rabbit muscle phosphoglycerate mutase with trinitrobenzenesulfonate and with
pyridoxal-5'-phosphate produces the concurrent loss of the three activites of the enzyme:phosphoglycerate mutase, glycerate-2,3-P: synthase and glycerate-2,3-P2 phosphatase. 2. With both reagents complete inactivation occurs with modification of about 3 moles of lysine per mole of enzyme. 3. Inactivated phosphoglycerate mutase is unable to form the functionally active phosphoenzyme when mixed with glycerate-2,3-P2. 4. Substrate (glycerate-3-P) protects against pyridoxal-5'-phosphate inactivation, and offers some protection against TNBS inactivation. Cofactor (glycerate-2,3-P2) does not prevent inactivation. 5. These results provide additional evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase and favour their location at the same active site. 6. In addition, they suggest that the essential lysyl residues are located at or near the substrate binding site.
INTRODUCTION
Glycerate-2,3-P2-dependent phosphoglycerate mutase (EC 2.7.5.3), in addition to the main enzymatic activity, possesses glycerate-2,3-P2 synthase activity and glycerate-2,3-P2 phosphatase activity which is stimulable by glycolate-2-P (Sasaki et al., 1975, 1976; Carreras et al., 1981; Bosch et al., 1982; Bartrons and Carreras, 1982). It has been shown that rabbit muscle phosphoglycerate mutase loses its mutase, synthase and phosphatase activities with the specific modification of residues of cysteine (Bartrons and Carreras, 1982; Tauler et al., 1983), arginine (Berrocal and Carreras, 1983a) and histidine (Berrocal and Carreras, 1983b). This paper demonstrates that treatment of rabbit muscle phosphoglycerate mutase with trinitrobenzenesulfonatet and with pyridoxalY-phosphate results in the loss of the main and of the collateral enzymatic activities. TNBS is considered to be an amino-specific reagent although at high concentrations it will react with other residues, particularly thiol groups (Okuyama and Satake, 1960; Satake et al., 1960; Goldfarb, 1966; Freedman and Radda, 1968). Pyridoxal-5'-phosphate in combination with reduction by sodium borohydride has been *To whom correspondence should be addressed. tAbbreviations used: TNBS, trinitrobezenesulfonate; PGM, phosphoglycerate mutase; BPGS, glycerate-2,3P2 synthase; BPGP, glycerate-2,3-P2 phosphatase; BPGP*, glycolate-2-P-stimulated glycerate-2,3-P2 phosphatase. CB.P. 77/3I~ D
475
used as a specific reagent for modifying lysine residues (Rippa et al., 1967). It has been suggested that pyridoxal-5'-phosphate can be used specifically for modifying lysine residues at or near phosphate binding sites of enzymes (Raetz and Auld, 1972; Colombo and Marcus, 1974; Milhausen and Levy, 1975). Therefore it can be postulated that lysine is implicated in the active site of rabbit muscle phosphoglycerate mutase, like it is in the active site of chicken muscle (Carne and Flynn, 1977) and of yeast (Sasaki et al., 1971a, 1971b, 1976) phosphoglycerate mutases.
MATERIALS AND METHODS
Chemicals Crystalline phosphoglycerate mutase, enolase, pyruvate kinase and lactate dehydrogenase from rabbit muscle, and phosphoglycerate kinase from yeast, glycerate-3-P (trisodium salt), glycerate-2,3-P2 (pentacyclohexylammonium salt), ATP (disodium salt) and NADH (disodium salt) were obtained from Boehringer Mannheim. Glycerate-3-P (barium salt), glycerate-2,3-P2(pentasodium salt), glycolate-2-P (tri-monocyclohexylaminesalt), TNBS (picrylsulfonic acid, grade I), pyridoxal-5'-phosphate, sodium borohydride and lysine (monohydrochloride form) were from Sigma. Dithiothreitol and bovine serum albumin were from Calbiochem. 2-Mercaptoethanol was from Merck, Darmstadt. All other chemicals were reagent grade. Dowcx AGI-X8 (chloride form) was from Bio-Rad. Sephadex G-25 was from Pharmacia. Glycerate-3-P free of contaminant glycerate-2,3-P2 was prepared from the barium salt by
476
F~LIX BERROCALand JOsl~ CARRERAS
purification on a Dowex AG1-X8 column as described by Grisolia et al. (1969), but the purified solution was neutralized with sodium hydroxide.
Assay methods' The protein concentration of phosphoglycerate mutase solutions was determined from the extinction at 280 nm by using the coefficient 1.25 cmZ/mg protein (Zwaig and Milstein, 1966) and by the method of Mokrasch and McGilvery (1956) using bovine serum albumin as a standard. Phosphoglycerate mutase, glycerate-2,3-F 2 synthase and glycerate-2,3-P 2 phosphatase activities were assayed as previously described (Carreras et al., 1981).
Enzyme modification The commercial preparation of rabbit muscle phosphoglycerate mutase was found to be practically homogenous and free of contaminant enzymes with glycerate2,3-P2 synthase and glycerate-2,3-P 2 phosphatase activities (Berrocal and Carreras, 1983a). Prior to the modification experiments, the enzyme was reactivated as previously described (Carreras et al., 1982). The ammonium sulfate suspension of phosphoglycerate mutase was centrifuged and dissolved in 50mM borate buffer, pH 8.2, containing 40 mM dithiothreitol. After 20 min incubation at 4~C, the solution was filtered through a Sephadex G-25 column equilibrated with buffer without dithiothreitol. Reaction with TNBS was carried out at 25~C and in the dark in 50 mM borate buffer pH 8.2 under the conditions given in the figure legends. At the desired time intervals, aliquots were removed from the incubation mixture, diluted about 20-fold in 50 mM borate buffer, pH 8.2, containing 1~o bovine serum albumin and 1 mM lysine, and assayed for enzymatic activities. In the experiments done to determine the number of trinitrophenylated lysyl residues, reaction with TNBS was carried out in a cell thermostated to 25'C of a Gilford 250 spectrophotometer equipped with a KippZonen BD 40 recorder. The reaction was followed by the increase in absorbance at 367 nm. A blank without phosphoglycerate mutase was used. The extent of reaction was calculated by assuming a molar extinction coefficient of 1.08 x 104 (Goldfarb, 1966; Coffee et al., 1971). As the reaction proceeded samples were removed, diluted in borate buffer containing lysine and bovine serum albumin, and assayed for phosphoglycerate mutase activity.
Reaction with pyridoxal-5'-phosphate was carried out at 25°C and in the dark in 50 mM borate buffer, pH 8.2, under the conditions given in the figure legends. As the reaction progressed, aliquots were removed and diluted 5-fold in 50 mM borate buffer, pH 8.2, containing l"J;~bovine serum albumin, l mM lysine and l mM sodium borohydride to reduce the pyridoxal-enzyme. After 30min the enzymatic activities were assayed as described. To test the reversibility of the reaction, phosphoglycerate mutase was incubated with pyridoxal-5'-phosphate under the described conditions, and after 40 min incubation the enzymatic activities were determined. The incubation mixture was dialysed against 20 mM Tris HCI buffer pH 7.5 overnight and assayed again. To determine the number of pyridoxal-P groups incorporated into the enzyme, 12 #M (0.8 mg/ml) phosphoglycerate mutase was incubated at 25C and in the dark with pyridoxal-5'-phosphate (60pM) in 50mM borate buffer pH 8.2. At intervals aliquots were removed from the incubation mixture and diluted 5-fold in 50 mM borate buffer pH 8.2 containing 200 ~M lysine and I mM sodium borohydride. After 30 rain the samples were dialysed against 20mM Tris-HCl buffer pH 7.5, and assayed for phosphoglycerate mutase activity. The absorbance at 325 nm was determined and the extent of reaction was calculated by assuming a molar extinction coefficient of 1.07 × 104 for reduced phosphopyridoxal-lysine (McKinley-McKec and Morris, 1972). A control sample of untreated enzyme was used.
Formation of the phosphoenzyme The formation of the phosphoenzyme from phosphoglycerate mutase and glycerate-2,3-P2 was determined spectrophotometrically as previously described (Britton et al., 1972). Prior to the assay the inactivated and the control phosphoglycerate mutase was dialysed against 20mM Tris-HCl buffer, pH 7.5, containing l mM 2-mercaptoethanol and 1 mM EDTA. RESULTS
Reaction with T N B S Trinitrophenylation and loss of enzymatic actit, ity. Preliminary test d e m o n s t r a t e d that rabbit muscle phosphoglycerate mutase is very sensitive to TNBS
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mutase by TNBS. The enzyme (12#M) in 50raM borate buffer, pH 8.2, was incubated at 25C with 240 #M TNBS, in the absence and in the presence of 5 mM glycerate-3-P. The enzymatic activity (solid symbols) and the number of trinitrophenylated lysyl residues (open symbols) were determined as described under Materials and Methods. (O, Q) incubation without glycerate-3-P; ([23, m) incubation with glycerate-3-P.
Metabolism of glycerate-2,3-P2--Vl
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Fig. 2. Stoichiometry of inactivation of phosphoglycerate mutase by modification with TNBS. The experimental conditions were as described in the legend to Fig. 1. (O) incubation without glycerate-3-P: (A) incubation with glycerate-3-P.
treatment, therefore a 20-fold molar excess of reagent was used in the modification experiments. As shown in Fig. 1, reaction with TNBS resulted in the loss of phosphoglycerate mutase activity. Complete inactivation occurred with incorporation of an average of 3 moles of trinitrophenyl groups per mole of enzyme (Fig. 2). Glycerate-2,3-P2 synthase, glycerate-2,3-P2 phosphatase and glycolate-2-P-stimulated glycerate2,3-P2 phosphatase activities were concurrently lost with the main enzymatic activity (Fig. 3). Effect of substrate and cofactor. The presence of glycerate-3-P reduced both the rate of reaction with TNBS and the rate of inactivation (Fig. 1). However,
100
complete inactivation still occurred with the incorporation of about 3 moles of trinitrophenyl groups per mole of phosphoglycerate mutase, indicating that the extent of modification was not changed despite the reduction in the rate of reaction (Fig. 2). The main and the collateral enzymatic activities were similarly protected by the substrate (Fig. 3). The cofactor did not offer protection against TNBS treatment. Phosphoglycerate mutase was inactivated by TNBS at the same rate in the absence and in the presence of 5 mM glycerate-2,3-P2 (Fig. 3).
Fa£lure of the inactivated phosphoglycerate inulase to form the phosphoenzyme. The formation of the
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Fig. 3. Time course of the inactivation of rabbit muscle phosphoglycerate mutase by TNBS. The enzyme (3~M) in 50raM borate buffer, pH 8.2, was incubated at 25°C with 60#M TNBS in the absence of substrate and cofactor (O) or in the presence of 5 mM glycerate-2,3-P2 (11) or 5 mM glycerate-3-P (A). At intervals aliquots were removed and assayed for phosphoglycerate mutase (PGM), glycerate-2,3-P2 synthase (BPGS), glycerate-2,3-P2 phosphatase (BPGP) and glycolate~2-P-stimulatedglycerate-2,3-P2 phosphatase (BPGP*) activities as described in Materials and Methods.
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FI~LIX BERROCALand Josl~ CARRERAS
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Fig. 4. Changes in absorbance at 340 nm on addition of glycerate-2,3-P 2 to substrate quantities of native and inactivated rabbit muscle phosphoglycerate mutase. The reaction mixture (in a l-cm lightpath quartz cuvette) contained in a total volume of 1 ml: phosphoglycerate mutase (2 mg/ml); enolase and pyruvate kinase, 0.7 mg/ml each; lactate dehydrogenase, 0.35 mg/ml; NADH, 0.3 raM; ADP, 0.3 mM; KCI, 6 mM: MgC12, 6mM; Tris-HC1 buffer, pH5.5, 25mM. After 10min incubation at 30C, glycerate-2,3-P~ (0.5~mol) was added in a volume of 5~1, and the decrease in A340nm was followed. After 10min, glycolate-2-P (0.5/~mol) was added in a volume of 5 #1. (O) Native phosphoglycerate mutase; (11) TNBS-inactivated phosphoglycerate mutase; (&) pyridoxal-5'-phosphate-inactivated phosphoglycerate mutase.
phosphoenzyme was examined spectrophotometrically by adding glycerate-2,3-P2 to substrate quantities of phosphoglycerate mutase. As shown in Fig. 4, the addition of the cofactor to the reaction mixture containing untreated enzyme caused an immediate drop of 0.285 in the absorbance at 340 nm followed by a linear decline. From the initial burst in
absorbance 0.6 tool phosphoenzyme per enzyme subunit was calculated to be formed. The linear decline in absorbance, interpreted as the hydrolysis of the phosphoenzyme (Britton et al., 1972), was increased by glycolate-2-P, which acts as an activator of the glycerate-2,3-P2 phosphatase activity of the enzyme. When the incubation mixture contained TNBS-
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Fig. 5. Time course of the inactivation and degree of modification of rabbit muscle phosphoglycerate mutase by pyridoxal-5'-phosphate. The enzyme (12 ~M) in 50 mM borate buffer, pH 8.2, was incubated at 25c'C with 60 pM pyridoxal-5'-phosphate. The enzymatic activity (O) and the number of pyridoxal-P groups incorporated into the enzyme (,A) were determined as described in Materials and Methods.
Metabolism of glycerate-2,3-P_,--VI
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Fig. 6. Stoichiometry of inactivation of phosphoglycerate mutase by modification with pyridoxal-5'phosphate. The experimental conditions were as described in the legend to Fig. 5. inactivated phosphoglycerate mutase the initial drop was undetectable, showing the unability of the treated enzyme to form the phosphoenzyme from the co factor.
Reaction with pyridoxal-5'-phosphate Loss of enzymatic activity. As shown in Fig. 5, reaction of rabbit muscle phosphoglycerate mutase with pyridoxal-5'-phosphate and NaBH4 resulted in a loss of about 80% of the enzymatic activity. Reaction with pyridoxal-5'-phosphate and enzyme inactivation showed a linear relationship up to 80% inactivation, and extrapolation of the linear portion of the activity curve revealed that complete inactivation of the enzyme is the result of the incorporation of an
average of 3 moles of pyridoxal-5'-phosphate group per mole of phosphoglycerate mutase (Fig. 6). Glycerate-2,3-P2 synthase, glycerate-2,3-P2 phosphatase and glycolate-2-P-stimulated glycerate-2,3-P2 phosphatase activities were concurrently lost with the mutase activity (Fig. 7). Treatment of the enzyme with pyridoxal-5'-phosphate in the absence of NaBH4 resulted also in the concurrent loss of the main and of the collateral enzymatic activities. All activities were completely recovered when the inactivated enzyme was either dialysed or filtered through Sephadex G-25 (Table 1). Effect of substrate and cofactor. As shown in Fig. 7, in the presence of glycerate-3-P the three enzymatic activities of rabbit muscle phosphoglycerate mutase were only reduced by 20% with
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Fig. 7. Time course of the inactivation of phosphoglycerate mutase by pyridoxal-5'-phosphate. The enzyme (6/~M) in 50raM borate buffer, pH 8.2, was incubated at 25°C with 30raM pyridoxal-5'phosphate in the absence of substrate and cofactor (O) or in the presence of 5 mM glycerate-2,3-P2( . ) or 5 mM glycerate-3-P(A). At intervals aliquots were removed and assayed for phosphoglycerate mutase (PGM), glycerate-2,3-P2 synthase (BPGS), glycerate-2,3-P., phosphatase (BPGP) and glycolate-2-Pstimulated glycerate-2,3-P2 phosphatase (BPGP*) activities as described in Materials and Methods.
480
FEL1X BERROCALand Jos~ CARRERAS Table 1. Reversible inactivation of rabbit muscle phosphoglycerate mutase by pyridoxal-5'-phosphate % of initial activity PGM BPGS BPGP BPGP* Before dialysis 20 18 24 25 After dialysis 100 100 100 100 The enzyme (6 ~M) in 50 mM borate buffer, pH 8.2, was incubated at 25C and in the dark with 30 ~ M pyridoxal-5'-phosphate. After 40 rain the enzymatic activities were determined. The incubation mixture was dialysed overnight at 4 C against 20mM Tris HCI buffer, pH 7.5, containing 1 mM 2-mercaptoethanol, and assayed again.
pyridoxal-5'-phosphate treatment. In the presence of glycerate-2,3-P2 inactivation was not prevented, although the rate of inactivation was reduced. In the presence of cofactor 40 min incubation were required to produce the same level of inactivation as that produced in 15 min incubation in the absence of glycerate-2,3-P2. Failure o f the treated phosphoglycerate mutase to form the phosphoenzyme. Similarly to the TNBSinactivated phosphoglycerate mutase, the enzyme irreversibly-inactivated with pyridoxal-5'-phosphate and NaBH4 was unable to form the active phosphoenzyme when mixed with glycerate-2,3-P2 (Fig. 4). DISCUSSION Lysyl residues have been implicated in the mechanism of yeast phosphoglycerate mutase (Sasaki et al., 1971a,b, 1976). Chemical modification experiments with trinitrobenzenesulfonate demonstrated that yeast phosphoglycerate mutase possesses four reactive amino groups (one group per enzyme subunit) located at the monophosphoglycerate banding site (Sasaki et al., 1971a). These groups are essential for the glycerate-2,3-P2 synthase and the glycerate2,3-P 2 phosphatase as well the phosphoglycerate mutase activity of the enzyme (Sasaki et al., 1971b, 1976). Chicken muscle phosphoglycerate mutase also has essential lysyl residues. Trinitrophenylation resulted in the loss of the enzymatic activity, complete inactivation occurring with the incorporation of about four moles of trinitrophenyl group per mole of enzyme. Glycerate-3-P offered some protection to TNBS-inactivation but glycerate-2,3-P2 did not. Reaction of the Schiff's base complex between pyridoxal-5'-phosphate and chicken muscle phosphoglycerate mutase gave irreversible inactivation. Inactivation was due to incorporation of 1 mole of pyridoxal-5'-phosphate per mole of enzyme dimer. Since neither large change in conformation nor dissociation of the dimer enzyme was observed, a "halfof-the-sites" type effect was assumed to occur. Glycerate-3-P protected the enzyme against both reaction and inactivation. Glycerate-2,3-P2 did not show protective effect, although reduced the rate at which inactivation occurred. Pyridoxal-5'-phosphate treatment interferred with formation of the functionally active phosphoenzyme but did not completely prevent its formation. F r o m these results it was concluded that a lysyl residue is located at or
near the active site of chicken muscle phosphoglycerate mutase, being probably involved in the binding of substrate (Came and Flynn, 1977). The experiments reported in this paper demonstrate that lysyl residues are also essential for rabbit muscle phosphoglycerate mutase. Treatment of the enzyme with both T N B S and pyridoxal-5'-phosphate and NaBH4 produces the loss of the main and of the collateral enzymatic activities. With both reagents complete inactivation occurs with modification of about 3 moles of lysine per mole of enzyme. The concurrent loss of the mutase, synthase and phosphatase activities of rabbit muscle phosphoglycerate mutase provide additional evidence to prove the intrinsic character of the three enzymatic activities and to support their location at the same active site (Berrocal and Carreras, 1983a,b). The different effectiveness of glycerate-2,3-P 2 and glycerate-3-P to protect the enzyme against both reaction and inactivation favours the existence of separate binding sites for monophosphoglycerates and for biphosphoglycerates suggested by others (Sasaki et al., 1971b, 1976) and supported by experiments of specific modification of residues of arginine (Berrocal and Carreras, 1983a), cysteine (Tauler et al., 1983) and histidine (Berrocal and Carreras, 1983b). The protective effect of glycerate-3-P suggests that in rabbit muscle phosphoglycerate mutase, like in chicken muscle phosphoglycerate mutase, the essential lysyl residues are located at or near the substrate binding site. The inability of the inactivated enzyme to form the phosphoenzyme when mixed with glycerate-2,3-P 2 suggests a close proximity between the binding sites for substrate and for cofactor. It may be concluded that the active sites of yeast, chicken muscle and rabbit muscle phosphoglycerate mutases seem to be structurally related.
Acknowledgements--This work has been supported by the Spanish Comisidn Asesora de lnvestigaci6n Cientifica y T6cnica (Grant No 3974/79). F. Berrocal is recipient of a research fellowship from the Fundacidn Pedro Pons. REFERENCES
Bartrons R. and Carreras J. (1982) Purification and characterization of phosphoglycerate mutase isozymes from pig heart. Biochim. biophys. Acta 708, 167 177. Berrocal F. and Carreras J. (1983a) Metabolism of glycerate-2,3-P2--III. Arginine-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase activities of rabbit muscle phosphoglycerate mutase. Comp. Biochem. Physiol. 76B, 9-14. Berrocal F. and Carreras J. (1983b) Metabolism of glycerate-2,3-P2--V. Histidine-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P2 phosphatase activities of rabbit muscle phosphoglycerate mutase. Comp. Biochem. Physiol. 76B, 795-799. Bosch J., Pons G. and Carreras J. (1982) Metabolism of glycerate-2,3-P2--H. Enzymes involved in the glycerate2,3-P 2 metabolism in chicken skeletal muscle. Comp. Biochem. Physiol. 72B, 39 48. Britton H. G., Carreras J. and Grisolia S. (1972) Formation of an active phosphoenzyme by diphosphoglyceratedependent phosphoglyceromutases from muscle, kidney and yeast. Biochim. biophys. Acta 289, 311-322.
Metabolism of glycerate-2,3-P:~--VI Carne T. J. and Flynn T. G. (1977) Studies on phosphoglyceromutase from chicken breast muscle: chemical modification of lysyl residues. Can. J. Biochem. 55, 856-864. Carreras J., Bartrons R., Bosch J. and Pons G. (1981) Metabolism of glycerate-2,3-P,--I. Distribution of the enzymes involved in the glycerate-2,3-P2 metabolism in pig tissues. Comp. Biochem. Physiol. 70B, 477-485. Carreras J., Mezquita J. and Pons G. (1982) Phylogeny and ontogeny of the phosphoglycerate mutases--V. Inactivation of phosphoglycerate mutase isozymes by histidine-specific reagents. Comp. Biochem. Physiol. 72B, 401-407. Coffee C. J., Bradshaw R. A., Goldin B. R. and Frieden C. (1971) Identification of the sites of modification of bovine liver glutamate dehydrogenase reacted with trinitrobenzenesulfonate. Biochemistry 10, 3 5 1 6 3526. Colombo G. and Marcus F. (1974) Modification of fructose-l,6-diphosphatase with pyridoxal-5'-phosphate. Evidence for the participation of lysyl residues at the active site. Biochemistry 13, 3085-3091. Freedman R. B. and Radda G. K. (1968) The reaction of 2,4,6-trinitrobenzenesulphonic acid with amino acids, peptides and proteins. Biochem. J. 108, 383-391. Goldfarb A. R. (1966) A kinetic study of the reaction of amino acids and peptides with trinitrobenzenesulfonic acid. Biochemistry 5, 2570-2574. Grisolia S., Moore L., Luque J. and Gardy H. (1969) Automatic procedure for the microestimation of 2,3-diphosphoglycerate. Analyt. Biochem. 31, 235-245. McKinley-McKee J. S. and Morris D. L. (1972) The lysines in liver alcohol dehydrogenase. Chemical modification with pyridoxal-5'-phosphate and methyl picolinimidate. Eur. J. Biochem. 28, 1 11. Milhausen M. and Levy H. R. (1975) Evidence for an essential lysine in glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Eur. J. Biochem. 50, 453-461.
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Mokrasch L. C. and McGilvery R. W. (1956) Purification and properties of fructose-l,6-diphosphatase. J. biol. Chem. 221, 909-917. Okuyama T. and Satake K. (1960) On the preparation and properties of 2,4,6-trinitrophenyl-amino acids and peptides. J. Biochem. 47, 454~466. Raetz C. R. H. and Auld D. S. (1974) Schiff bases of pyridoxal phosphate with active center lysines of ribonuclease A. Biochemistry 11, 2229-2236. Rippa M., Spanio L. and Pontremoli S. (1967) A specific interaction of pyridoxal-5'-phosphate and 6-phosphogluconic dehydrogenase. Archs Biochem. Biophys. 118, 48-57. Sasaki R., Sugimoto E. and Chiba H. (1971a) Studies on the active site of yeast phosphoglycerate mutase. Biochim. biophys. Acta 227, 584-594. Sasaki R., Hirose M., Sugimoto E. and Chiba H. (1971b) Studies on a role of the 2,3-diphosphoglycerate activity in the yeast phosphoglycerate mutase reaction. Biochim. biophys. Acta 227, 595-607. Sasaki R., lkura K., Sugimoto E. and Chiba H. (1975) Purification of bisphosphoglycex&te phosphatase and phosphoglyceromutase from human erythrocytes. Eur. J. Biochem. 50, 581-593. Sasaki R., Utsimi S., Sugimoto E. and Chiba H. (1976) Subunit structure and multifunctional properties of yeast phosphoglyceromutase. Eur. J. Biochem. 66, 523-533. Satake K., Okuyama T., Ohashi M. and Shinoda T. (1960) The spectrophotometric determination of amine, amino acid and peptide with 2,4,6-trinitrobenzene 1-sulfonic acid. J. Biochem. 47, 654-660. Tauler A., Bartrons R., Pons G. and Carreras J. (1983) Metabolism of glycerate-2,3-P2--IV. Effects of Hg 2+ on the enzymes involved in the metabolism of glycerate-2,3P2 in pig skeletal muscle. Comp. Biochem. Physiol. 76B, 789-793. Zwaig N. and Milstein C. (1966) The phosphorylated intermediate in the phosphoglyceromutase reaction. Biochem. J. 98, 360-368.
0305-0491/84 $3.00+0.00 ~2 1984PergamonPress Ltd
METABOLISM OF GLYCERATE-2,3-P2--VI. LYSYL-SPECIFIC REAGENTS INACTIVATE THE PHOSPHOGLYCERATE MUTASE, GLYCERATE-2,3-P2 SYNTHASE AND GLYCERATE-2,3-P2 PHOSPHATASE ACTIVITIES OF RABBIT MUSCLE PHOSPHOGLYCERATE MUTASE FI~LIX BERROCAL and Jos~ CARRERAS* Departamento de Bioquimica, Facultad de Medicina, Universidad de Barcelona, Casanova 143, Barcelona (36), Spain (Received 25 July 1983) A b s t r a c t - - 1 . Treatment of rabbit muscle phosphoglycerate mutase with trinitrobenzenesulfonate and with
pyridoxal-5'-phosphate produces the concurrent loss of the three activites of the enzyme:phosphoglycerate mutase, glycerate-2,3-P: synthase and glycerate-2,3-P2 phosphatase. 2. With both reagents complete inactivation occurs with modification of about 3 moles of lysine per mole of enzyme. 3. Inactivated phosphoglycerate mutase is unable to form the functionally active phosphoenzyme when mixed with glycerate-2,3-P2. 4. Substrate (glycerate-3-P) protects against pyridoxal-5'-phosphate inactivation, and offers some protection against TNBS inactivation. Cofactor (glycerate-2,3-P2) does not prevent inactivation. 5. These results provide additional evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase and favour their location at the same active site. 6. In addition, they suggest that the essential lysyl residues are located at or near the substrate binding site.
INTRODUCTION
Glycerate-2,3-P2-dependent phosphoglycerate mutase (EC 2.7.5.3), in addition to the main enzymatic activity, possesses glycerate-2,3-P2 synthase activity and glycerate-2,3-P2 phosphatase activity which is stimulable by glycolate-2-P (Sasaki et al., 1975, 1976; Carreras et al., 1981; Bosch et al., 1982; Bartrons and Carreras, 1982). It has been shown that rabbit muscle phosphoglycerate mutase loses its mutase, synthase and phosphatase activities with the specific modification of residues of cysteine (Bartrons and Carreras, 1982; Tauler et al., 1983), arginine (Berrocal and Carreras, 1983a) and histidine (Berrocal and Carreras, 1983b). This paper demonstrates that treatment of rabbit muscle phosphoglycerate mutase with trinitrobenzenesulfonatet and with pyridoxalY-phosphate results in the loss of the main and of the collateral enzymatic activities. TNBS is considered to be an amino-specific reagent although at high concentrations it will react with other residues, particularly thiol groups (Okuyama and Satake, 1960; Satake et al., 1960; Goldfarb, 1966; Freedman and Radda, 1968). Pyridoxal-5'-phosphate in combination with reduction by sodium borohydride has been *To whom correspondence should be addressed. tAbbreviations used: TNBS, trinitrobezenesulfonate; PGM, phosphoglycerate mutase; BPGS, glycerate-2,3P2 synthase; BPGP, glycerate-2,3-P2 phosphatase; BPGP*, glycolate-2-P-stimulated glycerate-2,3-P2 phosphatase. CB.P. 77/3I~ D
475
used as a specific reagent for modifying lysine residues (Rippa et al., 1967). It has been suggested that pyridoxal-5'-phosphate can be used specifically for modifying lysine residues at or near phosphate binding sites of enzymes (Raetz and Auld, 1972; Colombo and Marcus, 1974; Milhausen and Levy, 1975). Therefore it can be postulated that lysine is implicated in the active site of rabbit muscle phosphoglycerate mutase, like it is in the active site of chicken muscle (Carne and Flynn, 1977) and of yeast (Sasaki et al., 1971a, 1971b, 1976) phosphoglycerate mutases.
MATERIALS AND METHODS
Chemicals Crystalline phosphoglycerate mutase, enolase, pyruvate kinase and lactate dehydrogenase from rabbit muscle, and phosphoglycerate kinase from yeast, glycerate-3-P (trisodium salt), glycerate-2,3-P2 (pentacyclohexylammonium salt), ATP (disodium salt) and NADH (disodium salt) were obtained from Boehringer Mannheim. Glycerate-3-P (barium salt), glycerate-2,3-P2(pentasodium salt), glycolate-2-P (tri-monocyclohexylaminesalt), TNBS (picrylsulfonic acid, grade I), pyridoxal-5'-phosphate, sodium borohydride and lysine (monohydrochloride form) were from Sigma. Dithiothreitol and bovine serum albumin were from Calbiochem. 2-Mercaptoethanol was from Merck, Darmstadt. All other chemicals were reagent grade. Dowcx AGI-X8 (chloride form) was from Bio-Rad. Sephadex G-25 was from Pharmacia. Glycerate-3-P free of contaminant glycerate-2,3-P2 was prepared from the barium salt by
476
F~LIX BERROCALand JOsl~ CARRERAS
purification on a Dowex AG1-X8 column as described by Grisolia et al. (1969), but the purified solution was neutralized with sodium hydroxide.
Assay methods' The protein concentration of phosphoglycerate mutase solutions was determined from the extinction at 280 nm by using the coefficient 1.25 cmZ/mg protein (Zwaig and Milstein, 1966) and by the method of Mokrasch and McGilvery (1956) using bovine serum albumin as a standard. Phosphoglycerate mutase, glycerate-2,3-F 2 synthase and glycerate-2,3-P 2 phosphatase activities were assayed as previously described (Carreras et al., 1981).
Enzyme modification The commercial preparation of rabbit muscle phosphoglycerate mutase was found to be practically homogenous and free of contaminant enzymes with glycerate2,3-P2 synthase and glycerate-2,3-P 2 phosphatase activities (Berrocal and Carreras, 1983a). Prior to the modification experiments, the enzyme was reactivated as previously described (Carreras et al., 1982). The ammonium sulfate suspension of phosphoglycerate mutase was centrifuged and dissolved in 50mM borate buffer, pH 8.2, containing 40 mM dithiothreitol. After 20 min incubation at 4~C, the solution was filtered through a Sephadex G-25 column equilibrated with buffer without dithiothreitol. Reaction with TNBS was carried out at 25~C and in the dark in 50 mM borate buffer pH 8.2 under the conditions given in the figure legends. At the desired time intervals, aliquots were removed from the incubation mixture, diluted about 20-fold in 50 mM borate buffer, pH 8.2, containing 1~o bovine serum albumin and 1 mM lysine, and assayed for enzymatic activities. In the experiments done to determine the number of trinitrophenylated lysyl residues, reaction with TNBS was carried out in a cell thermostated to 25'C of a Gilford 250 spectrophotometer equipped with a KippZonen BD 40 recorder. The reaction was followed by the increase in absorbance at 367 nm. A blank without phosphoglycerate mutase was used. The extent of reaction was calculated by assuming a molar extinction coefficient of 1.08 x 104 (Goldfarb, 1966; Coffee et al., 1971). As the reaction proceeded samples were removed, diluted in borate buffer containing lysine and bovine serum albumin, and assayed for phosphoglycerate mutase activity.
Reaction with pyridoxal-5'-phosphate was carried out at 25°C and in the dark in 50 mM borate buffer, pH 8.2, under the conditions given in the figure legends. As the reaction progressed, aliquots were removed and diluted 5-fold in 50 mM borate buffer, pH 8.2, containing l"J;~bovine serum albumin, l mM lysine and l mM sodium borohydride to reduce the pyridoxal-enzyme. After 30min the enzymatic activities were assayed as described. To test the reversibility of the reaction, phosphoglycerate mutase was incubated with pyridoxal-5'-phosphate under the described conditions, and after 40 min incubation the enzymatic activities were determined. The incubation mixture was dialysed against 20 mM Tris HCI buffer pH 7.5 overnight and assayed again. To determine the number of pyridoxal-P groups incorporated into the enzyme, 12 #M (0.8 mg/ml) phosphoglycerate mutase was incubated at 25C and in the dark with pyridoxal-5'-phosphate (60pM) in 50mM borate buffer pH 8.2. At intervals aliquots were removed from the incubation mixture and diluted 5-fold in 50 mM borate buffer pH 8.2 containing 200 ~M lysine and I mM sodium borohydride. After 30 rain the samples were dialysed against 20mM Tris-HCl buffer pH 7.5, and assayed for phosphoglycerate mutase activity. The absorbance at 325 nm was determined and the extent of reaction was calculated by assuming a molar extinction coefficient of 1.07 × 104 for reduced phosphopyridoxal-lysine (McKinley-McKec and Morris, 1972). A control sample of untreated enzyme was used.
Formation of the phosphoenzyme The formation of the phosphoenzyme from phosphoglycerate mutase and glycerate-2,3-P2 was determined spectrophotometrically as previously described (Britton et al., 1972). Prior to the assay the inactivated and the control phosphoglycerate mutase was dialysed against 20mM Tris-HCl buffer, pH 7.5, containing l mM 2-mercaptoethanol and 1 mM EDTA. RESULTS
Reaction with T N B S Trinitrophenylation and loss of enzymatic actit, ity. Preliminary test d e m o n s t r a t e d that rabbit muscle phosphoglycerate mutase is very sensitive to TNBS
100
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i
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Fig, I. Time course o f the inactivation and degree o f modification o f rabbit muscle phosphoglycerate
mutase by TNBS. The enzyme (12#M) in 50raM borate buffer, pH 8.2, was incubated at 25C with 240 #M TNBS, in the absence and in the presence of 5 mM glycerate-3-P. The enzymatic activity (solid symbols) and the number of trinitrophenylated lysyl residues (open symbols) were determined as described under Materials and Methods. (O, Q) incubation without glycerate-3-P; ([23, m) incubation with glycerate-3-P.
Metabolism of glycerate-2,3-P2--Vl
477
ook >
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Fig. 2. Stoichiometry of inactivation of phosphoglycerate mutase by modification with TNBS. The experimental conditions were as described in the legend to Fig. 1. (O) incubation without glycerate-3-P: (A) incubation with glycerate-3-P.
treatment, therefore a 20-fold molar excess of reagent was used in the modification experiments. As shown in Fig. 1, reaction with TNBS resulted in the loss of phosphoglycerate mutase activity. Complete inactivation occurred with incorporation of an average of 3 moles of trinitrophenyl groups per mole of enzyme (Fig. 2). Glycerate-2,3-P2 synthase, glycerate-2,3-P2 phosphatase and glycolate-2-P-stimulated glycerate2,3-P2 phosphatase activities were concurrently lost with the main enzymatic activity (Fig. 3). Effect of substrate and cofactor. The presence of glycerate-3-P reduced both the rate of reaction with TNBS and the rate of inactivation (Fig. 1). However,
100
complete inactivation still occurred with the incorporation of about 3 moles of trinitrophenyl groups per mole of phosphoglycerate mutase, indicating that the extent of modification was not changed despite the reduction in the rate of reaction (Fig. 2). The main and the collateral enzymatic activities were similarly protected by the substrate (Fig. 3). The cofactor did not offer protection against TNBS treatment. Phosphoglycerate mutase was inactivated by TNBS at the same rate in the absence and in the presence of 5 mM glycerate-2,3-P2 (Fig. 3).
Fa£lure of the inactivated phosphoglycerate inulase to form the phosphoenzyme. The formation of the
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i
20
40
60
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60
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Fig. 3. Time course of the inactivation of rabbit muscle phosphoglycerate mutase by TNBS. The enzyme (3~M) in 50raM borate buffer, pH 8.2, was incubated at 25°C with 60#M TNBS in the absence of substrate and cofactor (O) or in the presence of 5 mM glycerate-2,3-P2 (11) or 5 mM glycerate-3-P (A). At intervals aliquots were removed and assayed for phosphoglycerate mutase (PGM), glycerate-2,3-P2 synthase (BPGS), glycerate-2,3-P2 phosphatase (BPGP) and glycolate~2-P-stimulatedglycerate-2,3-P2 phosphatase (BPGP*) activities as described in Materials and Methods.
478
FI~LIX BERROCALand Josl~ CARRERAS
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Fig. 4. Changes in absorbance at 340 nm on addition of glycerate-2,3-P 2 to substrate quantities of native and inactivated rabbit muscle phosphoglycerate mutase. The reaction mixture (in a l-cm lightpath quartz cuvette) contained in a total volume of 1 ml: phosphoglycerate mutase (2 mg/ml); enolase and pyruvate kinase, 0.7 mg/ml each; lactate dehydrogenase, 0.35 mg/ml; NADH, 0.3 raM; ADP, 0.3 mM; KCI, 6 mM: MgC12, 6mM; Tris-HC1 buffer, pH5.5, 25mM. After 10min incubation at 30C, glycerate-2,3-P~ (0.5~mol) was added in a volume of 5~1, and the decrease in A340nm was followed. After 10min, glycolate-2-P (0.5/~mol) was added in a volume of 5 #1. (O) Native phosphoglycerate mutase; (11) TNBS-inactivated phosphoglycerate mutase; (&) pyridoxal-5'-phosphate-inactivated phosphoglycerate mutase.
phosphoenzyme was examined spectrophotometrically by adding glycerate-2,3-P2 to substrate quantities of phosphoglycerate mutase. As shown in Fig. 4, the addition of the cofactor to the reaction mixture containing untreated enzyme caused an immediate drop of 0.285 in the absorbance at 340 nm followed by a linear decline. From the initial burst in
absorbance 0.6 tool phosphoenzyme per enzyme subunit was calculated to be formed. The linear decline in absorbance, interpreted as the hydrolysis of the phosphoenzyme (Britton et al., 1972), was increased by glycolate-2-P, which acts as an activator of the glycerate-2,3-P2 phosphatase activity of the enzyme. When the incubation mixture contained TNBS-
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Fig. 5. Time course of the inactivation and degree of modification of rabbit muscle phosphoglycerate mutase by pyridoxal-5'-phosphate. The enzyme (12 ~M) in 50 mM borate buffer, pH 8.2, was incubated at 25c'C with 60 pM pyridoxal-5'-phosphate. The enzymatic activity (O) and the number of pyridoxal-P groups incorporated into the enzyme (,A) were determined as described in Materials and Methods.
Metabolism of glycerate-2,3-P_,--VI
479
10C
75
< Z
50
0
0
8
25
2
4
MOLES P Y R I D O X A L - L Y S I N E / MOLE PGM
Fig. 6. Stoichiometry of inactivation of phosphoglycerate mutase by modification with pyridoxal-5'phosphate. The experimental conditions were as described in the legend to Fig. 5. inactivated phosphoglycerate mutase the initial drop was undetectable, showing the unability of the treated enzyme to form the phosphoenzyme from the co factor.
Reaction with pyridoxal-5'-phosphate Loss of enzymatic activity. As shown in Fig. 5, reaction of rabbit muscle phosphoglycerate mutase with pyridoxal-5'-phosphate and NaBH4 resulted in a loss of about 80% of the enzymatic activity. Reaction with pyridoxal-5'-phosphate and enzyme inactivation showed a linear relationship up to 80% inactivation, and extrapolation of the linear portion of the activity curve revealed that complete inactivation of the enzyme is the result of the incorporation of an
average of 3 moles of pyridoxal-5'-phosphate group per mole of phosphoglycerate mutase (Fig. 6). Glycerate-2,3-P2 synthase, glycerate-2,3-P2 phosphatase and glycolate-2-P-stimulated glycerate-2,3-P2 phosphatase activities were concurrently lost with the mutase activity (Fig. 7). Treatment of the enzyme with pyridoxal-5'-phosphate in the absence of NaBH4 resulted also in the concurrent loss of the main and of the collateral enzymatic activities. All activities were completely recovered when the inactivated enzyme was either dialysed or filtered through Sephadex G-25 (Table 1). Effect of substrate and cofactor. As shown in Fig. 7, in the presence of glycerate-3-P the three enzymatic activities of rabbit muscle phosphoglycerate mutase were only reduced by 20% with
8PGS
PGM
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i
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Fig. 7. Time course of the inactivation of phosphoglycerate mutase by pyridoxal-5'-phosphate. The enzyme (6/~M) in 50raM borate buffer, pH 8.2, was incubated at 25°C with 30raM pyridoxal-5'phosphate in the absence of substrate and cofactor (O) or in the presence of 5 mM glycerate-2,3-P2( . ) or 5 mM glycerate-3-P(A). At intervals aliquots were removed and assayed for phosphoglycerate mutase (PGM), glycerate-2,3-P2 synthase (BPGS), glycerate-2,3-P., phosphatase (BPGP) and glycolate-2-Pstimulated glycerate-2,3-P2 phosphatase (BPGP*) activities as described in Materials and Methods.
480
FEL1X BERROCALand Jos~ CARRERAS Table 1. Reversible inactivation of rabbit muscle phosphoglycerate mutase by pyridoxal-5'-phosphate % of initial activity PGM BPGS BPGP BPGP* Before dialysis 20 18 24 25 After dialysis 100 100 100 100 The enzyme (6 ~M) in 50 mM borate buffer, pH 8.2, was incubated at 25C and in the dark with 30 ~ M pyridoxal-5'-phosphate. After 40 rain the enzymatic activities were determined. The incubation mixture was dialysed overnight at 4 C against 20mM Tris HCI buffer, pH 7.5, containing 1 mM 2-mercaptoethanol, and assayed again.
pyridoxal-5'-phosphate treatment. In the presence of glycerate-2,3-P2 inactivation was not prevented, although the rate of inactivation was reduced. In the presence of cofactor 40 min incubation were required to produce the same level of inactivation as that produced in 15 min incubation in the absence of glycerate-2,3-P2. Failure o f the treated phosphoglycerate mutase to form the phosphoenzyme. Similarly to the TNBSinactivated phosphoglycerate mutase, the enzyme irreversibly-inactivated with pyridoxal-5'-phosphate and NaBH4 was unable to form the active phosphoenzyme when mixed with glycerate-2,3-P2 (Fig. 4). DISCUSSION Lysyl residues have been implicated in the mechanism of yeast phosphoglycerate mutase (Sasaki et al., 1971a,b, 1976). Chemical modification experiments with trinitrobenzenesulfonate demonstrated that yeast phosphoglycerate mutase possesses four reactive amino groups (one group per enzyme subunit) located at the monophosphoglycerate banding site (Sasaki et al., 1971a). These groups are essential for the glycerate-2,3-P2 synthase and the glycerate2,3-P 2 phosphatase as well the phosphoglycerate mutase activity of the enzyme (Sasaki et al., 1971b, 1976). Chicken muscle phosphoglycerate mutase also has essential lysyl residues. Trinitrophenylation resulted in the loss of the enzymatic activity, complete inactivation occurring with the incorporation of about four moles of trinitrophenyl group per mole of enzyme. Glycerate-3-P offered some protection to TNBS-inactivation but glycerate-2,3-P2 did not. Reaction of the Schiff's base complex between pyridoxal-5'-phosphate and chicken muscle phosphoglycerate mutase gave irreversible inactivation. Inactivation was due to incorporation of 1 mole of pyridoxal-5'-phosphate per mole of enzyme dimer. Since neither large change in conformation nor dissociation of the dimer enzyme was observed, a "halfof-the-sites" type effect was assumed to occur. Glycerate-3-P protected the enzyme against both reaction and inactivation. Glycerate-2,3-P2 did not show protective effect, although reduced the rate at which inactivation occurred. Pyridoxal-5'-phosphate treatment interferred with formation of the functionally active phosphoenzyme but did not completely prevent its formation. F r o m these results it was concluded that a lysyl residue is located at or
near the active site of chicken muscle phosphoglycerate mutase, being probably involved in the binding of substrate (Came and Flynn, 1977). The experiments reported in this paper demonstrate that lysyl residues are also essential for rabbit muscle phosphoglycerate mutase. Treatment of the enzyme with both T N B S and pyridoxal-5'-phosphate and NaBH4 produces the loss of the main and of the collateral enzymatic activities. With both reagents complete inactivation occurs with modification of about 3 moles of lysine per mole of enzyme. The concurrent loss of the mutase, synthase and phosphatase activities of rabbit muscle phosphoglycerate mutase provide additional evidence to prove the intrinsic character of the three enzymatic activities and to support their location at the same active site (Berrocal and Carreras, 1983a,b). The different effectiveness of glycerate-2,3-P 2 and glycerate-3-P to protect the enzyme against both reaction and inactivation favours the existence of separate binding sites for monophosphoglycerates and for biphosphoglycerates suggested by others (Sasaki et al., 1971b, 1976) and supported by experiments of specific modification of residues of arginine (Berrocal and Carreras, 1983a), cysteine (Tauler et al., 1983) and histidine (Berrocal and Carreras, 1983b). The protective effect of glycerate-3-P suggests that in rabbit muscle phosphoglycerate mutase, like in chicken muscle phosphoglycerate mutase, the essential lysyl residues are located at or near the substrate binding site. The inability of the inactivated enzyme to form the phosphoenzyme when mixed with glycerate-2,3-P 2 suggests a close proximity between the binding sites for substrate and for cofactor. It may be concluded that the active sites of yeast, chicken muscle and rabbit muscle phosphoglycerate mutases seem to be structurally related.
Acknowledgements--This work has been supported by the Spanish Comisidn Asesora de lnvestigaci6n Cientifica y T6cnica (Grant No 3974/79). F. Berrocal is recipient of a research fellowship from the Fundacidn Pedro Pons. REFERENCES
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Mokrasch L. C. and McGilvery R. W. (1956) Purification and properties of fructose-l,6-diphosphatase. J. biol. Chem. 221, 909-917. Okuyama T. and Satake K. (1960) On the preparation and properties of 2,4,6-trinitrophenyl-amino acids and peptides. J. Biochem. 47, 454~466. Raetz C. R. H. and Auld D. S. (1974) Schiff bases of pyridoxal phosphate with active center lysines of ribonuclease A. Biochemistry 11, 2229-2236. Rippa M., Spanio L. and Pontremoli S. (1967) A specific interaction of pyridoxal-5'-phosphate and 6-phosphogluconic dehydrogenase. Archs Biochem. Biophys. 118, 48-57. Sasaki R., Sugimoto E. and Chiba H. (1971a) Studies on the active site of yeast phosphoglycerate mutase. Biochim. biophys. Acta 227, 584-594. Sasaki R., Hirose M., Sugimoto E. and Chiba H. (1971b) Studies on a role of the 2,3-diphosphoglycerate activity in the yeast phosphoglycerate mutase reaction. Biochim. biophys. Acta 227, 595-607. Sasaki R., lkura K., Sugimoto E. and Chiba H. (1975) Purification of bisphosphoglycex&te phosphatase and phosphoglyceromutase from human erythrocytes. Eur. J. Biochem. 50, 581-593. Sasaki R., Utsimi S., Sugimoto E. and Chiba H. (1976) Subunit structure and multifunctional properties of yeast phosphoglyceromutase. Eur. J. Biochem. 66, 523-533. Satake K., Okuyama T., Ohashi M. and Shinoda T. (1960) The spectrophotometric determination of amine, amino acid and peptide with 2,4,6-trinitrobenzene 1-sulfonic acid. J. Biochem. 47, 654-660. Tauler A., Bartrons R., Pons G. and Carreras J. (1983) Metabolism of glycerate-2,3-P2--IV. Effects of Hg 2+ on the enzymes involved in the metabolism of glycerate-2,3P2 in pig skeletal muscle. Comp. Biochem. Physiol. 76B, 789-793. Zwaig N. and Milstein C. (1966) The phosphorylated intermediate in the phosphoglyceromutase reaction. Biochem. J. 98, 360-368.