108
Biochimica et Biophysica Acta, 4 3 8 ( 1 9 7 6 ) 1 0 8 - - 1 1 8 © E l s e v i e r S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 67838
SECONDARY KINASE REACTIONS CATALYZED BY YEAST PYRUVATE KINASE *
D A V I D J. L E B L O N D
** a n d J A M E S L. R O B I N S O N
Division of Biochemistry, Department of Dairy Science, University of Illinois, Urbana, Ill. 61801 (U.S.A.) (Received November 24th, 1975)
Summary 1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATPdependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. These reactions are accelerated by fructose-l,6-bisphosphate, the aUosteric activator of the primary reaction. With Mg2÷ as the required divalent cation, none of these reactions are observed in the absence of fructose-bisphosphate. With Mn 2÷, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. The effect of other divalent cations and pH on the three secondary kinase reactions was also examined. 4. Results are compared with those obtained for muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed.
Introduction The primary, physiologically important reaction catalyzed by rabbit muscle pyruvate kinase (EC 2.7.1.40) involves phosphoryl transfer from phosphoenolpyruvate to ADP, forming ATP and enolpyruvate, and proton transfer from water to enolpyruvate forming ketopyruvate. The phosphoryl and proton * F r o m thesis b y D.J. L e B l o n d in partial fulfillment o f the r e q u i r e m e n t s for the Master of S c i e n c e d e g r e e f r o m the University o f Illinois. ** Current address: D e p a r t m e n t o f B i o c h e m i s t r y , Michigan S t a t e University, East Lansing, MI 4 8 8 2 3 U.S.A. A b b r e v i a t i o n s u s e d are: MES, 2(N-morpholino)ethane sulfonate; TES, N-tris(hydroxymethyl)methyl-2-aminoethane sulfonate ; TAPS, t r i s ( h y d r o x y m e t h y l ) m e t h y l a m l n o p r o p a n e s u l f o n a t e .
109
transfers are likely to be sequential during the course of the primary reaction [ 1 ]. Proton transfer can proceed in the absence of phophoryl transfer, as in the enolization of pyruvate activated by Pi-like dianions [2]. Furthermore, phosphoryl transfer can be separate and independent of p r o t o n transfer, as in three secondary kinase reactions: the fluorokinase reaction, in which ATP phosphorylates fluoride ion [3] ; the hydroxylamine kinase reaction, whereby ATP phosphorylates the oxygen of hydroxylamine [4]; and the glycolate kinase reaction, in which the hydroxyl of glycolate is phosphorylated by ATP [5]. Each of these reactions requires, as does the primary reaction, a monovalent and divalent cation activator (e.g. K ÷ or NH4 ÷ and Mg ~÷, Mn 2+, or Co 2÷ respectively). In addition, the fluorokinase and hydroxylamine kinase reactions require bicarbonate as an effector. Yeast pyruvate kinase, unlike the rabbit muscle enzyme, it susceptible to allosteric activation b y Fru-l,6-P: [6,7]. As such, it represents a class of pyruvate kinases subject to feedforward regulation, a class which includes the predominant liver enzyme in mammals [8,9], the enzyme from many tissues in lower animals [10--13] and from some bacteria [14,15]. The present communication reports the existance of the three secondary kinase reactions catalyzed b y yeast pyruvate kinase, their requirements for Fru-l,6-P: and other characteristics. An accompanying paper [16] deals with the proton transfer reactions catalyzed by yeast pyruvate kinase. Experimental procedure The preparation of yeast pyruvate kinase was based on the procedure of RSschlau and Hess [ 1 7 ] . The procedure was modified in the following ways: Red Star Baker's Yeast (obtained fresh locally) was used as starting material, the extract was fractionated between 45 and 50% saturated ammonium sulfate, rather than 40--50%, valine (1 mM) was included in all chromatography buffers (suggested b y the effects of valine in renaturing the yeast enzyme [18] ), chromatography on DEAE-cellulose was at pH 7.0 instead of 6.5, and the G100 Sephadex chromatography was replaced by G-200. The enzyme was not crystallized b u t was homogeneous on polyacrylamide gel electrophoresis in presence and absence of sodium dodecyl sulfate and on cellulose acetate sheets. The following activities were not present at more than 0.01% that of pyruvate kinase, aldolase, fructose diphosphatase, hexokinase, adenosine triphosphatase, adenylate kinase, phosphoenolpyruvate phosphatase, pyruvate decarboxylase, adenosine diphosphatase, and lactate dehydrogenase. A specific activity of 382 pmol ATP synthesized/min per mg protein was obtained, which approaches that recently reported [19,20]. The kinetic parameters of the forward reaction agree quantitatively with those reported b y Haeckel et al. [21] and Hunsley and Suelter [7]. The extinction coefficient at 280 nm used for the purified enzyme was that of the latter investigators. The enzyme was stable for over six months as a precipitate in 80% saturated ammonium sulfate at 5°C and enzyme so stored was used within six months of preparation. The enzyme was unstable upon dilution in buffer, with valine (lmM), bovine serum albumin (1 mg/ml), and glycerol (50%) providing increasing protection against inactivation. The diluent used routinely contained 50%
110 glycerol, 0.1 mM EDTA, and 10 mM MES (pH 6.2). The enzyme was stable for at least a week at 5°C when stored under these conditions. All assays were performed in the presence of 1 mg/ml bovine serum albumin to ensure stability of the enzyme during the course of the assays. Assays of the primary reaction were performed at 26°C using spectrophotometric coupling to lactate dehydrogenase. The [~/_32p]ATP was prepared according to the method of Glynn and ChappeU [22] and stored in aliquots at --22°C until needed. The preparation was spectrally pure and less than 2% of the label was in the fl-position, assessed by reaction with hexokinase and glucose, followed by polyethyleneimine-cellulose chromatography. All secondary kinase reactions were measured at 30°C as 32p transferred from [7.32p] ATP to [32p].non.nucleotide (not charcoal adsorbable), as described by Switzer [23]. Experimental values were corrected for controls omitting either fluoride, hydroxylamine, glycolate or enzyme, as no differences were observed between any of these controls. For each secondary reaction, the assay was shown to be linear with time and enzyme concentration in the region used. Between 50 000 and 100 000 cpm of [7.32p] ATP were used in each assay. The time intervals used routinely were 15 to 30 min. All other components were the best grade available commercially and were used w i t h o u t further purification. Results
Yeast pyruvate kinase was tested for its ability to catalyze the fluoro-, hydroxylamine, and glycolate kinase reactions in the presence of either Mg 2÷ or Mn 2÷. The results of such a study (Table I) indicate that yeast pyruvate kinase catalyzes all reactions in the presence of Fru-l,6-P2, b u t no reaction is observed in the absence of Fru-l,6-P2 for any of the Mg2÷-dependent reactions or for Mn2+-dependent glycolate kinase. In all other Mn2+-dependent reactions, Fru-1,
TABLE
I
VELOCITY
OF YEAST
PYRUVATE
KINASE
SECONDARY
KINASE
REACTIONS
Effects of fructose-bisphosphate and M g 2+ or M n 2+. btrnol A T P c o n s u m e d / m i n p e r m g Fru-l,6-P 2 Finorokinase a
Hydroxylamine kinase a
+ -+ --
Glycolate
kinase b
+ --
Mg 2+
Mn 2+
0.277
Biochimica et Biophysica Acta, 4 3 8 ( 1 9 7 6 ) 1 0 8 - - 1 1 8 © E l s e v i e r S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 67838
SECONDARY KINASE REACTIONS CATALYZED BY YEAST PYRUVATE KINASE *
D A V I D J. L E B L O N D
** a n d J A M E S L. R O B I N S O N
Division of Biochemistry, Department of Dairy Science, University of Illinois, Urbana, Ill. 61801 (U.S.A.) (Received November 24th, 1975)
Summary 1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATPdependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. These reactions are accelerated by fructose-l,6-bisphosphate, the aUosteric activator of the primary reaction. With Mg2÷ as the required divalent cation, none of these reactions are observed in the absence of fructose-bisphosphate. With Mn 2÷, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. The effect of other divalent cations and pH on the three secondary kinase reactions was also examined. 4. Results are compared with those obtained for muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed.
Introduction The primary, physiologically important reaction catalyzed by rabbit muscle pyruvate kinase (EC 2.7.1.40) involves phosphoryl transfer from phosphoenolpyruvate to ADP, forming ATP and enolpyruvate, and proton transfer from water to enolpyruvate forming ketopyruvate. The phosphoryl and proton * F r o m thesis b y D.J. L e B l o n d in partial fulfillment o f the r e q u i r e m e n t s for the Master of S c i e n c e d e g r e e f r o m the University o f Illinois. ** Current address: D e p a r t m e n t o f B i o c h e m i s t r y , Michigan S t a t e University, East Lansing, MI 4 8 8 2 3 U.S.A. A b b r e v i a t i o n s u s e d are: MES, 2(N-morpholino)ethane sulfonate; TES, N-tris(hydroxymethyl)methyl-2-aminoethane sulfonate ; TAPS, t r i s ( h y d r o x y m e t h y l ) m e t h y l a m l n o p r o p a n e s u l f o n a t e .
109
transfers are likely to be sequential during the course of the primary reaction [ 1 ]. Proton transfer can proceed in the absence of phophoryl transfer, as in the enolization of pyruvate activated by Pi-like dianions [2]. Furthermore, phosphoryl transfer can be separate and independent of p r o t o n transfer, as in three secondary kinase reactions: the fluorokinase reaction, in which ATP phosphorylates fluoride ion [3] ; the hydroxylamine kinase reaction, whereby ATP phosphorylates the oxygen of hydroxylamine [4]; and the glycolate kinase reaction, in which the hydroxyl of glycolate is phosphorylated by ATP [5]. Each of these reactions requires, as does the primary reaction, a monovalent and divalent cation activator (e.g. K ÷ or NH4 ÷ and Mg ~÷, Mn 2+, or Co 2÷ respectively). In addition, the fluorokinase and hydroxylamine kinase reactions require bicarbonate as an effector. Yeast pyruvate kinase, unlike the rabbit muscle enzyme, it susceptible to allosteric activation b y Fru-l,6-P: [6,7]. As such, it represents a class of pyruvate kinases subject to feedforward regulation, a class which includes the predominant liver enzyme in mammals [8,9], the enzyme from many tissues in lower animals [10--13] and from some bacteria [14,15]. The present communication reports the existance of the three secondary kinase reactions catalyzed b y yeast pyruvate kinase, their requirements for Fru-l,6-P: and other characteristics. An accompanying paper [16] deals with the proton transfer reactions catalyzed by yeast pyruvate kinase. Experimental procedure The preparation of yeast pyruvate kinase was based on the procedure of RSschlau and Hess [ 1 7 ] . The procedure was modified in the following ways: Red Star Baker's Yeast (obtained fresh locally) was used as starting material, the extract was fractionated between 45 and 50% saturated ammonium sulfate, rather than 40--50%, valine (1 mM) was included in all chromatography buffers (suggested b y the effects of valine in renaturing the yeast enzyme [18] ), chromatography on DEAE-cellulose was at pH 7.0 instead of 6.5, and the G100 Sephadex chromatography was replaced by G-200. The enzyme was not crystallized b u t was homogeneous on polyacrylamide gel electrophoresis in presence and absence of sodium dodecyl sulfate and on cellulose acetate sheets. The following activities were not present at more than 0.01% that of pyruvate kinase, aldolase, fructose diphosphatase, hexokinase, adenosine triphosphatase, adenylate kinase, phosphoenolpyruvate phosphatase, pyruvate decarboxylase, adenosine diphosphatase, and lactate dehydrogenase. A specific activity of 382 pmol ATP synthesized/min per mg protein was obtained, which approaches that recently reported [19,20]. The kinetic parameters of the forward reaction agree quantitatively with those reported b y Haeckel et al. [21] and Hunsley and Suelter [7]. The extinction coefficient at 280 nm used for the purified enzyme was that of the latter investigators. The enzyme was stable for over six months as a precipitate in 80% saturated ammonium sulfate at 5°C and enzyme so stored was used within six months of preparation. The enzyme was unstable upon dilution in buffer, with valine (lmM), bovine serum albumin (1 mg/ml), and glycerol (50%) providing increasing protection against inactivation. The diluent used routinely contained 50%
110 glycerol, 0.1 mM EDTA, and 10 mM MES (pH 6.2). The enzyme was stable for at least a week at 5°C when stored under these conditions. All assays were performed in the presence of 1 mg/ml bovine serum albumin to ensure stability of the enzyme during the course of the assays. Assays of the primary reaction were performed at 26°C using spectrophotometric coupling to lactate dehydrogenase. The [~/_32p]ATP was prepared according to the method of Glynn and ChappeU [22] and stored in aliquots at --22°C until needed. The preparation was spectrally pure and less than 2% of the label was in the fl-position, assessed by reaction with hexokinase and glucose, followed by polyethyleneimine-cellulose chromatography. All secondary kinase reactions were measured at 30°C as 32p transferred from [7.32p] ATP to [32p].non.nucleotide (not charcoal adsorbable), as described by Switzer [23]. Experimental values were corrected for controls omitting either fluoride, hydroxylamine, glycolate or enzyme, as no differences were observed between any of these controls. For each secondary reaction, the assay was shown to be linear with time and enzyme concentration in the region used. Between 50 000 and 100 000 cpm of [7.32p] ATP were used in each assay. The time intervals used routinely were 15 to 30 min. All other components were the best grade available commercially and were used w i t h o u t further purification. Results
Yeast pyruvate kinase was tested for its ability to catalyze the fluoro-, hydroxylamine, and glycolate kinase reactions in the presence of either Mg 2÷ or Mn 2÷. The results of such a study (Table I) indicate that yeast pyruvate kinase catalyzes all reactions in the presence of Fru-l,6-P2, b u t no reaction is observed in the absence of Fru-l,6-P2 for any of the Mg2÷-dependent reactions or for Mn2+-dependent glycolate kinase. In all other Mn2+-dependent reactions, Fru-1,
TABLE
I
VELOCITY
OF YEAST
PYRUVATE
KINASE
SECONDARY
KINASE
REACTIONS
Effects of fructose-bisphosphate and M g 2+ or M n 2+. btrnol A T P c o n s u m e d / m i n p e r m g Fru-l,6-P 2 Finorokinase a
Hydroxylamine kinase a
+ -+ --
Glycolate
kinase b
+ --
Mg 2+
Mn 2+
0.277
