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Neurospora crassa glycogen phosphorylase: Interconversion and kinetic properties of the “active” form
.4RCHIVES
OF
BIOCHEMISTRY
Neurospora
AND
crassa
BIOPHYSICS
Glycogen
Kinetic MICHAEL
Department
Properties
H. GOLD,2
of Biochemistry
ROBERT IRWIN and
161, 515-527
Phosphorylase: of the
University October
interconversion
“Active”
J. FARR.AND, H. SEGEL4
Biophysics, Received
(1974)
and
Form’ JOHN
of California,
P. LIVON13
Davis,
California
AND
96616
26, 1973
Glycogen phosphorylase in cell-free extracts of Xeurospora crassa is activated loto 15-fold by incubation with MgATP2-. When the MgATP” is removed, the active form (a form) reverts to the inactive form (b form). The inactivation requires Mg2+ and is inhibited by NaF. The results confirm that Neurospora crassa glycogen phosphorylase exists in two interconvertible forms and strongly suggests that the interconversion is catalyzed by a kinase and phosphatase. The a form was partially purified. The enzyme has a molecular weight of 320,000. Uridine diphosphate glucose is a linear competitive inhibitor with respect to glucose-l-phosphate and a linear noncompetitive inhibitor with respect to glycogen. Glucose-6-phosphate is a hyperbolic (partial) noncompetitive inhibitor with respect to all substrates in both directions. The b form of the enzyme in crude cell-free extracts is stimulated 2- to a-fold by 5’AMP. As the b form is purified, the 5’-AMP activation is diminished. The molecular weight of the partially purified “b” form was also 320,000.
Glycogen phosphorylase (a-1,4-glucan: orthophosphate glucosyltransferase, EC 2.4.1.1) has been isolated from a wide variety of organisms (1, 2). The Neurospora crassa enzyme was first studied by Shepherd and Segel (3, 4) and by Tellez-Inon and Torres (5). The latter workers demonstrated that the enzyme exists in two interconvertible forms called a (active) and b (inactive). In this paper, the interconversion of phosphorylase a and b in cell-free extract’s is confirmed, and some physical and kinetic properties of the partially purified a form are reported. In addition, the kinetic consequences of a “two-mode” model for glycogen binding are discussed.
MATERIALS
5 Abbreviations; Pi = G-l-P = glucose-l-phosphate; g-phosphate. 515
@ 1974 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Culture conditions. All experiments described in this paper were carried out with a wild type strain of N. crassa (CM+). The organism was grown aerobically on the synthetic medium previously described (3). The cells were grown in 2-liter Erlenmeyer flasks containing 1 liter of medium on a New Brunswick shaker (New Brunswick Scientific Co., Inc.) operating at a speed of 250 rpm and describing a 2-in. circle. These cultures were maintained by transferring some of mycelial suspension to fresh medium every 2 days. Cells which had been allowed to grow for 48 hr were used for extraction of enzyme. Protein determination. Protein was determined either by the biuret procedure (6), or, in later stages of purification, by its absorbance at 280 nm. Enzyme assays. Glycogen synthesis direction: Phosphorylase activity was determined by following the rate of incorporation of [r%]glucose from [%]G-l-P6 into glycogen. Activity was meas-
1 The research described in this paper was supported by grant AI-07560 from the United States Public Health Service. 2 Present address: Laboratory of Biochemical Genetics, The Rockefeller University, New York, N. Y. 10021. 0 Undergraduate Honors Research Student. 4 Addressee for reprints. Copyright All rights
AND
inorganic G-6-P
phosphate; = glucose-
GOLD
MINUTES
of Neurospora glycogen FIG. 1. Activation phosphorylase b. The crude extract was incubated at 15°C with 10 mM ATP and 20 mM MgCl:. Periodically, aliquots were removed and assayed in the synthesis direction in the absence of 5’AMP.
ured in 0.1 ml of reaction mixture containing 50 mM Tris-Cl (pH 7.2), 2.0-20 mg/ml glycogen, 0.5-5 mM [l%]G-1-P (10”lo6 cpm/amole), 10 mM EDTA, potential inhibitors and activators as described below, and enzyme. The reaction mixtures were incubated at 30°C for 10 min. Reactions were terminated by adding 2 ml of 75% ethanol containing 1% KCl. The precipitated glycogen was centrifuged in a clinical centrifuge for 2 min. After the supernatant was decanted, 0.2 ml of Hz0 was added to dissolve the precipitate. The glycogen was precipitated two more times and finally redissolved in 0.5 ml of water and counted in 5 ml of scintillation fluid. The scintillation fluid contained 6 g of 2,5-diphenyloxazole and 100 g of naphthalene per liter of dioxane. Glycogen degradation direction: The assay was essentially the same as that described by Shepherd et al. (4). Activity was measured in 0.1 ml of reaction mixture containing the same reactants described above except that 2-20 mM “Pi replaced the [‘“ClG-1-P. The reaction mixtures were incubated at 30°C for 10 min. The [32P]G-1-P formed was measured after precipitating the “Pi with cadmium acetate. Enzyme extraction. The mycelium from 1 liter was harvested by suction filtration, washed sev-
ET
AL.
eral times with deionized water, and weighed. The pad was mixed with glass beads (1 mm, Braun) equal to twice the weight of mycelium and a volume of 50 mM Tris-Cl buffer (pH 7.5) equal to twice the weight of mycelium. The mixture was homogenized for 2 min in a Bronwill homogenizer (Bronwill Scientific). The homogenate was centrifuged at 20,000 g for 15 min at 4’C (crude extract). Purifcation of phosphorylase a. Phosphorylase in the crude cell-free extract was first fully activated by incubation with 10 mM ATP and 20 M MgCl: for 30 min at 15°C (Fig. 1). Ammonium sulfate fractionation: Solid ammonium sulfate was added to bring the crude extract to 400% saturation. The solution was stirred for 30 min and centrifuged at 20,000 g for 15 min. The supernatant was brought to 65% saturation, maintaining the pH above 7.0, and centrifuged as before. The precipitate was then taken up in 10 mxu succinate buffer, pH 5.8, containing 1 mM EDTA (buffer A), and dialyzed against several changes of the same buffer for 16 hr. DE-62 chromatography: The enzyme was adsorbed to a column (2.0 X 20 cm) of DE-52 previously equilibrated with buffer A. Aft,er adsorption of the enzyme, the column was washed with 100 ml of the equilibration buffer. Finally, the enzyme was eluted at pH 5.8 with a linear gradient consisting of buffer A in the mixing chamber and buffer A + 0.5 M NaCl in the reservoir. The total volume of the gradient was 400 ml. Four-milliliter fractions were collected and assayed. The enzyme activity peak was pooled and dialyzed against several changes of buffer A for 16 hr. DEAE Sephadex A-60 chromatography: The enzyme was adsorbed to a column (1.0 X 30 cm) of A-50 previously equilibrated with buffer A. After adsorption of the enzyme, the column was washed with 50 ml of buffer A. The enzyme was eluted with the same gradient as used in the DE-52 chromatography. Four-milliliter fractions were collected and assayed. The enzyme activity peak was pooled and concentrated to 5 ml by ultrafiltration through a Diaflo membrane (PMlO, Amicon Corporation). Sephadex G-300 gel jiltration: The concentrate was applied to a 2.5 X 37 cm column of Sephadex G-200 previously equilibrated with 25 mM succinate + 100 mM NaCl, pH 6.0. Four-milliliter fractions were assayed for protein and activity. The fractions containing the enzyme were pooled, dialyzed against 50 mM Tris buffer (pH 7.2), and stored at -20°C. Sucrose gradient centrifugation. Sucrose density gradient, studies were conducted by the method of Martin and Ames (7) using a Beckman SW40 rotorat35,0OOrpmfor16hrat5”C.Linearsucrose gradients from 6 to 30% sucrose in 25 mM succinate
Neurospora
GLYCOGEN
517
PHOSPHORYLASE
rapid and could be followed conveniently only by lowering the incubation temperature to 15”. Figure 2 shows t’he inactivation of phosphorylase a in the absence of ATP (which was removed by ammonium sulfate precipitation and gel filtration). The results are similar to those reported by Tellez-Inon and Torrcs (5). There seemsno doubt that N. cmssa glycogen phosphorylase exists in two forms, and that their interconversion involves a phosphorylase b kinase and a phosphorylase a phosphatase. Purif?,ation
5
IO
15
20
25
30
MINUTES
FIG. 2. Inactivation of Neurospora glycogen phosphorylase a. The enzyme was first activated as described in Fig. 1. Solid ammonium sulfate (Mann, enzyme grade) was then added to the extract to 65yo saturat’ion. The solution was stirred for 30 min and centrifuged at 20,000 g for 15 min. The precipitate was then taken up in 50 mM TrisCl, pH 7.2, and a 0.5-ml aliquot was filtered on a 1.0 X 25 cm column of Sephadex G-25 equilibrated in 50 mM Tris-Cl, pH 7.2. The fractions containing phosphorylase activity (breakthrough volume) were pooled. Aliquots were incubated at 30°C in 50 mM Tris-Cl, pH 7.2, containing 5 mM MgCls with the following additions: none (O ) ; 50 mM NaF (0); 15 mM EDTA (0); 5 mM 5’-AMP (II). Periodically enzyme activity was measured in the synthesis direction in the absence of 5’AMP. The arrows show the preactivation activity of ATeurospora phosphorylase b in the presence and absence of 5’-AMP. + I mM EDTA, pH 6.0, were used. Fractions of 0.3 ml were collected. p-Galactosidase and skeletal muscle phosphorylase b (assayed in the presence of 5’-AMP) were used as markers. RESULTS
Interconversion
of Neurospora b and a
Phosphorylase
Figure 1 shows the activation of Neurospora glycogen phosphorylase b in the pres-
enee of ATP and Mg*+. The activation is
of Phosphorylase
a
It seemed likely that the Neurospora glycogcn “phosphorylase” studied by Shepherd et al. (3, 4) was a mixture of a and b forms, since no preactivation or inactivation step was included before purification. Consequent’ly, in t,he present study, we concentrated on the a form. Figure 3 shows t’he elution profile of phosphorylase a from Whatman DE-52. The enzyme elutes at 0.22 M XaCl as a single activity peak. The activity of each fraction did not change when 5 mM 5’-AMP was added to the reaction mixture. The enzyme also eluted as a single peak from DEAF Sephadex A-50. Figure 4 shows a gel filtration profile. The enzyme elutes as a single symmet’rical peak. The molecular weight of the enzyme as determined by the method of Andrews (8) was calculated to be 320,000 (Fig. 4, insert). The specific activities at various stages of the preparation are described in Table I. After the gel filtration step, the enzyme was purified approximately 124-fold relative to the specific activity of the activated crude extract. Sucrose density gradient centrifugation was used to study the sedimentation behavior of the enzyme and as an independent check on the number of forms in our preparation. Figure 5 illustrates the activity profile for Neurospora phosphorylase a, skeletal muscle phosphorylase b (8.4S), and the A2Bonm profile for fi-galactosidase (16.0 S). From this figure the sedimentation coefficient of Neurospora phosphorylase a was
518
GOLD
FRACTION
ET
AL.
NUMBER
FIG. 3. Chromatography
of Neurospora phosphorylase a on DE-52. The enzyme was applied to a 2.0 X 20 cm column of Whatman DE-52 equilibrated with 10 mM succinate buffer, pH 5.8, containing 1 mM EDTA. The column was washed with 100 ml of the same buffer. The enzyme was eluted at pH 5.8 with a linear gradient consisting of 200 ml of the above buffer in the mixing chamber and 200 ml of the same buffer containing 100 mM NaCl in the reservoir. Absorbancy at 280 nm (0) and phosphorylase activity (0) are shown. I
PHOSPHORYLASE a
PHOSPHORYLASE b
I f f 2 Y 8 .G E 5 i= Y
IO
50 FRA&ON
&FIG.
l&BE~O
4. Sephadex G-200 gel filtration of Neurospora phosphorylase a. A 2.5 X 37 cm column of Sephadex G-200 equilibrated with 25 mM succinate mM NaCl. Absorbancy at 280 nm (0) and phosphorylase activity (0) are weight versus VJVO using ferritin, muscle phosphorylase b, hemoglobin,
5-ml sample was applied to a buffer, pH 6.0, containing 100 shown. Insert: log molecular and myoglobin as standards.
determined to be 11.6 S, which corresponds equilibrium random kinetic mechanism to an approximate molecular weight of (9-11). The data reported in this paper are consistent with a random mechanism al325,000. though an ordered sequence in which Pi or Initial Velocity Studies G-1-P adds first cannot be excluded. The All polyglucose phosphorylases that have binding sequencecan be visualized as shown been investigated appear to have a rapid below:
Areurospora
GLYCOGEN
K, = K,,
Kc, = K,,
E+Ar EA + + B (phosphorolysis) B K,
= K:,
11
aK,
= K,, ‘
EB + A ’
II
EQ + B
aK,,
_ EAB
’ h,
= Kk,, k,
where A = I!‘<, B = glycogcn, and Q = G-1-P. a and b represent’ the interaction factors describing the cffcct’ of the binding of one ligand on the dissociation constant for the other. The above scheme assumes that there is only one mode of glycogen binding (which may not be true). Figure 6A shows the family of l/v versus l/[G-l-P] plots obtained at different’ fixed glycogen concentrations. The plots intersect on the horizontal axis. The insert shows a replot of the l/v-axis intercepts versus l/[glycogen]. The horizontal-axis intercept gives bKu or K,,. The replot yields a K, for glycogcn of 3.2 mg/ml. Since the family of plots intersect on the horizontal axis, the slope rcplot (which gives KB = Kib) yields essentially the samevalue. Thus, for the assumedmodel, the binding of glycogen has no effect on the binding of G-1-P and vice versa (i.e., b = 1). Figure 6B shows the data plotted as l/v versus l/[glycogen] at different fixed G-l-P
PURIFICATION
Fraction
1. Crude
con-
Q+E
L
+
(synthesis) bK,,
B
= Km, bK,t = Km,
EQB ’
Ka = Ktb
it
r Q + EB
concentrations. The insert showsthe l/v-axis intercept replot, which yields bKQ = K,,. The K, for G-1-P is 1.4 nw. A slope replot gives K, = K, = 1.3 mM. Figures 7A and B show the kinetics of the phosphorolysis reaction. The families of plots intersect beion- the horizont,al axis. The l/v-axis intercept replots yield uK~ = K,, (i.e., K, for glycogen) = 1.33 mg/ml, aK, = K, (i.e., K, for Pi) = 7.4 mM. The slope replots yield Kg = Kib = 0.66 mg/ml and K, = Ki, = 4.2 mM. Thus, for the phosphorolysis direction, the binding of one ligand does affect the binding of the other. The interaction factor, a, is 1.8-2.0.
Inhibition
by UDPG
Figure 8A shows that UDPG is a linear competitive inhibitor with respect to G-l-P. The slope replot (insert) yields a Kx’,zpe of 3.2 mM at 20 mg/ml glycogen. Uridine diphosphate glucose is a linear noncompetiI
oli PHOSPHORYLASE Total protein bd*
extract
2. Activated crude extract 3. (NH&S04 (4@65’%) precipitation 4. DE-52 chromatography and Diaflo centration 5. DEAE-Sephadex A-50 chromatography 6. G-200 gel filtration
.
II
TABLE PARTIAL
519
PHOSPHOItYLASE
a FROM
Volume (ml)
Ar. crassaa
Specific activity &moles min-’ m g-l) c
1550
50
0.014
1550 455 40
51 26 20
0.11 0.263 2.19
10.3 4.3
a For details see text. * Protein was determined with biuret reagent in fractions at 280 = 10.0) was used for fractions 4, 5, and 6. c Standard assay conditions including 20 mg/ml glycogen;
23 20
l-3;
6.33 13.7
absorbance
5 mM G-1-P.
Purification factor
Yield (%I
-
-
1.0 2.39 19.9
100 70 51
57.5 124.5
at 280 nm
38 34
(assuming
e:&
520
ET AL.
GOLD
a a5
LO [G-I-P]-’
1.5
2.0
hM)-’
FRACTION NUMBER
FIG. 5. Sucrose density gradient centrifugation of Neurospora phosphorylase a. Centrifugation was done at 35,000 rpm for 16 hr at 5°C in a Beckman SW-40 rotor. The gradient contained 6-30yo sucrose in 25 mrw succinate buffer, pH 7.0, containing 1 InM EDTA. Absorbancy at 280 nm (A) and phosphorylase act.ivity ( l , 0) are shown.
tive inhibitor with respect to glycogen (Fig. SB). The replot gives IX;: = K$zpe = 9.5 mM at 5 mM G-l-P. The Kis are given by: K:;ype
= cKu
(1 +
KBABI
>/ (1 +
CKB/[BI
>,
[&l/&J, zsl.,,,e = Ku0 + [Ql/Kd,
KY,:
= cKu
(1 +
b
KY/B
where KU represents the dissociation constant for the enzyme-UDPG complex and cKU represents the dissociation constant for UDPG from the enzyme-UDPG-glycogen complex. Thus, c is an interaction factor analogous to a and b. The symbol KE{,‘p, means “the Ki calculated from the slope replot of l/v versus l/[Q] plots with U = UDPG as an inhibitor.” Similarly, KxE means “the Ki calculated from the l/u-axis intercept replot of l/v versus l/[B] plots with U as an inhibitor.” All Kis are apparent values depending on the concentration of the nonvaried substrate. The fact that KxE =
0
0.1
0.3
0.2 [GLYCOGEN]-1
0.4
(MG/ML)-I
FIG. 6. (a) l/v versus l/[G-l-P] at several fixed concentrations of glycogen. The glycogen concentrations were (mg/ml) : 20 (0); 7.0 (u); 4.0 (0); 2.5 (A). Insert: replot of the l/o-axis intercepts versus l/[glycogen]. (b) l/v versus l/[glycogen] at several fixed concentrations of G-l-P. The G-1-P concentrations used were (millimolar): 5.0 (0); 2.0 (C); 1.0 (0); 0.66 (A). Insert: replot of the l/v-axis intercepts versus l/[G-l-P]. G/IfptT
Kt{zpe
KXL
suggests that c = a = 1. Kxz or yields a value for Ku of 2.1 mM. yields a K, of 3.2 mM. Uridine
Neurospora
GLYCOGEN
521
PHOSPHORYLASE
[GLVCGGEN]
-’ IMG/MLi
-’
FIG. 7. (a) l/v versus l/[phosphate] at several fixed concentrations of glycogen. The glycogen concentrations were (mg/ml) : 5.0 (a); 2.0 (m); 1.0 (0) ; 0.66 (A); 0.5 (0). Insert: replot of the l/v-axis interl/[glycogen] at several fixed concentrations of phosphate. The cepts versus l/[glycogen]. (b) 1/ ZJversus phosphate concentrations were (millimolar): 20 (a); 6.67 (m); 4.0 (0); 2.65 (A); 2.0 (Cl).
diphosphate glucose has been shown to inhibit glycogen phosphorylase from a number of sources (1%16), akhough Fosset et al. (17) reported that the yeast enzyme was not affected. Inhibition
by Glucose-6-phosphate
Glucose-6-phosphate was a noncompetitive inhibitor with respect to G-1-P (Fig.
9A). The family of plots intersect on the horizont’al axis showing no effect of G-6-P on G-l-P binding. The l/v-axis intercept replot was hyperbolic, indicating partial inhibition. A secondary replot of l/A intercept versus l/[G-6-P] was linear. The horizontal-axis intercept of the secondary replot, gives -f/Ki, where fV,,, is the maximal velocity at saturating G-6-P and tbe
GOLD
1.0 [G-l-P]-'
1.5
ET AL.
20
(&I)-'
FIG. 8. Inhibition by UDPG. (a) Linear competitive inhibition with G-l-P as the varied substrate and glycogen held constant at 20 mg/ml. The UDPG concentrations were (millimolar): 0.00 (a); 1.0 (m); 3.0 (0); 6.0 (A). Insert: replot of the slopes against the concentration of UDPG. (b) Linear noncompetitive inhibition with glycogen as the varied substrate and G-l-P held constant at 5 mM. The UDPG concentrations were (millimolar) 0.00 (a); 2.0 (I); 4.0 (0); 6.0 (A). Insert: replot of the l/vaxis intercepts against the concentration of UDPG.
constant glycogen concentration. The vertical axis intercept of the secondary replot gives fV,,,/ (1 - f). The replot yields f = 0.47 and K; = 2.1 mM. Figure 9B showsthat G-6-P is also a partial noncompetitive inhibitor with respect to glycogen. The replot yields f = 0.42 and Ki = 1.6 mM. In the phosphorolysis direction (Figs. 10A and B) G-6-P behaved in the same partial manner (f = 0.42-0.52, Ki = 1.6-4.0 mM>. The results indicate that Neurospora glycogen phosphorylase possessesa specific, modifier site for G-6-P that is distinct from the G-l-P and glycogen sites. Partial inhibition by G-6-P was also noted by Wang et al., with muscle glycogen phosphorylase b (18). The inhibition was competitive with respect to 5’-AMP and was ascribed to an allosteric effect at a site distinct from the nucleotide binding site. Yeast glycogen phosphorylase
is also noncompetitively inhibited by G-6-P (17). In this case, G-6-P was reported to be a linear inhibitor with respect to G-l-P and a parabolic inhibitor with respect to glycogen. The kinetic constants of the Neurospora glycogen phosphorylase a are summarized in Table II. The calculated K,, for the reaction in the synthesis direction is about 2, in good agreement with the calculated value for muscle glycogen phosphorylase a (11). Neurospora Glycogen Phosphorylase b Although the activity of the b form was quite low in cell-free extracts, an attempt was made to purify it using the same methods described above (ammonium sulfate precipitation, DE-52 chromatography, and gel filtration through Sephadex G-200). The enzyme eluted from Sephadex G-200 at exactly the same position as the a form
2.0 -
4
FIG. 9. Partial noncompetitive varied substrate. Glycogen was 0.00 (0); 2.0 @I); 5.0 (0); 10 concentration of G-6-P. Insert petitive inhibition with glycogen concentrations were (millimolar) l/v-axis intercepts against the l/[G-6-P].
/
inhibition held constant (A); 20 (D). (2): replot of as the varied : 0.00 (0); concentration
[G-W
,,,,I&
by G-6-P in the synthesis direction. (a) G-1-P was the at 40 mg/ml. The G-6-P concentrations were (millimolar) : Insert (1): replot of the l/v-axis intercepts against the the l/A intercept against l/[G-6-P]. (b) Partial noncomsubstrate and G-1-P held constant at 5 mM. The G-6-P 2.0 (m); 5.0 (0); 10 (A); 20 (0). Insert (1) : replot of the of G-6-P. Insert (2): replob of the I/A intercept against 523
524
ET AL.
GOLD
0.2 [PO,, =] -'
0
b
/
a5
I.0 [GLYCOGEN].1
FIG. 10. Partial noncompetitive inhibition in the was the varied substrate with glycogen held constant molar): 0.00 (0); 2.0 (m); 5.0 (0); 10 (A); 20 (0). the concentration of G-6-P. Insert (2): replot of the the varied substrate, and phosphate was held constant molar): 0.00 (a); 2.0 (m); 5.0 (0); 10 (A); 20 (0). the concentration of G-6-P. Insert (2) : replot of the
I.5
(mM) :"
1
2.0
(MC/WI
phosphorolysis direction by G-6-P. (a) Phosphate at 40 mg/ml. The G-6-P concentrations were (milliInsert (1): replot of the l/v-axis intercepts against l/A intercept against l/[G-G-P]. (b) Glycogen was at 20 mM. The G-G-P concentrations were (milliInsert (1): replot of the l/v-axis intercepts against l/A intercept against l/[G-6-P].
Xeurospora TABLE EXPERIMENTAL spora
KINETIC GLYCOGE~V
Synthesis K ng Kib K 7nQ Ki, Ir Ku Ki
= = = = =
Phosphorolysis K;, I &b Ii,, Ki, V
3.2-5.7 mg/ml 3.2-5.7 mg/ml 1.3-2.0 rnM 1.3-2.0 mnt 3.2 nmoles/minb
direction
1.3-2.1 mg/ml 0.66-0.92 mg/ml 6.0-7.8 mM 2.8-4.2 mM 2.0 nmoles/min*
Zii of G-G-P = 1.M.O rnM f = 0.42-0.52
N_ 2~
Q = G-I-P, aA = Pi, B = glycogen, UDPG. The values shown represent the observed for all the experiments reported paper. 6 V values for the same preparation. V,,,
CK “'I =
=V
X
K,
Tie,.,
x
Kia
phua
X K,, x
Kim,
x
Kn,
x
Kia
x
pim x
K,
X Km,
V,,., =V
U = ranges in this
X KmA X K:b
Tiphc,a
x
Kzb
Kin,
(M, = 320,000). $Adenosine monophosphate activated the b form 2- to 3-fold after ammonium sulfate fractionation, but this effect decreased after DE-52 chromatography and disappeared completely after Sephadex G-200 gel filtration. Thus, it’ seems as if the b form undergoes a slow, ATP-independent’ conversion to a more active form. This conclusion is supported
PHOSPHOROLYSIS
FIG. 11. Models thesis directions.
of the
by the observation that the total phosphorylasc activit,y increased markedly during purificat’ion. Because of this slow activation, no conclusion can be drawn concerning the molecular weight of the native b form.
~~:eU~lo-
mM
of G-G-P = 1.6-2.1 mM f = 0.42-0.47
K,,
= = = = =
OF
central
DISCUSSION
It is generally assumed that glycogen ran bind to phosphorylase in two distinct modes: me in which the terminal glucosyl residue is bound in a position suitable for phosphorolysis and one in which the terminal glucosyl residue is bound in a position that allows it] to act as a glucosyl acceptor. Although the two binding modes have been recognized by several workers (g-11), a complete analysis of the kinetic consequences has not, (to our knowledge) been pres&ed. Figure 11 shows a schematic reprcscntatlon of the two kinds of central complcxcs. The two modes of glycogen binding are obviously mut’ually exclusive. In the phosphorolysis direction, glycogen can bind in the two modes at all concentrations of inorganic phosphate. However, in the synthesis direction, G-1-P excludes glycogen bound in the phosphorolysis mode. Thus, at low G-l-P concentrations, glycogen can bind both ways, but at saturat’ing G-l-P, only one glycogen binding mode is possible. The various equilibria involved are shown below. EB represents glycogen bound in the phosphorolysis mode wit’h a dissociation constant Ke. BE represents glycogen bound in t’he synthesis mode with a dissociation const’ant ?Ks. A = Pi, and Q = G-l-P. The factors CX, ,6, and 6 represent possible intcract’ion fact’ors.
MODE
complexes
525
PHOSPHORYLASE
II CONST.4NTS PIIOSPHORYLASEn
direction
= 2.1-3.2
GLYCOGEN
SYNTHESIS
of glycogen
phosphorylase
MODE
in the phosphorolysis
and syn-
GOLD
526 BE+A=<
@K.\
ET AL.
BEA
EB
11KB
YKHII +
,I?\
+
YKH.
EA E +A-< + + B (phosphorolysis) B
KH11 EB+A’
aK.4
II aK, k, 6K, _ 11
c EAB
+
BE . B+E + + Q (synthesis) Q
-k,
BEQ .
Y~KH _
it
KQ
EQ
value. Kib determined kinetically must be identical to the dissociation constant determined by equilibrium binding whether or not there are two binding modes. The fact that Kib (synthesis) is almost five times Kia (phosphorolysis) for the Neurospora glycogen phosphorylase a suggeststhat the inter[QIBI -I conversion of the central complexes may not I LB1 + m+;J+KK [AI[Bl * YKB A B Q B> & be the sole rate-limiting step (i.e., the mechanism may be random but not truly In the phosphorolysis direction: rapid equilibrium random in both direcapparent IL = KB/(~ + l/r), tions). A similar discrepancy between the Kib values in the two directions was noted by apparent K,, = ~KB/ (1 + dpr); Gold et al. for rabbit muscle glycogen phosapparent Ki, = KA, phorylase a (11). (b) Any inequality between Kib and K,, in the phosphorolysis apparent K-,, = aKA(l + l/r)/ direction must result from interactions be(1 + 4P-f). tween the two substrates (i.e., a: # 1 or In the synthesis direction: @# 1, or both). If Q: = /3 = 1, then Kib = K mg,regardless of the value of y. However, apparent Kib = KB/’ (1 + l/r 1, it is also possible for Kia to equal K,, when (Y # 1 and fl # 1 if coincidentally, l/a = apparent K,, = y8K~; 1 + l/r + l/Or. (c) Engers, Bridger, and apparent Ki, = KQ, Madsen (9) concluded that the kinetic K,s apparent K,, = 6(1 + r)K,. for G-l-P and inorganic phosphate are both Several points are obvious: (a) The appar- twice the “theoretical” (intrinsic) values if ent Kib for glycogen (which represents t’he y = 1. While this is true for G-l-P if 6 = 1, apparent dissociation constant of the en- the apparent K,n for inorganic phosphate zyme-glycogen complex) should be the (apparent KmA) is not ~KA, UdeSSa = b = samein both directions, whether or not there 2. (d) An inequality of Kib and K,, in the are two binding modes. If there are indeed synthesis direction does not necessarily two binding modes, the apparent Kib is a indicate an interaction between substrates. composite dissociation constant. If glycogen If 6 = 1, Kib will still not equal K,,. For binds equally well in the two modes (i.e., the Neurospora glycogen phosphorylase a, the apparent K;b equals the apparent K,,. y = l), the apparent Kib is x the intrinsic The complete velocity equation is:
Neurospora
GLYCOGEN
This means either that there really are not, two nonequivalent modes of glycogen binding or coincidentally, 6 = l/(1 + y). REFERENCES 1. FISCHER, E. H., HEILMEYER, L. M. G., AND HASCHKE, R. H. (1971). in Current Topics in Cellular Regulation (HORECKER, B. L., AND STADTMAN, E. R., eds.), Vol. IV, p. 211, Academic Press, New York. 2. RYMAN, B. E., AND WHELAN, W. J. (1971) in Advances in Enzymology (NORD, F. F., ed.), Vol. 34, p. 285, Interscience, New York. 3. SHEPHERD, D., .\ND SEGEL, I. H. (1969) Arch. Bioehem. Biophys. 131, 609. 4. SHEPHERD, D., ROSENTH.~L, S., LUNDBLAD, G., .~ND SEGEL, I. H. (1968) Arch. Biochem. Biophys. 136, 334. 5. TELLEZ-INON, M. T., .&ND TORRES, H. N. (1970) Proc. Naf. Acad. Sci. USA 66, 459. 6. L~YNE, E. (1957) in Methods in Enzymology (COLOWICK, S. P., AND KAPLAN, N. O., eds ), Vol. III, p. 450, Academic Press, New York.
PHOSPHORYLASE
527
7. MARTIN, R. G., .~ND AMES, B. N. (1961) J. Biol. Chem. 236, 1372. 8. ANDRE~~S, P. (1965) Biochem. J. 96, 595. 9. ENGERS, H. D., BRIDGER, W. A., AND MADSEN, N. B. (1969) J. Biol. Chem. 244, 5936. 10. CHAO, J., JOHNSON, G. F., AND GRAVES, D. J. (1969) Biochemistry 8, 1459. 11. GOLD, A. M., JOHNSON, R. M., IND TSENG, J. K. (1970) J. Biol. Chem. 246, 2564. 12. ~~~~~~~~~~~ V. T., AND MADSEN, N. B. (1966) J. Biol. Chem. 241, 3873. 13. CHEN, G. S., AND SEGEL, I. H. (1968) Arch. Biochem. Biophys. 127, 175. 14. JONES, T. H. D., AND WRIGHT, B. E. (1970) J. Bncteriol. 104, 754. 15. KAHN, V., AND BLUM, J. J. (1971) Arch. Biothem. Biophys. 143, 92. 16. SAGARDIA, F., GoT.~~, I., AND RODRIQUEZ, M. (1971) Biochem. Biophys. Res. Commun. 42, 829. 17. FOSSET, M., MUIR, L. W., NIELSEN, L. I>., AND FISCHER, E. H. (1971) Biochemistry 10, 4105. 18. WANG, J. H., Tu, J., AND Lo, F. M. (1970) J. Biol. Chem. 246, 3115.
OF
BIOCHEMISTRY
Neurospora
AND
crassa
BIOPHYSICS
Glycogen
Kinetic MICHAEL
Department
Properties
H. GOLD,2
of Biochemistry
ROBERT IRWIN and
161, 515-527
Phosphorylase: of the
University October
interconversion
“Active”
J. FARR.AND, H. SEGEL4
Biophysics, Received
(1974)
and
Form’ JOHN
of California,
P. LIVON13
Davis,
California
AND
96616
26, 1973
Glycogen phosphorylase in cell-free extracts of Xeurospora crassa is activated loto 15-fold by incubation with MgATP2-. When the MgATP” is removed, the active form (a form) reverts to the inactive form (b form). The inactivation requires Mg2+ and is inhibited by NaF. The results confirm that Neurospora crassa glycogen phosphorylase exists in two interconvertible forms and strongly suggests that the interconversion is catalyzed by a kinase and phosphatase. The a form was partially purified. The enzyme has a molecular weight of 320,000. Uridine diphosphate glucose is a linear competitive inhibitor with respect to glucose-l-phosphate and a linear noncompetitive inhibitor with respect to glycogen. Glucose-6-phosphate is a hyperbolic (partial) noncompetitive inhibitor with respect to all substrates in both directions. The b form of the enzyme in crude cell-free extracts is stimulated 2- to a-fold by 5’AMP. As the b form is purified, the 5’-AMP activation is diminished. The molecular weight of the partially purified “b” form was also 320,000.
Glycogen phosphorylase (a-1,4-glucan: orthophosphate glucosyltransferase, EC 2.4.1.1) has been isolated from a wide variety of organisms (1, 2). The Neurospora crassa enzyme was first studied by Shepherd and Segel (3, 4) and by Tellez-Inon and Torres (5). The latter workers demonstrated that the enzyme exists in two interconvertible forms called a (active) and b (inactive). In this paper, the interconversion of phosphorylase a and b in cell-free extract’s is confirmed, and some physical and kinetic properties of the partially purified a form are reported. In addition, the kinetic consequences of a “two-mode” model for glycogen binding are discussed.
MATERIALS
5 Abbreviations; Pi = G-l-P = glucose-l-phosphate; g-phosphate. 515
@ 1974 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Culture conditions. All experiments described in this paper were carried out with a wild type strain of N. crassa (CM+). The organism was grown aerobically on the synthetic medium previously described (3). The cells were grown in 2-liter Erlenmeyer flasks containing 1 liter of medium on a New Brunswick shaker (New Brunswick Scientific Co., Inc.) operating at a speed of 250 rpm and describing a 2-in. circle. These cultures were maintained by transferring some of mycelial suspension to fresh medium every 2 days. Cells which had been allowed to grow for 48 hr were used for extraction of enzyme. Protein determination. Protein was determined either by the biuret procedure (6), or, in later stages of purification, by its absorbance at 280 nm. Enzyme assays. Glycogen synthesis direction: Phosphorylase activity was determined by following the rate of incorporation of [r%]glucose from [%]G-l-P6 into glycogen. Activity was meas-
1 The research described in this paper was supported by grant AI-07560 from the United States Public Health Service. 2 Present address: Laboratory of Biochemical Genetics, The Rockefeller University, New York, N. Y. 10021. 0 Undergraduate Honors Research Student. 4 Addressee for reprints. Copyright All rights
AND
inorganic G-6-P
phosphate; = glucose-
GOLD
MINUTES
of Neurospora glycogen FIG. 1. Activation phosphorylase b. The crude extract was incubated at 15°C with 10 mM ATP and 20 mM MgCl:. Periodically, aliquots were removed and assayed in the synthesis direction in the absence of 5’AMP.
ured in 0.1 ml of reaction mixture containing 50 mM Tris-Cl (pH 7.2), 2.0-20 mg/ml glycogen, 0.5-5 mM [l%]G-1-P (10”lo6 cpm/amole), 10 mM EDTA, potential inhibitors and activators as described below, and enzyme. The reaction mixtures were incubated at 30°C for 10 min. Reactions were terminated by adding 2 ml of 75% ethanol containing 1% KCl. The precipitated glycogen was centrifuged in a clinical centrifuge for 2 min. After the supernatant was decanted, 0.2 ml of Hz0 was added to dissolve the precipitate. The glycogen was precipitated two more times and finally redissolved in 0.5 ml of water and counted in 5 ml of scintillation fluid. The scintillation fluid contained 6 g of 2,5-diphenyloxazole and 100 g of naphthalene per liter of dioxane. Glycogen degradation direction: The assay was essentially the same as that described by Shepherd et al. (4). Activity was measured in 0.1 ml of reaction mixture containing the same reactants described above except that 2-20 mM “Pi replaced the [‘“ClG-1-P. The reaction mixtures were incubated at 30°C for 10 min. The [32P]G-1-P formed was measured after precipitating the “Pi with cadmium acetate. Enzyme extraction. The mycelium from 1 liter was harvested by suction filtration, washed sev-
ET
AL.
eral times with deionized water, and weighed. The pad was mixed with glass beads (1 mm, Braun) equal to twice the weight of mycelium and a volume of 50 mM Tris-Cl buffer (pH 7.5) equal to twice the weight of mycelium. The mixture was homogenized for 2 min in a Bronwill homogenizer (Bronwill Scientific). The homogenate was centrifuged at 20,000 g for 15 min at 4’C (crude extract). Purifcation of phosphorylase a. Phosphorylase in the crude cell-free extract was first fully activated by incubation with 10 mM ATP and 20 M MgCl: for 30 min at 15°C (Fig. 1). Ammonium sulfate fractionation: Solid ammonium sulfate was added to bring the crude extract to 400% saturation. The solution was stirred for 30 min and centrifuged at 20,000 g for 15 min. The supernatant was brought to 65% saturation, maintaining the pH above 7.0, and centrifuged as before. The precipitate was then taken up in 10 mxu succinate buffer, pH 5.8, containing 1 mM EDTA (buffer A), and dialyzed against several changes of the same buffer for 16 hr. DE-62 chromatography: The enzyme was adsorbed to a column (2.0 X 20 cm) of DE-52 previously equilibrated with buffer A. Aft,er adsorption of the enzyme, the column was washed with 100 ml of the equilibration buffer. Finally, the enzyme was eluted at pH 5.8 with a linear gradient consisting of buffer A in the mixing chamber and buffer A + 0.5 M NaCl in the reservoir. The total volume of the gradient was 400 ml. Four-milliliter fractions were collected and assayed. The enzyme activity peak was pooled and dialyzed against several changes of buffer A for 16 hr. DEAE Sephadex A-60 chromatography: The enzyme was adsorbed to a column (1.0 X 30 cm) of A-50 previously equilibrated with buffer A. After adsorption of the enzyme, the column was washed with 50 ml of buffer A. The enzyme was eluted with the same gradient as used in the DE-52 chromatography. Four-milliliter fractions were collected and assayed. The enzyme activity peak was pooled and concentrated to 5 ml by ultrafiltration through a Diaflo membrane (PMlO, Amicon Corporation). Sephadex G-300 gel jiltration: The concentrate was applied to a 2.5 X 37 cm column of Sephadex G-200 previously equilibrated with 25 mM succinate + 100 mM NaCl, pH 6.0. Four-milliliter fractions were assayed for protein and activity. The fractions containing the enzyme were pooled, dialyzed against 50 mM Tris buffer (pH 7.2), and stored at -20°C. Sucrose gradient centrifugation. Sucrose density gradient, studies were conducted by the method of Martin and Ames (7) using a Beckman SW40 rotorat35,0OOrpmfor16hrat5”C.Linearsucrose gradients from 6 to 30% sucrose in 25 mM succinate
Neurospora
GLYCOGEN
517
PHOSPHORYLASE
rapid and could be followed conveniently only by lowering the incubation temperature to 15”. Figure 2 shows t’he inactivation of phosphorylase a in the absence of ATP (which was removed by ammonium sulfate precipitation and gel filtration). The results are similar to those reported by Tellez-Inon and Torrcs (5). There seemsno doubt that N. cmssa glycogen phosphorylase exists in two forms, and that their interconversion involves a phosphorylase b kinase and a phosphorylase a phosphatase. Purif?,ation
5
IO
15
20
25
30
MINUTES
FIG. 2. Inactivation of Neurospora glycogen phosphorylase a. The enzyme was first activated as described in Fig. 1. Solid ammonium sulfate (Mann, enzyme grade) was then added to the extract to 65yo saturat’ion. The solution was stirred for 30 min and centrifuged at 20,000 g for 15 min. The precipitate was then taken up in 50 mM TrisCl, pH 7.2, and a 0.5-ml aliquot was filtered on a 1.0 X 25 cm column of Sephadex G-25 equilibrated in 50 mM Tris-Cl, pH 7.2. The fractions containing phosphorylase activity (breakthrough volume) were pooled. Aliquots were incubated at 30°C in 50 mM Tris-Cl, pH 7.2, containing 5 mM MgCls with the following additions: none (O ) ; 50 mM NaF (0); 15 mM EDTA (0); 5 mM 5’-AMP (II). Periodically enzyme activity was measured in the synthesis direction in the absence of 5’AMP. The arrows show the preactivation activity of ATeurospora phosphorylase b in the presence and absence of 5’-AMP. + I mM EDTA, pH 6.0, were used. Fractions of 0.3 ml were collected. p-Galactosidase and skeletal muscle phosphorylase b (assayed in the presence of 5’-AMP) were used as markers. RESULTS
Interconversion
of Neurospora b and a
Phosphorylase
Figure 1 shows the activation of Neurospora glycogen phosphorylase b in the pres-
enee of ATP and Mg*+. The activation is
of Phosphorylase
a
It seemed likely that the Neurospora glycogcn “phosphorylase” studied by Shepherd et al. (3, 4) was a mixture of a and b forms, since no preactivation or inactivation step was included before purification. Consequent’ly, in t,he present study, we concentrated on the a form. Figure 3 shows t’he elution profile of phosphorylase a from Whatman DE-52. The enzyme elutes at 0.22 M XaCl as a single activity peak. The activity of each fraction did not change when 5 mM 5’-AMP was added to the reaction mixture. The enzyme also eluted as a single peak from DEAF Sephadex A-50. Figure 4 shows a gel filtration profile. The enzyme elutes as a single symmet’rical peak. The molecular weight of the enzyme as determined by the method of Andrews (8) was calculated to be 320,000 (Fig. 4, insert). The specific activities at various stages of the preparation are described in Table I. After the gel filtration step, the enzyme was purified approximately 124-fold relative to the specific activity of the activated crude extract. Sucrose density gradient centrifugation was used to study the sedimentation behavior of the enzyme and as an independent check on the number of forms in our preparation. Figure 5 illustrates the activity profile for Neurospora phosphorylase a, skeletal muscle phosphorylase b (8.4S), and the A2Bonm profile for fi-galactosidase (16.0 S). From this figure the sedimentation coefficient of Neurospora phosphorylase a was
518
GOLD
FRACTION
ET
AL.
NUMBER
FIG. 3. Chromatography
of Neurospora phosphorylase a on DE-52. The enzyme was applied to a 2.0 X 20 cm column of Whatman DE-52 equilibrated with 10 mM succinate buffer, pH 5.8, containing 1 mM EDTA. The column was washed with 100 ml of the same buffer. The enzyme was eluted at pH 5.8 with a linear gradient consisting of 200 ml of the above buffer in the mixing chamber and 200 ml of the same buffer containing 100 mM NaCl in the reservoir. Absorbancy at 280 nm (0) and phosphorylase activity (0) are shown. I
PHOSPHORYLASE a
PHOSPHORYLASE b
I f f 2 Y 8 .G E 5 i= Y
IO
50 FRA&ON
&FIG.
l&BE~O
4. Sephadex G-200 gel filtration of Neurospora phosphorylase a. A 2.5 X 37 cm column of Sephadex G-200 equilibrated with 25 mM succinate mM NaCl. Absorbancy at 280 nm (0) and phosphorylase activity (0) are weight versus VJVO using ferritin, muscle phosphorylase b, hemoglobin,
5-ml sample was applied to a buffer, pH 6.0, containing 100 shown. Insert: log molecular and myoglobin as standards.
determined to be 11.6 S, which corresponds equilibrium random kinetic mechanism to an approximate molecular weight of (9-11). The data reported in this paper are consistent with a random mechanism al325,000. though an ordered sequence in which Pi or Initial Velocity Studies G-1-P adds first cannot be excluded. The All polyglucose phosphorylases that have binding sequencecan be visualized as shown been investigated appear to have a rapid below:
Areurospora
GLYCOGEN
K, = K,,
Kc, = K,,
E+Ar EA + + B (phosphorolysis) B K,
= K:,
11
aK,
= K,, ‘
EB + A ’
II
EQ + B
aK,,
_ EAB
’ h,
= Kk,, k,
where A = I!‘<, B = glycogcn, and Q = G-1-P. a and b represent’ the interaction factors describing the cffcct’ of the binding of one ligand on the dissociation constant for the other. The above scheme assumes that there is only one mode of glycogen binding (which may not be true). Figure 6A shows the family of l/v versus l/[G-l-P] plots obtained at different’ fixed glycogen concentrations. The plots intersect on the horizontal axis. The insert shows a replot of the l/v-axis intercepts versus l/[glycogen]. The horizontal-axis intercept gives bKu or K,,. The replot yields a K, for glycogcn of 3.2 mg/ml. Since the family of plots intersect on the horizontal axis, the slope rcplot (which gives KB = Kib) yields essentially the samevalue. Thus, for the assumedmodel, the binding of glycogen has no effect on the binding of G-1-P and vice versa (i.e., b = 1). Figure 6B shows the data plotted as l/v versus l/[glycogen] at different fixed G-l-P
PURIFICATION
Fraction
1. Crude
con-
Q+E
L
+
(synthesis) bK,,
B
= Km, bK,t = Km,
EQB ’
Ka = Ktb
it
r Q + EB
concentrations. The insert showsthe l/v-axis intercept replot, which yields bKQ = K,,. The K, for G-1-P is 1.4 nw. A slope replot gives K, = K, = 1.3 mM. Figures 7A and B show the kinetics of the phosphorolysis reaction. The families of plots intersect beion- the horizont,al axis. The l/v-axis intercept replots yield uK~ = K,, (i.e., K, for glycogen) = 1.33 mg/ml, aK, = K, (i.e., K, for Pi) = 7.4 mM. The slope replots yield Kg = Kib = 0.66 mg/ml and K, = Ki, = 4.2 mM. Thus, for the phosphorolysis direction, the binding of one ligand does affect the binding of the other. The interaction factor, a, is 1.8-2.0.
Inhibition
by UDPG
Figure 8A shows that UDPG is a linear competitive inhibitor with respect to G-l-P. The slope replot (insert) yields a Kx’,zpe of 3.2 mM at 20 mg/ml glycogen. Uridine diphosphate glucose is a linear noncompetiI
oli PHOSPHORYLASE Total protein bd*
extract
2. Activated crude extract 3. (NH&S04 (4@65’%) precipitation 4. DE-52 chromatography and Diaflo centration 5. DEAE-Sephadex A-50 chromatography 6. G-200 gel filtration
.
II
TABLE PARTIAL
519
PHOSPHOItYLASE
a FROM
Volume (ml)
Ar. crassaa
Specific activity &moles min-’ m g-l) c
1550
50
0.014
1550 455 40
51 26 20
0.11 0.263 2.19
10.3 4.3
a For details see text. * Protein was determined with biuret reagent in fractions at 280 = 10.0) was used for fractions 4, 5, and 6. c Standard assay conditions including 20 mg/ml glycogen;
23 20
l-3;
6.33 13.7
absorbance
5 mM G-1-P.
Purification factor
Yield (%I
-
-
1.0 2.39 19.9
100 70 51
57.5 124.5
at 280 nm
38 34
(assuming
e:&
520
ET AL.
GOLD
a a5
LO [G-I-P]-’
1.5
2.0
hM)-’
FRACTION NUMBER
FIG. 5. Sucrose density gradient centrifugation of Neurospora phosphorylase a. Centrifugation was done at 35,000 rpm for 16 hr at 5°C in a Beckman SW-40 rotor. The gradient contained 6-30yo sucrose in 25 mrw succinate buffer, pH 7.0, containing 1 InM EDTA. Absorbancy at 280 nm (A) and phosphorylase act.ivity ( l , 0) are shown.
tive inhibitor with respect to glycogen (Fig. SB). The replot gives IX;: = K$zpe = 9.5 mM at 5 mM G-l-P. The Kis are given by: K:;ype
= cKu
(1 +
KBABI
>/ (1 +
CKB/[BI
>,
[&l/&J, zsl.,,,e = Ku0 + [Ql/Kd,
KY,:
= cKu
(1 +
b
KY/B
where KU represents the dissociation constant for the enzyme-UDPG complex and cKU represents the dissociation constant for UDPG from the enzyme-UDPG-glycogen complex. Thus, c is an interaction factor analogous to a and b. The symbol KE{,‘p, means “the Ki calculated from the slope replot of l/v versus l/[Q] plots with U = UDPG as an inhibitor.” Similarly, KxE means “the Ki calculated from the l/u-axis intercept replot of l/v versus l/[B] plots with U as an inhibitor.” All Kis are apparent values depending on the concentration of the nonvaried substrate. The fact that KxE =
0
0.1
0.3
0.2 [GLYCOGEN]-1
0.4
(MG/ML)-I
FIG. 6. (a) l/v versus l/[G-l-P] at several fixed concentrations of glycogen. The glycogen concentrations were (mg/ml) : 20 (0); 7.0 (u); 4.0 (0); 2.5 (A). Insert: replot of the l/o-axis intercepts versus l/[glycogen]. (b) l/v versus l/[glycogen] at several fixed concentrations of G-l-P. The G-1-P concentrations used were (millimolar): 5.0 (0); 2.0 (C); 1.0 (0); 0.66 (A). Insert: replot of the l/v-axis intercepts versus l/[G-l-P]. G/IfptT
Kt{zpe
KXL
suggests that c = a = 1. Kxz or yields a value for Ku of 2.1 mM. yields a K, of 3.2 mM. Uridine
Neurospora
GLYCOGEN
521
PHOSPHORYLASE
[GLVCGGEN]
-’ IMG/MLi
-’
FIG. 7. (a) l/v versus l/[phosphate] at several fixed concentrations of glycogen. The glycogen concentrations were (mg/ml) : 5.0 (a); 2.0 (m); 1.0 (0) ; 0.66 (A); 0.5 (0). Insert: replot of the l/v-axis interl/[glycogen] at several fixed concentrations of phosphate. The cepts versus l/[glycogen]. (b) 1/ ZJversus phosphate concentrations were (millimolar): 20 (a); 6.67 (m); 4.0 (0); 2.65 (A); 2.0 (Cl).
diphosphate glucose has been shown to inhibit glycogen phosphorylase from a number of sources (1%16), akhough Fosset et al. (17) reported that the yeast enzyme was not affected. Inhibition
by Glucose-6-phosphate
Glucose-6-phosphate was a noncompetitive inhibitor with respect to G-1-P (Fig.
9A). The family of plots intersect on the horizont’al axis showing no effect of G-6-P on G-l-P binding. The l/v-axis intercept replot was hyperbolic, indicating partial inhibition. A secondary replot of l/A intercept versus l/[G-6-P] was linear. The horizontal-axis intercept of the secondary replot, gives -f/Ki, where fV,,, is the maximal velocity at saturating G-6-P and tbe
GOLD
1.0 [G-l-P]-'
1.5
ET AL.
20
(&I)-'
FIG. 8. Inhibition by UDPG. (a) Linear competitive inhibition with G-l-P as the varied substrate and glycogen held constant at 20 mg/ml. The UDPG concentrations were (millimolar): 0.00 (a); 1.0 (m); 3.0 (0); 6.0 (A). Insert: replot of the slopes against the concentration of UDPG. (b) Linear noncompetitive inhibition with glycogen as the varied substrate and G-l-P held constant at 5 mM. The UDPG concentrations were (millimolar) 0.00 (a); 2.0 (I); 4.0 (0); 6.0 (A). Insert: replot of the l/vaxis intercepts against the concentration of UDPG.
constant glycogen concentration. The vertical axis intercept of the secondary replot gives fV,,,/ (1 - f). The replot yields f = 0.47 and K; = 2.1 mM. Figure 9B showsthat G-6-P is also a partial noncompetitive inhibitor with respect to glycogen. The replot yields f = 0.42 and Ki = 1.6 mM. In the phosphorolysis direction (Figs. 10A and B) G-6-P behaved in the same partial manner (f = 0.42-0.52, Ki = 1.6-4.0 mM>. The results indicate that Neurospora glycogen phosphorylase possessesa specific, modifier site for G-6-P that is distinct from the G-l-P and glycogen sites. Partial inhibition by G-6-P was also noted by Wang et al., with muscle glycogen phosphorylase b (18). The inhibition was competitive with respect to 5’-AMP and was ascribed to an allosteric effect at a site distinct from the nucleotide binding site. Yeast glycogen phosphorylase
is also noncompetitively inhibited by G-6-P (17). In this case, G-6-P was reported to be a linear inhibitor with respect to G-l-P and a parabolic inhibitor with respect to glycogen. The kinetic constants of the Neurospora glycogen phosphorylase a are summarized in Table II. The calculated K,, for the reaction in the synthesis direction is about 2, in good agreement with the calculated value for muscle glycogen phosphorylase a (11). Neurospora Glycogen Phosphorylase b Although the activity of the b form was quite low in cell-free extracts, an attempt was made to purify it using the same methods described above (ammonium sulfate precipitation, DE-52 chromatography, and gel filtration through Sephadex G-200). The enzyme eluted from Sephadex G-200 at exactly the same position as the a form
2.0 -
4
FIG. 9. Partial noncompetitive varied substrate. Glycogen was 0.00 (0); 2.0 @I); 5.0 (0); 10 concentration of G-6-P. Insert petitive inhibition with glycogen concentrations were (millimolar) l/v-axis intercepts against the l/[G-6-P].
/
inhibition held constant (A); 20 (D). (2): replot of as the varied : 0.00 (0); concentration
[G-W
,,,,I&
by G-6-P in the synthesis direction. (a) G-1-P was the at 40 mg/ml. The G-6-P concentrations were (millimolar) : Insert (1): replot of the l/v-axis intercepts against the the l/A intercept against l/[G-6-P]. (b) Partial noncomsubstrate and G-1-P held constant at 5 mM. The G-6-P 2.0 (m); 5.0 (0); 10 (A); 20 (0). Insert (1) : replot of the of G-6-P. Insert (2): replob of the I/A intercept against 523
524
ET AL.
GOLD
0.2 [PO,, =] -'
0
b
/
a5
I.0 [GLYCOGEN].1
FIG. 10. Partial noncompetitive inhibition in the was the varied substrate with glycogen held constant molar): 0.00 (0); 2.0 (m); 5.0 (0); 10 (A); 20 (0). the concentration of G-6-P. Insert (2): replot of the the varied substrate, and phosphate was held constant molar): 0.00 (a); 2.0 (m); 5.0 (0); 10 (A); 20 (0). the concentration of G-6-P. Insert (2) : replot of the
I.5
(mM) :"
1
2.0
(MC/WI
phosphorolysis direction by G-6-P. (a) Phosphate at 40 mg/ml. The G-6-P concentrations were (milliInsert (1): replot of the l/v-axis intercepts against l/A intercept against l/[G-G-P]. (b) Glycogen was at 20 mM. The G-G-P concentrations were (milliInsert (1): replot of the l/v-axis intercepts against l/A intercept against l/[G-6-P].
Xeurospora TABLE EXPERIMENTAL spora
KINETIC GLYCOGE~V
Synthesis K ng Kib K 7nQ Ki, Ir Ku Ki
= = = = =
Phosphorolysis K;, I &b Ii,, Ki, V
3.2-5.7 mg/ml 3.2-5.7 mg/ml 1.3-2.0 rnM 1.3-2.0 mnt 3.2 nmoles/minb
direction
1.3-2.1 mg/ml 0.66-0.92 mg/ml 6.0-7.8 mM 2.8-4.2 mM 2.0 nmoles/min*
Zii of G-G-P = 1.M.O rnM f = 0.42-0.52
N_ 2~
Q = G-I-P, aA = Pi, B = glycogen, UDPG. The values shown represent the observed for all the experiments reported paper. 6 V values for the same preparation. V,,,
CK “'I =
=V
X
K,
Tie,.,
x
Kia
phua
X K,, x
Kim,
x
Kn,
x
Kia
x
pim x
K,
X Km,
V,,., =V
U = ranges in this
X KmA X K:b
Tiphc,a
x
Kzb
Kin,
(M, = 320,000). $Adenosine monophosphate activated the b form 2- to 3-fold after ammonium sulfate fractionation, but this effect decreased after DE-52 chromatography and disappeared completely after Sephadex G-200 gel filtration. Thus, it’ seems as if the b form undergoes a slow, ATP-independent’ conversion to a more active form. This conclusion is supported
PHOSPHOROLYSIS
FIG. 11. Models thesis directions.
of the
by the observation that the total phosphorylasc activit,y increased markedly during purificat’ion. Because of this slow activation, no conclusion can be drawn concerning the molecular weight of the native b form.
~~:eU~lo-
mM
of G-G-P = 1.6-2.1 mM f = 0.42-0.47
K,,
= = = = =
OF
central
DISCUSSION
It is generally assumed that glycogen ran bind to phosphorylase in two distinct modes: me in which the terminal glucosyl residue is bound in a position suitable for phosphorolysis and one in which the terminal glucosyl residue is bound in a position that allows it] to act as a glucosyl acceptor. Although the two binding modes have been recognized by several workers (g-11), a complete analysis of the kinetic consequences has not, (to our knowledge) been pres&ed. Figure 11 shows a schematic reprcscntatlon of the two kinds of central complcxcs. The two modes of glycogen binding are obviously mut’ually exclusive. In the phosphorolysis direction, glycogen can bind in the two modes at all concentrations of inorganic phosphate. However, in the synthesis direction, G-1-P excludes glycogen bound in the phosphorolysis mode. Thus, at low G-l-P concentrations, glycogen can bind both ways, but at saturat’ing G-l-P, only one glycogen binding mode is possible. The various equilibria involved are shown below. EB represents glycogen bound in the phosphorolysis mode wit’h a dissociation constant Ke. BE represents glycogen bound in t’he synthesis mode with a dissociation const’ant ?Ks. A = Pi, and Q = G-l-P. The factors CX, ,6, and 6 represent possible intcract’ion fact’ors.
MODE
complexes
525
PHOSPHORYLASE
II CONST.4NTS PIIOSPHORYLASEn
direction
= 2.1-3.2
GLYCOGEN
SYNTHESIS
of glycogen
phosphorylase
MODE
in the phosphorolysis
and syn-
GOLD
526 BE+A=<
@K.\
ET AL.
BEA
EB
11KB
YKHII +
,I?\
+
YKH.
EA E +A-< + + B (phosphorolysis) B
KH11 EB+A’
aK.4
II aK, k, 6K, _ 11
c EAB
+
BE . B+E + + Q (synthesis) Q
-k,
BEQ .
Y~KH _
it
KQ
EQ
value. Kib determined kinetically must be identical to the dissociation constant determined by equilibrium binding whether or not there are two binding modes. The fact that Kib (synthesis) is almost five times Kia (phosphorolysis) for the Neurospora glycogen phosphorylase a suggeststhat the inter[QIBI -I conversion of the central complexes may not I LB1 + m+;J+KK [AI[Bl * YKB A B Q B> & be the sole rate-limiting step (i.e., the mechanism may be random but not truly In the phosphorolysis direction: rapid equilibrium random in both direcapparent IL = KB/(~ + l/r), tions). A similar discrepancy between the Kib values in the two directions was noted by apparent K,, = ~KB/ (1 + dpr); Gold et al. for rabbit muscle glycogen phosapparent Ki, = KA, phorylase a (11). (b) Any inequality between Kib and K,, in the phosphorolysis apparent K-,, = aKA(l + l/r)/ direction must result from interactions be(1 + 4P-f). tween the two substrates (i.e., a: # 1 or In the synthesis direction: @# 1, or both). If Q: = /3 = 1, then Kib = K mg,regardless of the value of y. However, apparent Kib = KB/’ (1 + l/r 1, it is also possible for Kia to equal K,, when (Y # 1 and fl # 1 if coincidentally, l/a = apparent K,, = y8K~; 1 + l/r + l/Or. (c) Engers, Bridger, and apparent Ki, = KQ, Madsen (9) concluded that the kinetic K,s apparent K,, = 6(1 + r)K,. for G-l-P and inorganic phosphate are both Several points are obvious: (a) The appar- twice the “theoretical” (intrinsic) values if ent Kib for glycogen (which represents t’he y = 1. While this is true for G-l-P if 6 = 1, apparent dissociation constant of the en- the apparent K,n for inorganic phosphate zyme-glycogen complex) should be the (apparent KmA) is not ~KA, UdeSSa = b = samein both directions, whether or not there 2. (d) An inequality of Kib and K,, in the are two binding modes. If there are indeed synthesis direction does not necessarily two binding modes, the apparent Kib is a indicate an interaction between substrates. composite dissociation constant. If glycogen If 6 = 1, Kib will still not equal K,,. For binds equally well in the two modes (i.e., the Neurospora glycogen phosphorylase a, the apparent K;b equals the apparent K,,. y = l), the apparent Kib is x the intrinsic The complete velocity equation is:
Neurospora
GLYCOGEN
This means either that there really are not, two nonequivalent modes of glycogen binding or coincidentally, 6 = l/(1 + y). REFERENCES 1. FISCHER, E. H., HEILMEYER, L. M. G., AND HASCHKE, R. H. (1971). in Current Topics in Cellular Regulation (HORECKER, B. L., AND STADTMAN, E. R., eds.), Vol. IV, p. 211, Academic Press, New York. 2. RYMAN, B. E., AND WHELAN, W. J. (1971) in Advances in Enzymology (NORD, F. F., ed.), Vol. 34, p. 285, Interscience, New York. 3. SHEPHERD, D., .\ND SEGEL, I. H. (1969) Arch. Bioehem. Biophys. 131, 609. 4. SHEPHERD, D., ROSENTH.~L, S., LUNDBLAD, G., .~ND SEGEL, I. H. (1968) Arch. Biochem. Biophys. 136, 334. 5. TELLEZ-INON, M. T., .&ND TORRES, H. N. (1970) Proc. Naf. Acad. Sci. USA 66, 459. 6. L~YNE, E. (1957) in Methods in Enzymology (COLOWICK, S. P., AND KAPLAN, N. O., eds ), Vol. III, p. 450, Academic Press, New York.
PHOSPHORYLASE
527
7. MARTIN, R. G., .~ND AMES, B. N. (1961) J. Biol. Chem. 236, 1372. 8. ANDRE~~S, P. (1965) Biochem. J. 96, 595. 9. ENGERS, H. D., BRIDGER, W. A., AND MADSEN, N. B. (1969) J. Biol. Chem. 244, 5936. 10. CHAO, J., JOHNSON, G. F., AND GRAVES, D. J. (1969) Biochemistry 8, 1459. 11. GOLD, A. M., JOHNSON, R. M., IND TSENG, J. K. (1970) J. Biol. Chem. 246, 2564. 12. ~~~~~~~~~~~ V. T., AND MADSEN, N. B. (1966) J. Biol. Chem. 241, 3873. 13. CHEN, G. S., AND SEGEL, I. H. (1968) Arch. Biochem. Biophys. 127, 175. 14. JONES, T. H. D., AND WRIGHT, B. E. (1970) J. Bncteriol. 104, 754. 15. KAHN, V., AND BLUM, J. J. (1971) Arch. Biothem. Biophys. 143, 92. 16. SAGARDIA, F., GoT.~~, I., AND RODRIQUEZ, M. (1971) Biochem. Biophys. Res. Commun. 42, 829. 17. FOSSET, M., MUIR, L. W., NIELSEN, L. I>., AND FISCHER, E. H. (1971) Biochemistry 10, 4105. 18. WANG, J. H., Tu, J., AND Lo, F. M. (1970) J. Biol. Chem. 246, 3115.