(Received for publication, June 26, 1995; and in revised form, August 30, 1995)
From the
The mechanism of yeast glycogen phosphorylase activation by
covalent phosphorylation involves structural elements distinct from the
mammalian homologs. To understand the role of the amino-terminal
39-residue extension in the phosphorylation control mechanism, mutants
with 22 and 42 amino-terminal residues removed were expressed in Escherichia coli, and their properties were compared with the
wild-type (WT) enzyme. The unphosphorylated WT enzyme had a specific
activity of 0.1 unit/mg and was not activated significantly by the
substrate, glucose 1-phosphate. Phosphorylation by protein kinase
resulted in a 1300-fold activation. Glucose 6-phosphate inhibited the
unphosphorylated enzyme more effectively than the phosphorylated form,
and inhibition of the latter was cooperative. Glucose was a poor
inhibitor for both the unphosphorylated and phosphorylated WT enzyme
with K >300 mM. The rate of
phosphorylation by protein kinase depended on substrates and
interactions of the amino terminus. Maltoheptaose increased the rate of
phosphorylation of the WT enzyme by yeast phosphorylase kinase 5-fold.
The 22-residue deletion mutant (Nd22) had overall kinetic properties
similar to the WT enzyme, except that Nd22 was a better substrate for
the protein kinase and the rate of phosphorylation was unaffected by
maltoheptaose. The 42-residue deletion mutant (Nd42), which lacks the
phosphorylation site, was measurably active, although much less active
than phosphorylated WT. Sedimentation equilibrium analysis indicated
that the WT, Nd22, and Nd42 exist as tetramer, partially dissociated
tetramer, and dimer, respectively. Phosphorylation of the WT and Nd22
converted both to dimer. The results indicated that the amino terminus
affects quaternary structure and mediates activity regulation through
conformational transition.
Glycogen phosphorylase (EC 2.4.1.1) catalyzes the mobilization
of glucose 1-phosphate (Glc-1-P) ()from glycogen and hence
provides readily usable energy when needed. Present among diverse
tissues and organisms, the enzyme has evolved with different control
features to regulate activity. Among such features, only feedback
inhibition by the metabolite Glc-6-P is highly conserved within the
enzyme family and may represent the most ancient control
feature(1) . The mechanism of activation by phosphorylation
appears to occur only in eukaryotes.
Among eukaryotic glycogen
phosphorylases studied, the mammalian muscle isozyme shows the most
elaborate regulation. Biochemical and structural characterization has
defined the mechanisms by which activators and inhibitors regulate
enzyme activity(2, 3, 4) . The
unphosphorylated enzyme is inactive even in the presence of high
concentrations of substrates, Glc-1-P and glycogen. Activation requires
either phosphorylation of Ser by phosphorylase kinase or
AMP binding. AMP and phosphoserine bind nonoverlapping sites at the
subunit interface of the functional dimer, some 35 Å distant from
the active site(5, 6) . Both stabilize the active
conformation by causing the subunits to be drawn together at the
interface and triggering the active site to open. The inhibitors
Glc-6-P and ADP also bind at the AMP site, but their binding stabilizes
the inactive conformation by denying AMP or phosphoserine from this
site(7) . Glucose is an even more potent inhibitor of the
muscle enzyme, abrogating activation by phosphorylation. Glucose
stabilizes the inactive conformation by binding in the active site
cleft between the amino- and carboxyl-terminal domains(8) .
Glucose inhibition promotes dephosphorylation of the muscle
phosphorylase by protein phosphatase 1 as a result of freeing the
phosphorylated amino termini from their binding sites(9) .
Several properties distinguish the yeast and muscle enzymes. Yeast phosphorylase is neither activated by AMP nor inhibited by glucose at physiological concentration(10, 11) . Although phosphorylation activates the yeast enzyme, the phosphorylation site and the kinase involved are different from the muscle enzyme(12, 13, 14) . A threonine residue at the amino terminus is phosphorylated by either yeast phosphorylase kinase or cAMP-dependent protein kinase, neither of which recognize the muscle enzyme.
Sequence alignment of the yeast and muscle enzymes
showed that the former has an unique 39-residue amino-terminal
extension (numbered as -1 to -39 toward the amino terminus)
relative to residue 1 of the muscle enzyme(13, 15) .
After amino acid 80, the sequences exhibit 49% identity at alignable
positions. While some biochemical differences between the two enzymes
can be rationalized on the basis of nonconservation, others cannot. For
example, the lack of activation by AMP in yeast enzyme can be
attributed to poor conservation of residues involved in binding the
base moiety of AMP. However, it is not understood why glucose is a poor
inhibitor for the yeast enzyme because all residues involved in binding
glucose in the muscle enzyme are conserved. Also, the structural
mechanism by which phosphorylation activates muscle enzyme cannot apply
to the yeast enzyme because of differing structural contexts;
phosphorylation occurs on Ser in muscle enzyme and on
Thr
in yeast enzyme. In addition, the residues
involved in binding the phosphoserine in the muscle enzyme, Arg
and Arg
, are not conserved.
Previous characterization of yeast phosphorylase suggests that the amino-terminal extension functions to stabilize the enzyme in an inactive conformation. Becker et al.(16) have observed that a proteolytic product of the yeast phosphorylase lacking the amino-terminal 39 residues elutes in the active fractions during gel filtration. Furthermore, the crystal structure of the unphosphorylated yeast enzyme has revealed that the amino-terminal extension binds near the catalytic site of the neighboring subunit in the homodimer (Fig. 1), suggesting that it prevents the substrate from entering the active site(17) . Both observations are consistent with the notion that phosphorylation activates the yeast enzyme by displacing the amino terminus from the active site. An observation that the unphosphorylated enzyme forms a tetramer and that phosphorylation converts the enzyme to a dimer has not been linked to regulation(18) .
Figure 1: Structural model of the unphosphorylated yeast phosphorylase, based on Rath et al.(17) . A dimer is shown with x denoting the position of the 2-fold axis approximately perpendicular to the page. The amino-terminal domain is shown in light color and the carboxyl-terminal domain is shown in darker gray. The dimer interface is formed only by residues from the amino-terminal domains. The amino-terminal extension relative to the rabbit muscle enzyme (residues -1 to -39) is indicated in black. Residues -12 to -22 are disordered and are missing from the model, as indicated by dashed lines. The amino terminus of each subunit extends to the active site cleft (indicated by the cofactor pyridoxal phosphate, PLP) of the neighboring subunit.
In this study, the role of the amino-terminal extension and dimer-tetramer transition in activity regulation were investigated. Kinetic characterization of the Escherichia coliexpressed wild-type and amino-terminal deletion mutant enzymes, both unphosphorylated and phosphorylated, were carried out. The oligomerization states of the wild-type and mutant enzymes in the presence of different activators and inhibitors were determined by sedimentation equilibrium to establish the role of the dimer-tetramer equilibrium and the amino terminus in the regulatory mechanism of yeast phosphorylase.
Figure 2:
a, SDS-PAGE of the WT, Nd22, and Nd42
yeast glycogen phosphorylases showing the decrease in apparent
molecular weight for the two deletion mutants. The two outer lanes contain high molecular weight markers (HMW). The
molecular weight of protein standards are indicated on the right-hand side. b, schematic representation
of the amino acid sequences of the muscle and yeast glycogen
phosphorylases. The regions shaded light gray are about 50%
identical. The amino-terminal region, from residue 1 to 80, are about
20% identical. The yeast enzyme has an extended amino terminus, residue
-1 to -39, which is not present in the muscle enzyme. The
phosphorylation sites are marked with triangles (Thr in yeast enzyme, Ser
in muscle enzyme). The Nd42
mutant lost the phosphorylation site as a result of the
deletion.
Figure 3:
The Glc-1-P concentration-dependent
velocity of the unphosphorylated WT (filled square), Nd22 (filled triangle), and Nd42 (filled circle). The
curve for Nd42 was obtained by fitting the data to the Hill equation, v = VS
/(S
+ S
). The resultant fit has a Hill
coefficient of 1.5 ± 0.03, and V
and S
values of 110 ± 15 units/mg and 40
± 6 M, respectively. The glycogen concentration in the
assays is 1%.
Figure 4:
Time course of yeast phosphorylase kinase
catalyzed phosphorylation of WT (filled square), Nd22 (filled triangle), and Nd42 (filled circle) yeast
phosphorylases. The curves were obtained by fitting data to A = A
(1 -
exp
),
where A
is the amount of phosphate
incorporated at time t, A is the amplitude, and k is the initial rate. The reaction contained 12 µM
phosphorylase, 1 mM ATP, and 5 mM MgCl
.
The oligosaccharide, maltoheptaose, increased the rate of phosphorylation of the WT enzyme by yeast phosphorylase kinase (Fig. 5). In the presence of 20 mM maltoheptaose, the rate of phosphorylation of the WT enzyme mimicked that of the Nd22 mutant. Shorter oligosaccharide had less effect at the same concentration, with maltotriose showing no noticeable effect. Addition of oligosaccharides to the Nd22 mutant, however, did not change the rate of phosphorylation (data not shown).
Figure 5:
Effect of oligosaccharides on the rate of
phosphorylation of the WT enzyme by yeast phosphorylase kinase. The
curves shown are as follows: WT enzyme alone (empty circle),
WT + 10 mM maltotriose (filled triangle), WT
+ 10 mM maltotetraose (empty square), WT +
10 mM maltopentaose (filled diamond), and WT +
10 mM maltoheptaose (filled circle). The curves were
obtained by fitting data to A = A
(1 -
exp
),
where A
is the amount of phosphate
incorporated at time t, A is the amplitude, and k is the initial rate. The reaction contained 12 µM
phosphorylase, 1 mM ATP, and 5 mM MgCl
.
The concentration of the oligosaccharides used is 10
mM.
Figure 6:
The Glc-1-P concentration-dependent
velocity of the phosphorylated WT (filled square) and Nd22 (filled triangle) yeast phosphorylases. The curves were
obtained by fitting the velocity data to the Michaelis-Menten equation, v = VS/(K
+ S). The phosphorylated WT enzyme has V
and K
values of
142 ± 2 units/mg and 0.86 ± 0.02 mM,
respectively. The phosphorylated Nd22 mutant has V
and K
values of 137 ± 3
units/mg and 0.65 ± 0.04 mM, respectively. Glycogen
concentration in the assays is 1%.
The results are summarized in Table 1.
The apparent K for Glc-6-P of the unphosphorylated
WT and Nd22 was 0.29 ± 0.01 and 0.21 ± 0.01 mM,
respectively. The apparent K
for Glc-6-P of Nd42
was 12.5 ± 0.5 mM. Interestingly, in contrast to the
noncooperative inhibition pattern of the unphosphorylated enzymes, as
reflected by their Hill coefficients, inhibition of the phosphorylated
WT and Nd22 by Glc-6-P were cooperative, with Hill coefficients of 1.36
and 1.37, respectively. The cooperative character of Glc-6-P inhibition
for the phosphorylated enzymes is also indicated by Dixon plot in Fig. 7. Glucose is a poor inhibitor of all enzyme forms, with
apparent K
> 300 mM.
Figure 7: The cooperative effect of Glc-6-P inhibition. The figure shows 1/velocity versus Glc-6-P concentration plots on phosphorylated WT (a) and phosphorylated Nd22 (b) yeast phosphorylases. The Glc-1-P concentration used was 5 mM, and glycogen concentration was 1%.
Table 2summarizes the results of sedimentation analysis. In the absence of effector, the tetramer to dimer transition of the WT enzyme upon phosphorylation was confirmed (from 393 to 198 kDa). The Nd42 mutant behaved as a dimer under all conditions (ranging from 194 to 220 kDa). The unphosphorylated Nd22 mutant exhibited an apparent molecular mass of 322 kDa, indicating a mixture of dimer and tetramer; phosphorylation of Nd22 stabilizes the dimeric state (200 kDa). Interestingly, addition of 50 mM Glc-6-P to both the phosphorylated WT and Nd22 reverted the oligomerization state to that of the unphosphorylated form (from 198 to 370 kDa for the WT, and 200 to 320 kDa for the Nd22). Since 50 mM Glc-6-P would completely inhibit the phosphorylated enzyme, one could argue that the dimer-tetramer equilibrium correlates with the activation state of the enzyme, with dimer and tetramer being active and inactive, respectively. Somewhat contrary to this interpretation, Nd42 mutant remained dimeric in the presence of 50 mM Glc-6-P, which would also inhibit the enzyme based on kinetic studies.
Maltoheptaose destabilized the tetrameric state of the unphosphorylated enzyme. Addition of 20 mM maltoheptaose to the unphosphorylated WT enzyme resulted in a change of apparent molecular mass from 393 to 203 kDa. The tetramer to dimer transition might account for the effect of maltoheptaose in increasing the rate of phosphorylation of the WT enzyme by protein kinase (Fig. 5). This interpretation is consistent with the observation that Nd22 served as a better substrate for the protein kinase than the WT enzyme (Fig. 4) and that Nd22 exists as a mixture of dimer and tetramer. In addition, maltoheptaose overcame the effect of Glc-6-P to promote tetramer formation of the phosphorylated enzyme. In the presence of 20 mM maltoheptaose, phosphorylated WT and Nd22 remained dimeric (236 kDa) even upon addition of 50 mM Glc-6-P.
Studies on muscle enzyme showed that oligosaccharides
bind preferentially at the high affinity glycogen storage
site(26, 27) . The high sequence homology between the
muscle and yeast enzyme at this site implies functional homology. The
crystal structure of the unphosphorylated yeast phosphorylase tetramer
revealed that the glycogen storage site forms part of the dimer-dimer
contact. ()For maltoheptaose to bind, the dimer-dimer
contact must be disturbed. Maltotriose did not affect the
oligomerization state of any enzyme forms, presumably because it could
not provide enough binding energy to stabilize the dimer. A consistent
observation is that maltotriose did not enhance the rate of
phosphorylation of the WT by protein kinase (Fig. 5).
An extraordinary dimension of eukaryotic glycogen phosphorylases is the tailoring of isozymes for specific cell and tissue environments through evolution of regulatory features. Yeast phosphorylase represents an intermediate in the development of the intricate control features in mammalian phosphorylases as it shares a number of structural attributes of the mammalian regulatory apparatus (conservation of Glc-6-P site, glucose binding residues, glycogen storage site). However, its covalent phosphoregulatory apparatus is distinct. An important focus of yeast glycogen phosphorylase studies is to elucidate novel mechanistic roles of protein phosphorylation in regulation.
In rabbit muscle glycogen phosphorylase, phosphorylation results in greater than 200-fold enzyme activation. Phosphorylation of the yeast enzyme was previously reported to result in only 5-fold activation, from about 25 to 135 units/mg(18) , whereas we report a 1000-fold activation, from 0.1 to 115 units/mg. The discrepancy arises because the specific activity for the E. coli-expressed enzyme is 50-250-fold lower than values for the enzyme purified from yeast(15, 18) . The latter values are likely due to contamination by the phosphorylated enzyme form as a consequence of purifying from yeast, where the endogenous protein kinases activate a fraction of the enzyme.
The previously determined crystal structure of unphosphorylated yeast phosphorylase showed that the amino terminus of each subunit binds near the catalytic site of the neighboring subunit in the homodimer and suggested that inactivation could be a consequence of active site occlusion by the amino-terminal peptide(17) . However, the lack of activity in the Nd22 mutant, where 22 amino-terminal residues are removed, indicates otherwise. That Nd22 also requires phosphorylation for activation argues that phosphorylation of the WT enzyme functions other than to displace a steric hindrance to substrates. Although there is indication that the amino terminus serves to stabilize the inactive conformation of the enzyme, from the observation that eliminating the amino-terminal 42 residues leads to an appreciable activity, the Nd42 mutant falls far short of a fully activated WT enzyme and, structurally, may resemble more an inactive conformer. Whether or not the phosphopeptide region binds as a ligand to stabilize the active conformation, as observed for rabbit muscle phosphorylase, awaits the determination of the crystal structure of the phosphorylated enzyme.
The amino terminus is also involved in stabilizing the tetrameric form of the enzyme. While the unphosphorylated WT enzyme forms a stable tetramer, the Nd42 mutant remains dimeric under all conditions, and the Nd22 mutant exists as a mixture of dimer and tetramer in rapid equilibrium, indicating weakened dimer/dimer interaction. Results also indicate that the active conformation of the enzyme is incapable of forming stable tetrameric structure. The activated phosphorylated WT enzyme is dimeric, but the Glc-6-P-inhibited phosphorylated WT enzyme is tetrameric.
The crystal structure of unphosphorylated yeast
phosphorylase reveals that the tetrameric state of the enzyme is
inactive because the two dimers interact on opposing catalytic faces,
thereby limiting active site access to glycogen. Is tetramerization the
mechanism adopted by the yeast enzyme to switch off activity? Could the
enzyme be inhibited in the dimeric state? Two of our observations
indicate that the yeast enzyme could be inhibited as dimer. First,
Glc-6-P inhibits the Nd42 mutant with apparent K about 12.5 mM, while at 50 mM Glc-6-P, this
mutant remains dimeric. Second, Glc-6-P inhibits the phosphorylated WT
enzyme cooperatively with a K
of 5 mM,
yet the enzyme is dimeric at 50 mM Glc-6-P when 20 mM
maltoheptaose is present. Based on the assumption that maltoheptaose
mimics glycogen and binds to the glycogen storage site, the second
observation suggests that the dimer is the physiological relevant
inhibited form, as phosphorylase in action has to bind glycogen. It is
also worth noting that the cooperative effect of Glc-6-P inhibition on
the phosphorylated enzyme cannot be attributed to dimer to tetramer
transition, since glycogen in the assays assures the dimeric state of
the enzyme. The cooperative effect is more likely due to interaction of
the Glc-6-P binding sites within the dimer. It is possible that
tetramerization, which occurs only when the enzyme is not bound to the
glycogen particles, plays a role in stabilizing phosphorylase at rest.
It is interesting that tetramer formation in the absence of glycogen
also occurs for the muscle enzyme but only when it is phosphorylated.
The dimeric and tetrameric forms of yeast phosphorylase have distinct biochemical properties. The separation of the phosphorylated (peak 1) and the unphosphorylated (peak 2) forms by ion-exchange chromatography is likely due to different surface charge of the dimer and tetramer. The introduction of the phosphate group cannot alone account for the separation, as one would expect the phosphorylated enzyme to elute later than the unphosphorylated form on an anion exchanger. The two oligomerization states differ further in that the dimeric state is a better substrate for the protein kinase. This was demonstrated by the faster rate of phosphorylation of the Nd22 mutant, and the enhanced rate of phosphorylation of the WT enzyme in the presence of maltoheptaose. Hence, another possible role for tetramerization is allowing the enzyme population bound on the glycogen particles to be phosphorylated preferentially when energy supply is needed.
The results from this study indicate that yeast glycogen phosphorylase is an allosteric enzyme and can be partly described using the two-state model of Monod et al.(28) . The unphosphorylated form is in the low activity T state. Phosphorylation stabilizes the high activity R state, which exhibits a hyperbolic velocity curve with respect to substrate, Glc-1-P. Glc-6-P is an allosteric inhibitor. It inhibits the unphosphorylated enzyme noncooperatively by stabilizing the T state, and it inhibits the phosphorylated enzyme cooperatively, causing an R to T state transition. A model describing the allosteric transitions between glycogen bound and free forms is shown in Fig. 8. The unphosphorylated T state enzyme can exist as either dimer (glycogen bound) or tetramer (glycogen free). The dimeric T state enzyme is a better substrate for protein kinase than the tetrameric T state enzyme. Covalent phosphorylation results in the formation of dimeric R state, which can be converted, upon Glc-6-P binding, to either tetrameric T state (glycogen free) or dimeric T state (glycogen bound).
Figure 8: A model showing the activity and quaternary structure relationship of yeast glycogen phosphorylase between glycogen-free and glycogen-bound states under different conditions. Squares with rounded edges indicate unphosphorylated enzyme with low activity. Squares represent completely inactive enzyme (T state), and circles represent fully active (R state) yeast phosphorylase. The letter P represents the phosphorylated enzyme.
It is intriguing that despite the conservation of the glucose-binding residues between the rabbit muscle and yeast enzymes, glucose is a poor inhibitor for the yeast enzyme. One speculation is that protein structure surrounding the site could influence these residues to bind glucose effectively. Understanding this observation requires a structural determination of the glucose bound yeast phosphorylase. Diffraction quality crystals of the phosphorylated yeast phosphorylase in complex with glucose have recently been obtained in this laboratory, and the structure determination is underway.