(Received for publication, December 27, 1994; and in revised form, May 8, 1995)
From the
Phosphorylase kinase is a multimeric protein kinase
()
whose enzymatic activity is conferred by its
-subunit. A library
of 18 overlapping synthetic peptides spanning residues 277-386 of
the
-subunit has been prepared to use in identifying important
regulatory structures in the protein. In the present study, the library
was screened to identify regions that might function as autoinhibitory
domains. Peptides from two distinct regions were found to inhibit the
Ca
-activated holoenzyme. The same regions were
previously found to bind calmodulin (i.e. the
-subunit;
Dasgupta, M. Honeycutt, T., and Blumenthal, D. K.(1989) J. Biol.
Chem. 264, 17156-17163). The most potent substrate
antagonist peptides were PhK13 (residues 302-326; K
= 300 nM) and PhK5
(residues 342-366; K
= 20
µM). Both peptides inhibited the holoenzyme competitively
with respect to phosphorylase b and noncompetitively with
respect to Mg
ATP. When the pattern of inhibition with both
peptides present was analyzed, inhibition was observed to be
synergistic and modestly cooperative indicating that the two peptides
can simultaneously occupy the protein substrate-binding site(s). These
data are consistent with a model in which the regions of the
-subunit represented by PhK5 and PhK13 work in concert as
regulatory subdomains that transduce Ca
-induced
conformational changes in the
-subunit to the catalytic
-subunit through a pseudosubstrate autoinhibitory mechanism.
Phosphorylase kinase is among the largest and most complex of
the protein kinase superfamily (reviewed by Pickett-Gies and
Walsh(1) ). Because of its complex structure, the catalytic
activity of phosphorylase kinase is sensitive to a variety of effectors
and chemical modifications including pH, divalent metal ions, ionic
strength and composition, glycogen, calmodulin, nucleosides,
phosphorylation state, limited proteolysis, and substrate conformation.
The subunit composition of phosphorylase kinase is
,
where the
-,
-, and
-subunits are regulatory and the
-subunit harbors the catalytic site. The
- and
-subunits
can be phosphorylated, and this modification increases the
enzyme's catalytic activity, whereas the
-subunit is
identical to calmodulin and confers Ca
dependence to
the enzyme's activity. Although the
-,
-, and
-subunits are all thought to interact directly with the
-subunit, the molecular mechanisms by which these regulatory
subunits effect changes in catalytic activity is not well understood at
present.
A common mechanism by which the catalytic activity of many protein kinases appears to be regulated is through a pseudosubstrate autoinhibitory mechanism (see reviews by Soderling (2) and Kemp and Pearson(3) ). This intrasteric form of regulation was first described in the cAMP-dependent protein kinase, but there is now substantial evidence that this mechanism may also be operating in the cGMP-dependent protein kinases, myosin light chain kinases, calmodulin-dependent type II kinases, the protein kinase-Cs, as well as the protein phosphatase calcineurin(2, 3) . The simplest mechanistic model for this form of regulation consists of two conformational states: an autoinhibited enzymatically inactive (or less active) state and a deinhibited active (or more active) state. In the autoinhibited state, an autoinhibitory regulatory domain occupies the enzyme's substrate-binding site and prevents access of the protein substrate to the catalytic site. Upon binding of an allosteric activator to the regulatory domain, a conformational change is induced in the autoinhibitory domain that removes it from the substrate-binding site allowing free access of the substrate to the catalytic site.
Typically, the amino acid sequence of an autoinhibitory domain closely resembles the consensus sequence of that enzyme's preferred substrates and is then termed a pseudosubstrate domain. In a number of cases, protein kinase pseudosubstrate domains also contain phosphorylatable residues, and these residues can be autophosphorylated. Thus, identification of putative pseudosubstrate autoinhibitory domains often entails visual inspection of a protein kinase's regulatory domain for sequences that resemble consensus substrate sequences. Candidate pseudosubstrate sequences are then chemically synthesized and tested for their ability to inhibit the enzyme in question. Although the peptide representing an enzyme's pseudosubstrate domain should act as a competitive inhibitor of the protein (or peptide) substrate, this is not always the case(2, 3) . Moreover, it is conceivable that selecting a putative pseudosubstrate domain on the basis of similarity to a substrate consensus sequence could result in a false positive identification, particularly if the inhibitory capacity of the putative synthetic pseudosubstrate is low. Thus, positive identification of putative pseudosubstrate domains must be supported by other data including site-directed mutagenesis and x-ray crystallographic structures.
The present investigation is the second phase of a long
term effort to characterize the structure-function relationships of the
regulatory domain of the -subunit of phosphorylase kinase. The
regulatory domain of the
-subunit is defined here as the region
spanning amino acids 277-386 of this subunit. The first phase of
this investigative effort involved identification of putative
-subunit-binding domains in the regulatory domain by preparing a
library of 18 overlapping synthetic peptides spanning residues
277-386 and screening each peptide for its ability to bind
calmodulin(4) . Two noncontiguous peptides were identified as
being likely
-subunit-binding domains. In the present study, the
same synthetic peptide library has been used to screen for possible
-subunit autoinhibitory domains. Two noncontiguous regions in the
-subunit regulatory domain have been identified that display the
properties expected of pseudosubstrate autoinhibitory domains. These
results are being used to direct structural studies to further
elucidate the molecular mechanisms underlying the regulation of
phosphorylase kinase catalytic activity.
where i is the fractional inhibition observed in the
presence of peptide inhibitor, [I] is the concentration of
peptide inhibitor, [S] is the concentration of phosphorylase b (30 µM) used in the reaction mixture, K is the Michaelis constant for phosphorylase b (33 µM), and v
and v
are the reaction rates in the presence and
absence of peptide inhibitor, respectively. K
values were determined by Marquardt weighted non-linear
least-squares fit estimation using the MathView Professional (v 1.2)
program run on a Macintosh computer. The assumption of competitive
inhibition used in these calculations was validated by kinetic analysis
(see ``Results'').
The purpose of the present investigation was to determine the
possible location of autoinhibitory domain(s) within the C terminus of
the -subunit. The approach taken was analogous to that previously
used to identify
-subunit (integral calmodulin)-binding domains in
the
-subunit. This approach utilizes a library of eighteen
overlapping 25-residue synthetic peptides (the sequences and
nomenclature are shown in Table 1) based on the C-terminal 110
residues of the
-subunit. The library was constructed in such a
way that every 20-residue segment between residue 277 and the C
terminus (residue 386) of the
-subunit is represented in one of
the 18 peptides. Each peptide in the library was assayed for its
ability to inhibit the catalytic activity of nonactivated
(nonphosphorylated) phosphorylase kinase holoenzyme. The assay reaction
was performed at pH 8.2 in the presence of Ca
with
limiting concentrations of the substrate, phosphorylase b.
Under these conditions, a peptide that can compete with phosphorylase b for the substrate-binding site of the enzyme will inhibit
enzyme activity. The results of the assay are depicted in Table 1, where the K
value of each peptide
is compared with its previously determined K
for
calmodulin(4) . The K
value of each
peptide was calculated as described under ``Experimental
Procedures.'' Peptides that showed no appreciable inhibition of
phosphorylase kinase activity at a concentration of 50 µM are indicated as having K
values >100,000
µM.
The data in Table 1indicate that the
-subunit regulatory domain peptides which exhibit the most potent
inhibitory activity are located in two distinct regions that closely
correspond to the regions previously shown to be involved in
-subunit interactions(4) . Moreover, the peptides from
each region that most strongly inhibited phosphorylase kinase activity,
PhK5 and PhK13 (K
values of 20 µM and
300 nM, respectively), are the same peptides from each region
that were previously found to bind calmodulin with the highest affinity (K
values of 20 and 6.5 nM,
respectively(4) ). The K
values estimated
for PhK5 and PhK13 are both within the range of values reported for
autoinhibitory domain peptides from other protein kinases (40 nM to 25 µM; see reviews by Soderling (2) and
Kemp and Pearson(3) ).
To determine the mechanism(s) by
which PhK5 and PhK13 inhibit the catalytic activity of phosphorylase
kinase, the kinetics of inhibition for each peptide were analyzed as a
function of phosphorylase b and ATP concentration. The data
obtained with PhK13 and PhK5 are plotted in Fig. 1. Fig. 1A shows double-reciprocal plots of
phosphorylation rates as a function of phosphorylase b concentration obtained when nonactivated phosphorylase kinase was
assayed in the presence of Ca (100 µM),
a fixed concentration of ATP (1 mM), and various fixed
concentrations of PhK13 (0.3-3 µM). The intersection
of the double-reciprocal plots for the various concentrations of PhK13
on the (1/v)-axis at [phosphorylase b]
= 0 indicates that PhK13
acts as a competitive inhibitor of phosphorylase b. A replot
of the slopes in Fig. 1A as a function of PhK13
concentration (shown in Fig. 2A) yields an estimate of
300 nM for the value of the K
, the same
value as estimated by non-linear curve-fitting procedures (Table 1). Double-reciprocal plots of phosphorylase kinase
activity as a function of ATP concentration, at various fixed
concentrations of PhK13, and at a fixed concentration of phosphorylase b (30 µM) are shown in Fig. 1B.
In this case, the double-reciprocal plots intersect on the
[ATP]
-axis indicating that PhK13 acts as a
pure noncompetitive inhibitor with respect to ATP(15) .
Figure 1: Double-reciprocal plots of phosphorylase kinase inhibition by PhK5 and PhK13. Panels A and B show double-reciprocal plots of phosphorylase kinase inhibition by the indicated concentrations of PhK13 as a function of phosphorylase b and ATP concentration, respectively. Panels C and D show the corresponding plots for inhibition by PhK5. The concentration of ATP used in panels A and C was 1 mM. The concentration of phosphorylase b used in panels B and D was 30 µM. Other details of the reaction conditions are given under ``Experimental Procedures.''
Figure 2:
Determination of Kvalues for PhK13 and PhK5 from replots of slope versus peptide concentration. Slopes of lines determined from plots shown
in Fig. 1, A and C, were replotted as a
function of PhK13 and PhK5 concentration, respectively, to determine K
values for PhK5 and PhK13 with respect
to phosphorylase b.
The
double-reciprocal plots shown in Fig. 1indicate that the
peptide PhK5 inhibits the catalytic activity of phosphorylase kinase in
a manner similar to PhK13. Fig. 1C shows a series of
plots in double-reciprocal format where the x axis represents
the reciprocal of the concentration of phosphorylase b, the y axis represents the reciprocal of the reaction rate, and
each line represents data obtained at the indicated concentration of
PhK5. As in the case of PhK13, the lines converge on the y axis at [phosphorylase b] = 0, indicating competitive inhibition by PhK5 with
respect to phosphorylase b. A replot of the slopes of the
lines in Fig. 1C as a function of PhK5 concentration (Fig. 2B) yields a K
value for
PhK5 of 20 µM, the same value obtained by non-linear
curve-fitting procedures (Table 1). The double-reciprocal plots
shown in Fig. 1D, where the rate of phosphorylase
kinase activity is plotted as a function of ATP concentration at
various fixed concentrations of PhK5, indicate that PhK5 exhibits
hyperbolic (partial) mixed-type inhibition with respect to
ATP(15) . A hyperbolic mixed-type inhibition pattern indicates
that PhK5 causes a change in the K
for ATP, as
well as a change in the V
, and is thus acting as
a partial noncompetitive inhibitor. Intersection of the
double-reciprocal plots below the x axis (1/[ATP])
indicates that PhK5 causes a decrease in the K
for
ATP(15) . Secondary replots (not shown) of the reciprocal of
the y axis intercept (1/
intercept) as a function of the
reciprocal of PhK5 concentration indicate that binding of PhK5 to
phosphorylase kinase decreases the K
for ATP by
about 2-fold (
= 0.47) and reduces V
by about 5-fold (
= 0.18). A pure noncompetitive
inhibitor would have no effect on K
(
= 1) and would totally block catalysis when bound to the enzyme
(
= 0).
Thus, PhK5 and PhK13 both act as competitive inhibitors of phosphorylase b, and both are noncompetitive inhibitors of ATP, although unlike PhK13, PhK5 is not a pure noncompetitive inhibitor of ATP. The pattern of inhibition seen with PhK5 and PhK13 is consistent with the phosphorylation reaction mechanism being a sequential rapid-equilibrium random Bi-Bi mechanism, as previously reported(16, 17, 18) . The pattern of inhibition seen here is similar to what was reported using synthetic peptides based on the putative autoinhibitory domain of skeletal muscle myosin light chain kinase, an enzyme that also exhibits a rapid-equilibrium random Bi-Bi mechanism (19, 20) . Those peptides inhibited myosin light chain kinase competitively with respect to phosphate acceptor substrate and noncompetitively with respect to ATP(21) . As in the case of myosin light chain kinase(21) , addition of a molar equivalent of calmodulin relative to inhibitor peptide was able to overcome inhibition (data not shown).
The finding that both PhK5 and PhK13 are competitive
inhibitors of phosphorylase b phosphorylation raises the
question as to whether the peptides can act synergistically or whether
their inhibition is mutually exclusive. To answer this question, the
activity of phosphorylase kinase inhibited by mixtures of PhK5 and
PhK13 was analyzed graphically using Dixon plots, as shown in Fig. 3. In Fig. 3A, the reciprocal of the
reaction rate is plotted as a function of PhK13 concentration at
various fixed concentrations of PhK5. The same data are shown in Fig. 3B, but in this plot the reciprocal of the
reaction rate is plotted as a function of PhK5 concentration, with each
line representing a different concentration of PhK13. In Fig. 3, A and B, the lines converge indicating that
inhibition by PhK5 and PhK13 with respect to phosphorylase b is synergistic(15) . Parallel lines would have indicated
that the two peptides inhibited the enzyme in a mutually exclusive
manner. The point at which the lines converge in each plot provides
information regarding the degree of cooperativity between the two
inhibitors. The x axis value where the lines converge is equal
to -K
, where K
is
the inhibition constant for the inhibitor that was varied and
is
the coefficient of cooperativity. When
= 1, it indicates a
lack of cooperativity between the inhibitors. When
> 1, it
indicates that inhibitor binding is negatively cooperative, whereas
when
< 1, it indicates that inhibitor binding is positively
cooperative. The values of
calculated from Fig. 3, A and B, were 0.35 and 0.67, respectively, based on the
previously determined K
values for PhK13 and PhK5
(0.3 and 20 µM, respectively; cf. Table 1and Fig. 2). These two estimates for
are in
reasonable agreement with each other and indicate that the simultaneous
binding of PhK5 and PhK13 to the phosphorylase b substrate-binding site shows modest positive cooperativity (0.3
0.7). These values indicate that the binding of one
peptide to the enzyme enhances the binding of the other peptide by
about a factor of 2. Synergistic inhibition between PhK5 and PhK13 also
indicates that phosphorylase kinase contains a distinct binding site
for each peptide. Taken together with the data that both peptides are
competitive inhibitors of phosphorylase b suggests that
phosphorylase kinase may have two distinct binding sites for
phosphorylase b.
Figure 3: Inhibition of phosphorylase kinase activity by mixtures of PhK5 and PhK13. Phosphorylase kinase activity was determined at the indicated concentrations of PhK5 and PhK13 and represented in Dixon plots. Panel A depicts the effects on phosphorylase kinase activity of fixed concentrations of PhK5 as a function of PhK13 concentration. The same data are replotted in panel B, but as a function of PhK5 concentration at fixed concentrations of PhK13. The concentrations of phosphorylase b and ATP used in these reactions were 30 µM and 1 mM, respectively. Other assay conditions are detailed under ``Experimental Procedures.''
To better define the determinants in PhK5
and PhK13 critical for autoinhibitory function versus -subunit interaction, a series of truncated peptides was
prepared and assayed for phosphorylase kinase inhibitory activity and
calmodulin binding activity. Because standard solid-phase peptide
synthesis proceeds from the carboxyl terminus toward the amino terminus
of the peptide, a series of truncation peptides which share a common
carboxyl terminus can be easily prepared by removing aliquots of
peptide resin at different stages during the synthesis. Table 2shows the sequences, calmodulin-binding affinity,
estimated K
for autoinhibition, and nomenclature
for the eight peptides that were synthesized. The sequences of PhK5,
PhK6, and PhK13 and their respective data are also presented in Table 2for purposes of comparison.
Because PhK5 and PhK6
exhibit similar K values for autoinhibition and
similar affinities for calmodulin, it is reasonable to propose that the
minimum essential determinants for autoinhibition and
-subunit
binding function for this regulatory subdomain would be contained in
the sequence common to both peptides. This core sequence is represented
by the peptide termed PhK6E which has the same amino terminus as PhK5
and the same carboxyl terminus as PhK6, but which lacks the
carboxyl-terminal sequence of PhK5 (Gln-Gln-Gln-Asn-Arg) and the
amino-terminal sequence of PhK6 (Tyr-Ala-Leu-Arg-Pro). The K
value for autoinhibition with PhK6E is the same
as that for PhK6 and approximately 50% higher than for PhK5, suggesting
that the sequence Tyr-Ala-Leu-Arg-Pro (337-341) does not contain
determinants essential for autoinhibition, whereas determinants in the
sequence Gln-Gln-Gln-Asn-Arg (362-366) contribute modestly to the K
value. With regard to its affinity for
calmodulin, PhK6E binds calmodulin 5-fold weaker than PhK6 and 10-fold
weaker than PhK5, suggesting that the sequences Tyr-Ala-Leu-Arg-Pro
(337-341) and Gln-Gln-Gln-Asn-Arg (362-366) both contribute
significantly to the interactions of the
-subunit with the
-subunit. The peptide PhK6D, which is two residues shorter at its
amino terminus than PhK6E, had a K
value for
autoinhibition that was only about 30% greater than PhK6E, but bound
calmodulin about 8-fold weaker, indicating that Leu-Arg (342-343)
contributes significantly to
-subunit binding, but is only
marginally important for autoinhibition. Similar effects on affinity
for calmodulin and K
for autoinhibition were
observed when the sequence Arg-Leu-Ile (344-346) was removed on
going from PhK6D to PhK6C. The peptide PhK6B, which is three residues
shorter than PhK6C, has no detectable autoinhibitory activity at
concentrations as high as 50 µM, but still retains the
ability to bind calmodulin with low affinity (K
= 20 µM). The peptide PhK6A, which is two
residues shorter than PhK6B, has no detectable autoinhibitory activity
or affinity for calmodulin. Thus, it appears that the sequence
represented by PhK5 is the shortest sequence in the C-terminal
regulatory domain of the
-subunit with optimal autoinhibitory
activity. The differences in affinity for calmodulin observed between
PhK6 and PhK6E suggest that adding the sequence Tyr-Ala-Leu-Arg-Pro
(337-341) to the amino terminus of PhK5 to form a 30-residue
peptide (337-366) might increase the affinity for calmodulin
above that seen with PhK5, but this idea has not been tested.
The
studies with peptides based on PhK13 included two truncated peptides
that were shorter than PhK13 and one peptide that was one residue
longer at its amino terminus than PhK13 (Table 2). The longer
peptide PhK13C (301-326) had Arg added to its amino
terminus and showed a 6.5-fold higher affinity for calmodulin than
PhK13 and a K
for autoinhibition that was 33%
lower than for PhK13. These data suggest that this single Arg residue
is important for both
-subunit interactions and autoinhibition.
Removing the six residue sequence Arg-Gly-Lys-Phe-Lys-Val
(301-306) from the amino terminus of PhK13 resulted in a 10-fold
decrease in affinity for calmodulin and less than a 2-fold increase in
the K
for autoinhibition relative to PhK13.
Removing an additional five residues, Ile-Cys-Leu-Thr-Val
(307-311), resulted in a 15-residue peptide (312-326) that
had no detectable affinity for calmodulin and no detectable
autoinhibitory activity. These data suggest that the sequence
Ile-Cys-Leu-Thr-Val (307-311) is essential for both
autoinhibition and
-subunit interactions. Consistent with this
proposal is the finding that all of the PhK peptides that contain this
sequence (PhK12 through PhK16) have autoinhibitory and calmodulin
binding activity, whereas peptides immediately flanking these peptides
are lacking in both functional activities (Table 1).
Using a library of overlapping synthetic peptides based on
the regulatory domain of the -subunit of phosphorylase kinase, two
distinct noncontiguous regions, residues 302-326 (PhK13) and
342-356 (PhK5), have been identified as being putative
pseudosubstrate autoinhibitory domains. Peptides from each of these two
regions inhibited catalytic activity of phosphorylase kinase
competitively with respect to the phosphate acceptor substrate,
phosphorylase b, and noncompetitively with respect to the
phosphate donor substrate, Mg
ATP. Moreover, peptides from the two
regions inhibited catalytic activity in a synergistic manner,
indicating that both peptides can bind the
-subunit at the same
time. The inhibitory capacity of these peptides could be overcome by
the addition of a molar excess of calmodulin, consistent with their
previously being identified as calmodulin(
-subunit)-binding
domains(4) . This appears to be the first example of a
calmodulin-regulated protein kinase with multiple noncontiguous
synergistic autoinhibitory/calmodulin-binding subdomains.
Reimann et al.(22) first suggested that the C-terminal 110
amino acids of the -subunit (residues 277-386) might
constitute the regulatory domain of phosphorylase kinase. Subsequently,
limited proteolysis (23) and genetically engineered truncation
mutants(24, 25) of the
-subunit were used to
show that residues 298-386 contain an autoinhibitory domain. The
two putative autoinhibitory domain sequences identified in the present
study, 302-326 (PhK13) and 342-356 (PhK5), are both located
in this region of the
-subunit. Neither of these sequences
corresponds to the putative pseudosubstrate domain proposed by Kemp and
Pearson (3) who suggested that residues 332-353 might
represent the pseudosubstrate domain based upon inspection of the
-subunit sequence. Moreover, the peptide containing this latter
sequence, PhK7 (residues 332-356), showed virtually no inhibitory
activity in the present study (Table 1).
Because PhK5 and
PhK13 act as competitive inhibitors of phosphorylase b, some
sequence similarity between these peptides and phosphorylase might be
expected. Sequence alignment of phosphorylase with PhK5 and PhK13
reveals that both peptides contain short stretches of sequence that
closely resemble the sequence of phosphorylase (Fig. 4). The two
regions of highest sequence similarity correspond closely to the
regions required for substrate inhibitory activity based on the data
shown in Table 1and Table 2. It is particularly noteworthy
that a major portion of PhK13 shows sequence similarity with the
sequence surrounding Ser in phosphorylase b, the
phosphorylation site in phosphorylase b.
Figure 4:
Amino acid sequence alignment of
phosphorylase b(2-87), and putative phosphorylase kinase
pseudosubstrate autoinhibitory domains from the
-subunit(296-377) and the
-subunit(296-377).
Amino acid identities are indicated by (
), close chemical
similarities by (:), and distant similarities by (
). The
sequences of PhK5 and PhK13 are underlined in bold and the interconvertible Ser
of phosphorylase b is underlined. The alignment of
-subunit(296-377) is taken from Sanchez and
Carlson(32) .
The most
significant sequence similarity between PhK13 (Lys to
Arg
) and phosphorylase b (Lys
to
Lys
) spans 21 amino acids. This sequence is only found in
its entirety in PhK13, the peptide with the lowest K
value (0.3 µM) of the inhibitory peptides in the
region spanning PhK12 through PhK16 (Table 1). The sequence
Ile-Cys-Leu-Thr-Val appears to be especially important for inhibition
because it is shared by all of the inhibitory peptides in this group
and because truncation peptides based on PhK13 that lack this sequence
are not inhibitory (Table 2). Interestingly, the corresponding
pentapeptide from phosphorylase b is a competitive inhibitor
with a K
value of about 6 mM(26) , and Ile-Ser-Val-Arg-Gly-Leu (phosphorylase b(13, 14, 15, 16, 17, 18) )
is the shortest synthetic peptide substrate that can be phosphorylated
by phosphorylase kinase(16) . Other important determinants of
substrate recognition (26) which are identical in the alignment
of PhK13 and phosphorylase b include
Lys
(Lys
),
Lys
(Lys
),
Ile
(Ile
), and
Leu
(Leu
). Using
-subunit(1-300),
a truncated form of the
-subunit that lacks a regulatory domain,
Huang et al.(27) recently demonstrated that
Glu
and Glu
are involved in binding peptide
substrates at their P-3 and P-2 positions, respectively. Mutations of
these residues increased the K
value for PhK13 by
14- and 8-fold, respectively, indicating their involvement in binding
PhK13, as well as peptide substrates. Thus, the sequence corresponding
to PhK13 (residues 302-326) appears to represent a prototypical
pseudosubstrate domain that regulates the catalytic activity of
phosphorylase kinase by preventing the phosphorylation site of
phosphorylase b access to the catalytic site.
The ability
of PhK5 to act as a competitive inhibitor of phosphorylase kinase
holoenzyme (Fig. 1) and its sequence similarity to a region of
phosphorylase b approximately 50 residues C-terminal to
Ser (Fig. 4) suggest that phosphorylase b may have a major binding site on the holoenzyme in addition to the
binding site that recognizes the sequence around Ser
and
that this secondary binding site is also involved in autoinhibition.
The sequence in PhK5 showing the highest similarity to phosphorylase is
Arg-Ile-Tyr-Gly-His-Trp-Val-Lys (residues 352-359) which aligns
with His-Leu-Val-Gly-Arg-Trp-Ile-Arg (residues 62-69) in
phosphorylase (Fig. 4). This sequence is shared by PhK4, PhK5,
and PhK6, the only inhibitory peptides in this region of the
-subunit (Table 1). It is interesting to note that Graves
and co-workers have shown that synthetic peptides corresponding to the
sequence of phosphorylase b around its phosphorylation site
are poor substrates compared to the intact protein (26) and
that longer a CNBr fragment of phosphorylase b corresponding
to residues 1-99 (16) exhibited a K
value intermediate between that of the synthetic peptides and
native phosphorylase b. The data obtained in the present study
showing that PhK5 inhibits phosphorylase kinase activity
synergistically with PhK13 (Fig. 3) provides further evidence
that there are at least two distinct substrate binding sites for
phosphorylase b and that both of these substrate binding sites
might be used by autoinhibitory elements in the
-subunit to
regulate the catalytic activity of phosphorylase kinase. Inspection of
the several x-ray crystal structures of phosphorylase (28, 29) show that the phosphorylation site and the
PhK5-like region of phosphorylase are relatively close to one another
on the surface of the protein, adding support to the idea that these
two regions of phosphorylase might be simultaneously involved in
interactions with phosphorylase kinase.
The kinetics of inhibition
seen with PhK5 and PhK13 in the present study using phosphorylase
kinase holoenzyme are in basic agreement with studies done using a
-subunit truncation mutant,
-subunit(1-300), which
represents a minimal
-subunit catalytic subunit that lacks
calmodulin-binding and autoinhibitory pseudosubstrate
domains(27) . In the studies with
-subunit(1-300),
PhK5 and PhK13 were both found to be potent inhibitors, although both
PhK5 and PhK13 were slightly less potent (3-6-fold) inhibitors of
-subunit(1-300) than the holoenzyme. The patterns of
inhibition of PhK5 and PhK13 toward
-subunit(1-300) were
identical to those seen in the present study using holoenzyme, except
that PhK5 was a noncompetitive inhibitor with regard to phosphorylase b (as compared to being a competitive inhibitor in the case of
the holoenzyme) and a simple noncompetitive inhibitor with regard to
ATP (as compared to being a mixed noncompetitive inhibitor of the
holoenzyme). Because the
-subunit(1-300) represents a
minimal
-subunit catalytic subunit that lacks the many inter- and
intrasubunit interactions present in the holoenzyme, it is not
unexpected that some differences in potency and patterns of inhibition
might be observed for PhK5 and PhK13 between the two forms of the
enzyme.
In contrast to the findings of the present study, Newsholme et al.(30) have concluded that neither PhK5 nor PhK13 are likely to represent pseudosubstrate domains because both peptides appeared to be noncompetitive inhibitors of phosphorylase b in their experiments. However, the kinetic data obtained by these investigators for PhK13 could not conclusively discriminate between competitive and noncompetitive inhibition because of problems of solubility at high peptide concentrations. No such solubility problems were encountered in the experiments described here or in other studies involving relatively high concentrations of PhK5 and PhK13(27, 31) . The differences in results obtained cannot be readily explained but might be due to differences in assay conditions or the quality of peptides used in the two studies. PhK5 in particular has several amino acids that are especially sensitive to incomplete side chain deprotection and modification during synthesis, cleavage, and purification. All of the peptides used in the present study were subjected to peptide sequence analysis, quantitative amino acid analysis, and UV spectral analysis.
In addition to the two
putative autoinhibitory domains in the -subunit, Sanchez and
Carlson (32) have recently reported that residues 420-436
in the
-subunit of phosphorylase kinase may constitute a potential
autoinhibitory domain. The proposed sequence alignment relative to the
phosphorylation site in phosphorylase b is shown in Fig. 4. Kinetic analysis of a synthetic peptide based on the
-subunit 420-436 sequence indicated a pattern of inhibition
that was competitive with respect to phosphorylase b (K
= 921 µM) and
uncompetitive with respect to ATP using the
-
enzyme complex.
How these various autoinhibitory domains might interact in the
holoenzyme remains to be answered. One possibility is that each domain
acts independently to inhibit phosphorylase b binding. Another
possibility is that the domain on the
-subunit acts by inducing
conformational changes in the regions corresponding to PhK5 and PhK13,
which in turn alter catalytic activity. Elucidation of the precise
mechanisms by which these various autoinhibitory domains effect changes
in the enzymatic activity of phosphorylase kinase will require detailed
structural studies involving a variety of approaches including the use
of synthetic peptide analogs, site-directed mutagenesis, and x-ray
crystallography.