Int. J. Biochem. Cell Biol. Vol. 27, No. 9, pp.

91 l-916, 1995
Copyright 0 1995 Elsevier &ace Ltd
Printed in Great Britain. All rights reserved
1357-2725(9!JpOOS5
1357-2725/95 S9.50 + 0.00

Caffeine Inhibition of Glycogen Phosphwylase from

Mytilrrs gdoprotrincialis Mantle Tissue
FUENCISLA SAN JUAN SERRANO, JO?& LUIS S~NCXEZ LOPEZ,
L. OSCAR GARCIA MARTfN
Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad de Santiago
de Compostela, Avda. de las Ciencias s/n, 15706 Santiago de Compostela, Spain
A different caffeine inhibition of both phosphorylnted and unpbospborylated forms of giycogen
pbospboryiase from Mytilns mantle has been demonstrated. Caffeine increases the ahteric
constant of phosphorylaseb 3&fold, acting as POalloskric inhibitor (IQ,= 2) of mixed type with
respect to inorganic phospbate (Pi) and AMP, and of single competitive type with respect to
glycogen. Ibe Mytih
pbospborylated form is also caffeine inhibited Wougb competitive
iddbition in relation to Pi and glycogen. In this cawz,the inhibitor does not modify the alhteric
tmnstmt (near 2), neither does it display akasteric effects (nH = 1). Tbe results demonstrate the
notable modifkation of the nucleotide site promoted by tbe phospborylation process and the
existence of a functional inhibitory nucleoside site in My&s phosphorylase.
Keywords: Mytilus

Phosphorylase

Inhibition

Caffeine

Znt. J. Biochem. Cell Biol. (1995) 27, 911-916

loci; the nucleoside (or inhibitor) site and

the glycogen storage site (Johnson et al., 1989;
Dombrhdi,
1981; Newgard et al., 1989).
Nucleosides, purine base analogues and heterocyclic compounds bind to the nucleoside site,
thereby inducing an inactive conformation and
acting like inhibitors (Steiner et al., 1980). Such
compounds are able to bind the nucleotide site
secondarily in competition with the allosteric
effector, thus increasing their inhibitor effect
(Johnson et al., 1979).
In the present study, caffeine was used to
obtain a better understanding of the function of
two AMP binding sites (nucleotide and nucleoside) in phosphorylase from Mytilus mantle and
the role of free nucleosides described in molluscs
(Moal et al., 1987).

INTRODUCTION

Glycogen phosphorylase (E.C. 2.4.1.1) catalyses the degradation of glycogen to glucose- lphosphate
under physiological
conditions.
The enzyme can exist in two different forms,
designated phosphorylase a and b, being active
and inactive in the absence of AMP, respectively. Phosphorylase activity can be regulated
by covalent modification
(phosphorylationdephosphorylation
reactions) (Fischer et al.,
1959) and by allosteric modulation mediated by
conformational
changes induced by ligands
binding to specific sites within the enzyme structure (Johnson et al., 1989).
Four separate ligand binding sites were localized within the vertebrate glycogen phosphorylase structures: the active site which binds the
substrates inorganic phosphate (Pi), glycogen
and glucose- 1-phosphate; the nucleotide site
or AMP allosteric site, which consists of three
different subsites, namely the phosphoryl group,
the ribosyl residue and the purine base-binding

MATERIALS

AND

METHODS

Glycogen phosphorylase preparations were

obtained from mussel mantle tissue previously
stored at - 30C. Phosphorylase b was prepared
by homogenization in a 40 mM Tris-acetate
buffer pH 7.0, containing 5 mM EDTA, imidazole and 2-mercaptoethanol and 0.12 M KCl.

*To whom all correspondence

should be addressed.
Received
13 July 1994; accepted 1 May 1995.
911

912

Fuencisla San Juan Serrano et a/

Homogenate in the same conditions as above,

but without EDTA, was incubated with ATP,
MgCl, and CaCl, in a concentration of 2 mM in
order to obtain a phosphorylase a suspension.
In this medium, endogenous phosphorylase
kinase transforms phosphorylase b into the
a form (San Juan et al., 1995a). Both homogenates, containing b and a phosphorylase forms
separately, were centrifuged at 15,4OOg,, for
20 min and the pellet was discarded. The supernatans were dialysed with 30 vol of extraction
buffer containing IO mM NaF, but without
KCl.
Phosphorylase activity was examined in the
direction of glycogen breakdown with a couple
enzyme system, similar to the one described
in Childress and Sacktor (1970). The reaction
mixture for phosphorylase b contained 40 mM
Tris-acetate buffer pH 7.0, 5 mM imidazole,
2 mM EDTA,
1.4 mM 2-mercaptoethanol,
5 mM acetate-Mg, 5 p M glucose ld-diphosphate, 0.6 mM NADP, 80 mM phosphate buffer
pH 7.0, 1.6 mM AMP, 16 mg/ml glycogen,
0.5 IU glucose-6-phosphate
dehydrogenase,
0.1 IU phosphoglucomutase
and the enzyme
preparation in a total volume of 1.0 ml. EDTA
and AMP were not added when assaying
for phosphorylase a, and the glycogen and
phosphate buffer concentrations were half the
reaction mixture for the b form.
The substrates, enzymes and coenzymes were
obtained from Sigma Chemical Co. The salts
and other chemicals were purchased from
Merck.
The inhibitory effect caused by several concentrations of caffeine was determined by a
means of provoked modifications in the kinetic
constants of glycogen phosphorylase in equilibrium, in relation to its substrates and specific
effecters.
The inhibitor
action mechanism
was
determined, as a first approximation,
using
Lineweaver-Burk
double reciprocal plots.
When the enzyme activity no longer coincided
with Michaelis-Menten
kinetics and this plot
became non-linear, plots of reciprocal velocity
versus l/[S]* were used. When the inhibitory
effect responded to a simple competitive model,
then the inhibition constants were calculated
from this plot and when it responded to a mixed
inhibition model, then Ki was estimated from
a secondary plot of double reciprocal slopes
versus inhibitor concentrations (Segel, 1975).
The allosteric constant (L) was calculated
from the V,,,,, values obtained in the double

reciprocal plots. The data was analysed according to Buts equation (But, 1967) for the exclusive bound model.
RESULTS

AND

DISCUSSION

Caffeine inhibition of both forms (phosphorylated and unphosphorylated) of glycogen

phosphorylase from Myths
mantle tissue
has been proved. The inhibitor effect of this
molecule is different in each form of mussel
enzyme.
Phosphorylase b inhibition
The binding of this molecule to the unphosphorylated form (b form) stabilized an inactive
conformation. This is confirmed by an increase
in the allosteric constant L (constant equilibrium for the molar ratio between inactive and
active conformers) from values of 50, in the
absence of inhibitor (San Juan et al., 1995b),
to 1500 in the presence of increasing caffeine
concentrations (Fig. 1).
In relation to glycogen, caffeine appears
to affect phosphorylase b by competitive
inhibition, with a K, of 0.18 mM (Fig. 2). The
enzymes affinity for the substrate decreases
notably when the inhibitor concentration is
increased and V,,,,, is maintained constant at
all caffeine concentrations. However, a slight
decrease in maximum velocity is observed at
high caffeine concentrations (0.4 mM) (Table 1).
The phosphorylase b conformation, promoted
by caffeine binding to the nucleoside site, results

0.1

0.2

0.3

0.4

0.5

[Caffeine] mM
Fig. 1. Determination of allosteric constant for glycogen
phosphorylase b from Mytilus mantle at severalfixed levels
of Pi: (0) 60 n&I, (a) 40 mM, (A) 20 mM, (A) 15 mM,
and in the presence of increasing caffeine concentrations.

h4ytilu.s

I
0.6

I
1.8

I
1.2

l/[Cilycogen]

I
2.4

mg/ml

Fig. 2. Double reciprocal plots of initial velocity of phosphorylase b as a function of glycogen concentration in the
presence of several caffeine concentrations: (0) 0 mM, (0)
O.lmM, (A) 0.2mM, (A) 0.3mM, (0) 0.4mM.

in less affinity of the active centre for the

substrates. Caffeine is thus shown to be like
a competitive inhibitor, but mediated by negative heterotropic effects since caffeine does
not compete directly with the substrate for the
same binding site. The I,,,, decrease at high
inhibitor concentrations may be a consequence
of secondary inhibitory binding to the nucleoTable 1. Modification of the kinetic parameters of glycogen
phosphorylase b from Myths
mantle with respect to substrates (glycogen and Pi) and effector (AMP) in the presence
of several caffeine concentrations
Caffeine

(I$%)
Pi (SO.OmM);
K,,,

OmM
0.1 mM
0.2 mM
0.3 mM
0.4 mM

AMP

(24mgjml);

tide site, thereby competing with the allosteric

effector AMP.
With regard to the Pi cosubstrate and allosteric effector, the inhibitor effect of caffeine
is neither subject to the nomenclature of competitive inhibition described by Segel (1975),
nor to the concerted symmetry model of allostery proposed by Monod et al. (1965) for
vertebrate enzymes. In the aforementioned
model, the caffeine effect on phosphorylase may
be regarded as inhibition in an exclusive binding
K system. However, the Lineweaver-Burk plots
obtained from mussel phosphorylase b were
non-linear and the reciprocal velocity plots
versus l/[S]* did not show any classical competitive inhibition. Both affinity and V- decreased
in the presence of caffeine, which indicates
that caffeine was acting as a mixed inhibitor
(Figs 3 and 4; Table 1). Mixed type inhibition is
defined as a combination of competitive and
non-competitive inhibition. From our results,
we believe that this is due to, on the one hand,
a non-competitive inhibition by caffeine binding
at the nucleoside site, through negative cooperativity effects and, on the other hand, a
competitive inhibition by caffeine binding at the
nucleoside site: caffeine competes directly with
AMP for the allosteric activator site and, at the
same time, hinders the Pi access to its specific
subsite within this locus. Thus, at high inhibitor
concentrations, Pi is moved from the nucleotide
binding site to the catalytic centre, and AMP

(1.6mM)

(mg/ml) for glycogen

0.37
0.96
1.48
2.22
2.34

Glycogen

913

glycogen phosphorylase

AMP

0.098
0.102
0.098
0.106
0.087
(1.6mM)

S,, (mM) for Pi

OmM
0.1 mM
0.2 mM
0.3 mM
0.4 mM

OPM
25j~M
50pM
1OOpM
3OOpM
500nM

8.00
13.77
16.89
18.96
20.58

0.129
0.100
0.075
0.066
0.051

Glycogen (24 mg /ml); Pi (80 mM)

K, (pM) for AMP
0.162
112
129
0.152
0.144
227
0.110
339
425
0.077
673
0.074

1.8
1.8
2.1
2.3
2.5

1.2
1.2
1.1
1.1
1.8
2.2

0.1 0.2 0.3 0.4

[Caffeine] mM
I
0

l/[Pi]* x IO*
Fig. 3. Reciprocal plots of initial velocity of phosphorylase b as a function of reciprocals of Pi concentration
squared in the presence of several caffeine concentrations:
(0) OmM, (0) O.lmM, (A) 0.2mM, (A,) 0.3mM,
(0) 0.4mM. The Ki for caffeine was calculated from the
secondary plot of the slopes versus inhibitor concentration
(inset).

Fuencisia San Juan Serrano et al

ase b displays cooperative binding and that this

molecule acts as an allosteric inhibitor.
Phosphorylase a inhibition

I
10

20

30

lI[AMP]

lCaffeine1
1
40
50

mM

mM

Fig. 4. Reciprocal plots of initial velocity of phosphorylase

b as a function of reciprocals of AMP concentration squared
in the presence of several caffeine concentrations: (0)

0 mM, (0) 25 pm, (A)

50@,

(A)

0.1 mM,

(0)

0.3

mM,

(m) 0.5mM. The K, for caffeine was calculated from the

secondary plot of the slopes versus inhibitor concentration
(inset).

and caffeine compete for both binding sites,

nucleotide and nucleoside, as the increase of
Hill coefficients for Pi and AMP demonstrates
(Table 1).
The Ki values of this inhibitor, with regard to
Pi and AMP (0.3 and 0.1 mM, respectively), was
calculated from the inserted plots in Figs 3 and
4. A Hill plot (not shown), as a function of log
[caffeine], yields a slope of 2. All of this suggests
that the nucleoside site of Mytilus phosphoryl-

The phosphorylated form (a form) of Myti1u.s

phosphorylase is not stimulated by any AMP
concentrations (San Juan et al., 1995b). It is
also inhibited by nucleoside analogues such as
caffeine, thus differing from studied phosphorylases of other sources (Wang and Graves, 1964;
Birnbaum and Fain, 1977; Burges et al., 1983;
Deaciuc and Spitzer, 1986; Kamp, 1986).
Caffeine inhibition, in mussel phosphorylase
a, responds to a competitive inhibition as
regards both enzyme substrates (glycogen and
Pi). Such inhibition proceeds through negative
heterotropic effects, the same as in the b form
(Fig. 5). In both cases the inhibition only
affects the affinity for the substrates. In reiation
to glycogen, this inhibition results in a drastic
increase K,,, (up to 20-fold in the presence
of high caffeine concentrations). However, in
the case of Pi this parameter is only modified
slightly (Table 2).
The allosteric constant for this phosphorylase
form in the presence of caffeine was calculated
to be almost 2. This is the same value as in the
absence of the inhibitor (San Juan et al., 1995b).
Likewise, the slope of the Hill plot as a function
of caffeine concentration (not shown) is almost
1. The affinity of this form for caffeine is much
higher than the b form. The Ki values obtained
from Lineweaver-Burk
plots (Fig. 5) as a

(B)

10

20

30

40

l/[Pi] mM
l/[Glyc] mg/ml
Fig. 5. Double reciprocal plots of initiai velocity of phosphorylase a as a function of glycogen (A)
and Pi concentration (B) in the presence of several caffeine concentrations: (0) OmM, (#) 0.1 mM,
(A) 0.2 mM, (A) 0.3 mM, (0) 0.4 mM.

915

Mytilus glycogen phosphorylase

Table 2. Modification of the kinetic parameters
of glycogen phosphorylase a from Mytilus mantle
with respect to substrates (glycogen and Pi) in the
presence of several caffeine concentrations
V
Caffeine
(IU%l)
Pi (3044)
K, (mg/ml) for glycogen
OmM
0.05
0.106
0.1 mM
0.21
0.096
0.2 mM
0.43
0.103
0.3 mM
0.58
0.109
0.4 mM
0.96
0.109

OmM
0.1 mM
0.2 mM
0.3 mM
0.4 mM

Glycogen (8 mg /ml)
K,,, (mM) for Pi
1.06
2.06
2.24
2.47
2.45

inhibition constants found in Myths and free

nucleoside levels described in molluscs (Moal
et al., 1987).
In addition, this data confirms the secondary
binding of caffeine and analogues to the allosteric effector site in phosphorylase b, as well as
the important change in this site by covalent
modification, demonstrated by a different inhibition way and different values of Ki in both
enzymatic forms.
Acknowledgement-This
work was supported by a grant
(XUGA 20305 B90; Conselleria de Educacibn) from the
Autonomous Government of Galicia (Spain).

0.098
0.101
0.101
0.105
0.102

function of glycogen and Pi are 25 PM and

0.1 mM, respectively. Although AMP binding
to Mytiius phosphorylase a has not been verified, the presence of 1.6 mM AMP decreased
the caffeine inhibition by 55%.
These results obtained from Mytilu.s phosphorylase a, compared with the results of phosphorylase b, reflect the notable modification
provoked by the phosphorylation process in its
quaternary and tertiary structure (Bardford
et al., 1991). Furthermore, they suggest that the
purine base-binding subsite, within the allosteric
locus, is the most affected. There seems to
exist only one binding site for the nucleoside
analogues in the Myths phosphorylated form.
This hypothesis explains the pure competitive
inhibition, the inhibition decrease in the presence of AMP, which would therefore bind to the
nucleoside site, and the high affinity for caffeine.
In this enzyme form, we would only be measuring the affinity of the specific binding site for
the purine base analogues while, at the same
time, we would be measuring in the b form, the
affinity of its secondary binding site (nucleotide
site).
Despite the fact that caffeine cannot be considered to be a physiological effector for Mytilus
glycogen phosphorylase, the kinetic studies
of the present communication demonstrate the
existence of a functional nucleoside (caffeine)
site in Mytilus phosphorylase. Such a site, with
inhibitory responses of the enzyme activity, may
play an important regulatory physiological
role in vim, as a negative heterotropic effector
site, especially when we consider the low

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