1. them. Rid. (1977) 68, 365-383
Double-site Enzymes and Squatting A Study of the Regulation by One or Several Ligaads Binding at Two Different Classes of Site JEAN-PIERRE
MAZAT
Laboratoire de Biochimie B.B.C., UniversitP de Bordeaux II. 351 Cows de la Lib&ration, 33405 Talewe, France JOBL LANGLA
AND FRANCON
MAZAT
Ecole Nationale SupPrieure des Arrs et Me’tiers, Avenue de I’ Universitt!, 33405 Talencr, Franczc (Received 4 February) The existenceof two (generally different) binding sitesper protomer fol the same ligand has already been observed (“double-site enzymes”). Furthermore, in some instances,the two sites appear to be shared in common by two or more ligands (“squatting”). The theoretical implications of such caseshave been emphasizedhere using a generalization of an allosteric V model. This model, by taking into account the competition betweenligandswhich occursin viva, evidencesvarious, or even peculiarregulatory patterns,and could be of generalphysiologicalinterest. It has beenillustrated here by somereal systems.
1. Introduction The existence of enzymes containing two binding sites per protomer for the sa.me ligand has already been described. Table 1 shows that such “doublesite” enzymes are uncommon. However, threonine-sensitive and lysinesensitive aspartokinases of Escherichiu coli K12 (Falcoz-Kelley et al., 1972: Richaud, Mazat, Felenbok & Patte, 1974) are good examples in this classof proteins: both possesstwo different sites per protomer for their allosteric inhibitor. In somecases,particularly for some aminoacyl tRNA synthetases (Koch. Boulanger & Hartley, 1974; Koch & Bruton. 1974; Kula, 1973; Sutton & Brew, 1974; Waterson & Konigsberg, 1974), the possible occurrence of two sites per protomer can be the result of the duplication of the polypeptide chain. 365
Ribonucleoside diphosphate reductase (E. coli Threonine sensitive aspartokinase (E. coli K 12) Eysine sensitive aspartokinase (E. coli K 12) Glutamate dehydrogenase (beef liver)
Enzymes
B)
4 12 12
6
BNADH aNADH
1 enzJvnes
-
-
-
Redundant sequencest
of double-site
No. of binding sites
2
No. of subunits
Lysine
Threonine
ATP dATP
Ligand studied
Examples
TABLE
+ -t
-
-
I-
Squattingt
jIh4
C(M
201~ and 3rn~
__-
Falcoz-Kelly CI al., 1972 Veron et al.. 1973
Brown & Reichard, 19696
__
References
Pantaloni & Dessen, 1969; Pantaloni & Lecuyer, 1973 Koberstein et al.. 1973
8 and 100 /IM Richaud et al.. 1973, 1974
10
Unknown 0.3 and I
Apparent dissociation constants§
4
GDP GTP
Iron
ATP
Transferrin
Phosphofructokinase (rabbit muscle) 2
3 per subunit
2
8
4
(A&
5 to tLi /AM
0.52 mhr 0.34 mhl
25 to 3X jihi
et al., 1973
Kemp & Krebs, 1967 Seydoux & Laurent (personal communication)
Coffee
Mann et al., 1970 Sutton & Brew, 1974
Koch & Brucon, !974 Blanquet el al., I972 Waygood et al., 1976
‘1 + when redundant sequence(s) exist(s); - when absence of redundant sequence is proven; nothing when nothing is known. +.>“Squatting” means: binding of a ligand at a site of another ligand; +, - and nothing have the same signification as above. # The dissociation constants for each site are often difficult to obtain, because overlapping of binding on two sites. :/ Two of the four ATP sites could be the two tRNA sites. 7 Though nothing is known about the number of sites. many amino-acyl-tRNA synthetases have been shown to exhibit redundance of sequences (Koch er al., 1974: Waterson & Konigsberg. 1974; Kula. 1973). This summary is not claimed to be complete.
CE. co/i)
(E. coli)
2
ATP ’
hlethionyl-t-RNA synthetase (E. coli K I2)k Pyruvate kinase activated by FDP
368
J.-P.
MAZAT,
J.
LANGLA
AND
F.
MAZAT
Another way for a ligand to possess two sites per protomer is to “squat on” the site of another structurally related ligand; this has been called s*quatthg in this paper. An example of squatting is exhibited by glutamate dehydrogenase where NADH can, in addition to its own sites, bind to the ADP sites (Pantaloni & Dessen, 1969; Pantaloni & Lecuyer, 1973; Koberstein, Krause & Sund, 1973). A border-line case appears when several sites (per protomer) can bind several ligands each. The ribonucleoside diphosphate reductase is an example of such a situation (Brown, Canellakis, Lundin, Reichard & Theiander, 1969; Brown & Reichard, 1969u,b): this enzyme carries two sites per protomer called /I and I by the authors. These two sites can bind four ligands: ATP, dATP, dTTP and dGTP (Fig. 9). Though such examples have already been described, it seemsthat the various properties following these situations have not been fully analyzed, particularly as regard to the regulatory pattern of these enzymes. It is the aim of this paper to bring out some salient features following the existence of two sitesper protomer for the sameligand (doublmite) and of the competition at these sites between at least two ligands (squatting). Only regulatory ligands and regulatory effects have been considered here. In order to describe the binding and the rate curves, an allosteric V model (Monod, Wyman & Changeux, 1965; Mazat & Patte, 1976) have been chosen as shown in Fig. I. It must be pointed out that, in this paper, the allosteric V model is only used as a convenient tool, becausethe regulatory role of the ligands is well emphasized by changesin the allosteric equilibrium R $ T, and, in the particular case of a V system, the substrates have no effects on this equilibrium. Therefore, we obtain very simple equations to express the activity of the enzyme (see below).
2. Theory The regulatory ligands have been called X and Y, the two sitesper protomcr I and 2 (see Fig. I). In an allosteric model, WC have to introduce the following dissociation constants: h,l R, X3 K:, x, K:, x, K-f, x for the ligand X and Ki, ,.. Kz, y, K:,., KT,. for the ligand Y.
Ki, x is the dissociation constant of the ligand X for the site 1 in the R form etc.
DOUBLE-SITE
ENZYMES
AND
369
SQUATTING
FIG. 1. Allosteric V-model (Monod ef nl., 1965) for the ligand X and Y competing to the sites 1 and 2. The enzyme is supposed to be a dimer: there are then four sites for the ligands X and Y. In the study of the properties of double-site enzymes the ligand Y is absent.
For the whole study, the number of protomers has been taken as equal to 2, there are thenfour sires for the iigands X and Y on the enzyme molecule.
(A)
BINDING
EQUATION
The binding equation of one ligand (X for example) has been already, given (Mazat & Patte, 1976): p = &R)+L’P(T) ’ Q(Ii,+ L@(T)
(1)
whcrc
P(T) and Q(T) are symmetrical expressionsinvolving the constants K+%x and K+$ x instead of Ki+ x and Kk, x respectively; lz is the number of protomers of the oligomeric enzyme. In the casewhen Ki,. = Kc,. (the two sites in the T form are equivalent but not necessarily in the R form) equation (1) simplifies into the binding equation of the pseudo-conservative transition of Viratelle & Seydoux (1975). If K' T,X = KS,. and Ki,. = Ki,,. then equation (1) gives the classical binding equation of an allosteric model with 2n identical sites (Monod et al..
1965). T.B.
-..
~~,:,xl-tc~~~lf
Double-site Enzymes and Squatting A Study of the Regulation by One or Several Ligaads Binding at Two Different Classes of Site JEAN-PIERRE
MAZAT
Laboratoire de Biochimie B.B.C., UniversitP de Bordeaux II. 351 Cows de la Lib&ration, 33405 Talewe, France JOBL LANGLA
AND FRANCON
MAZAT
Ecole Nationale SupPrieure des Arrs et Me’tiers, Avenue de I’ Universitt!, 33405 Talencr, Franczc (Received 4 February) The existenceof two (generally different) binding sitesper protomer fol the same ligand has already been observed (“double-site enzymes”). Furthermore, in some instances,the two sites appear to be shared in common by two or more ligands (“squatting”). The theoretical implications of such caseshave been emphasizedhere using a generalization of an allosteric V model. This model, by taking into account the competition betweenligandswhich occursin viva, evidencesvarious, or even peculiarregulatory patterns,and could be of generalphysiologicalinterest. It has beenillustrated here by somereal systems.
1. Introduction The existence of enzymes containing two binding sites per protomer for the sa.me ligand has already been described. Table 1 shows that such “doublesite” enzymes are uncommon. However, threonine-sensitive and lysinesensitive aspartokinases of Escherichiu coli K12 (Falcoz-Kelley et al., 1972: Richaud, Mazat, Felenbok & Patte, 1974) are good examples in this classof proteins: both possesstwo different sites per protomer for their allosteric inhibitor. In somecases,particularly for some aminoacyl tRNA synthetases (Koch. Boulanger & Hartley, 1974; Koch & Bruton. 1974; Kula, 1973; Sutton & Brew, 1974; Waterson & Konigsberg, 1974), the possible occurrence of two sites per protomer can be the result of the duplication of the polypeptide chain. 365
Ribonucleoside diphosphate reductase (E. coli Threonine sensitive aspartokinase (E. coli K 12) Eysine sensitive aspartokinase (E. coli K 12) Glutamate dehydrogenase (beef liver)
Enzymes
B)
4 12 12
6
BNADH aNADH
1 enzJvnes
-
-
-
Redundant sequencest
of double-site
No. of binding sites
2
No. of subunits
Lysine
Threonine
ATP dATP
Ligand studied
Examples
TABLE
+ -t
-
-
I-
Squattingt
jIh4
C(M
201~ and 3rn~
__-
Falcoz-Kelly CI al., 1972 Veron et al.. 1973
Brown & Reichard, 19696
__
References
Pantaloni & Dessen, 1969; Pantaloni & Lecuyer, 1973 Koberstein et al.. 1973
8 and 100 /IM Richaud et al.. 1973, 1974
10
Unknown 0.3 and I
Apparent dissociation constants§
4
GDP GTP
Iron
ATP
Transferrin
Phosphofructokinase (rabbit muscle) 2
3 per subunit
2
8
4
(A&
5 to tLi /AM
0.52 mhr 0.34 mhl
25 to 3X jihi
et al., 1973
Kemp & Krebs, 1967 Seydoux & Laurent (personal communication)
Coffee
Mann et al., 1970 Sutton & Brew, 1974
Koch & Brucon, !974 Blanquet el al., I972 Waygood et al., 1976
‘1 + when redundant sequence(s) exist(s); - when absence of redundant sequence is proven; nothing when nothing is known. +.>“Squatting” means: binding of a ligand at a site of another ligand; +, - and nothing have the same signification as above. # The dissociation constants for each site are often difficult to obtain, because overlapping of binding on two sites. :/ Two of the four ATP sites could be the two tRNA sites. 7 Though nothing is known about the number of sites. many amino-acyl-tRNA synthetases have been shown to exhibit redundance of sequences (Koch er al., 1974: Waterson & Konigsberg. 1974; Kula. 1973). This summary is not claimed to be complete.
CE. co/i)
(E. coli)
2
ATP ’
hlethionyl-t-RNA synthetase (E. coli K I2)k Pyruvate kinase activated by FDP
368
J.-P.
MAZAT,
J.
LANGLA
AND
F.
MAZAT
Another way for a ligand to possess two sites per protomer is to “squat on” the site of another structurally related ligand; this has been called s*quatthg in this paper. An example of squatting is exhibited by glutamate dehydrogenase where NADH can, in addition to its own sites, bind to the ADP sites (Pantaloni & Dessen, 1969; Pantaloni & Lecuyer, 1973; Koberstein, Krause & Sund, 1973). A border-line case appears when several sites (per protomer) can bind several ligands each. The ribonucleoside diphosphate reductase is an example of such a situation (Brown, Canellakis, Lundin, Reichard & Theiander, 1969; Brown & Reichard, 1969u,b): this enzyme carries two sites per protomer called /I and I by the authors. These two sites can bind four ligands: ATP, dATP, dTTP and dGTP (Fig. 9). Though such examples have already been described, it seemsthat the various properties following these situations have not been fully analyzed, particularly as regard to the regulatory pattern of these enzymes. It is the aim of this paper to bring out some salient features following the existence of two sitesper protomer for the sameligand (doublmite) and of the competition at these sites between at least two ligands (squatting). Only regulatory ligands and regulatory effects have been considered here. In order to describe the binding and the rate curves, an allosteric V model (Monod, Wyman & Changeux, 1965; Mazat & Patte, 1976) have been chosen as shown in Fig. I. It must be pointed out that, in this paper, the allosteric V model is only used as a convenient tool, becausethe regulatory role of the ligands is well emphasized by changesin the allosteric equilibrium R $ T, and, in the particular case of a V system, the substrates have no effects on this equilibrium. Therefore, we obtain very simple equations to express the activity of the enzyme (see below).
2. Theory The regulatory ligands have been called X and Y, the two sitesper protomcr I and 2 (see Fig. I). In an allosteric model, WC have to introduce the following dissociation constants: h,l R, X3 K:, x, K:, x, K-f, x for the ligand X and Ki, ,.. Kz, y, K:,., KT,. for the ligand Y.
Ki, x is the dissociation constant of the ligand X for the site 1 in the R form etc.
DOUBLE-SITE
ENZYMES
AND
369
SQUATTING
FIG. 1. Allosteric V-model (Monod ef nl., 1965) for the ligand X and Y competing to the sites 1 and 2. The enzyme is supposed to be a dimer: there are then four sites for the ligands X and Y. In the study of the properties of double-site enzymes the ligand Y is absent.
For the whole study, the number of protomers has been taken as equal to 2, there are thenfour sires for the iigands X and Y on the enzyme molecule.
(A)
BINDING
EQUATION
The binding equation of one ligand (X for example) has been already, given (Mazat & Patte, 1976): p = &R)+L’P(T) ’ Q(Ii,+ L@(T)
(1)
whcrc
P(T) and Q(T) are symmetrical expressionsinvolving the constants K+%x and K+$ x instead of Ki+ x and Kk, x respectively; lz is the number of protomers of the oligomeric enzyme. In the casewhen Ki,. = Kc,. (the two sites in the T form are equivalent but not necessarily in the R form) equation (1) simplifies into the binding equation of the pseudo-conservative transition of Viratelle & Seydoux (1975). If K' T,X = KS,. and Ki,. = Ki,,. then equation (1) gives the classical binding equation of an allosteric model with 2n identical sites (Monod et al..
1965). T.B.
-..
~~,:,xl-tc~~~lf
