235
Moh, cular and Biochemical Parasitology. 50 (1992) 235 244 ¢~' 1992 Elsevier Science Publishers B.V. All rights reserved.
O166-6851/92/$05.00
MOLBIO 01658
Some kinetic properties of pyruvate kinase from Trypanosoma brucei Mia Callens and Fred R. Opperdoes International Institute O/('el/ular and Moh'cular Pathology. Research Unit./or Tropical Diseases. Brussel.s. Belgium (Received 10 June 1991; accepted 28 August 1991)
We have studied the kinetics of the allosteric interactions of pyruvate kinase from I)'ypanosoma hrucei. The kinetics ['or phosphoenolpyruvate depended strongly on the nature of the bivalent metal ions. Pyruvate kinase activated by Mg 2 ' had the highest catalytic activity, but also the highest So 5 for phosphoenolpyruvate, while the opposite was true for pyruvate kinase activated by Mn 2 ' . The reaction rates of Mg 2 " -pyruvate kinase and Mn e +-pyruwtte kinase were clearly allosteric with respect to phosphoenolpyruvatc, while the kinetics with Co 2 -pyruvate kinase were hyperbolic. However, Co 2 ~-pyruvate kinase was still sensitive to heterotropie activation. Trypanosomal pyruvate kinase is unique in that the best activator was fructose 2,6-bisphosphate. Ribulose 1,5-bisphosphate and 5-phosphorylribose I-pyrophosphate were also strong heterotropic activators, which were much more effective than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. In the presence of the heterotropic activators, the sigmoidal kinetics with respect to phosphoenolpyruw~te and the bivalent metal ions were modified as were the concentrations of phosphoenolpyruvate and the bivalent metal ions needed to attain the maximal activity. Maximal activities were not significantly changed with Mg 2 ' and Mn 2 t as the activating metal ions. Moreover, with Co -~' and fructose 2,6-bisphosphate or ribulose 1,5-bisphosphate or 5-phosphorylribose 1-pyrophosphate, the maximal activity was significantly reduced. Ribulose 1,5-bisphosphate and 5-phosphorylribose I-pyrophosphate resembled fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate in their action in that the Ko5 wdues l`or the former 3 compounds increased when Mg :~ was replaced by C o 2 ' , while the Ko~ for fructose 1,6-bisphosphate and glucose 1,6-bisphosphate increased. The effect of pH on the cooperativity of the reaction rate of pyruwttc kinasc from T. hrucei towards phosphoenolpyruwtte and fructose 2,6-bisphosphate was unique. At alkaline pH the positive cooperativity in the conversion of phosphoenolpyruvatc was abolished while the apparent affinity for phosphoenolpyruwtte was not altered and the enzyme was still sensitive to heterotropic actiwttion. Optimal catalysis occurred at pH 6.0. At acidic pH the velocity versus fructose 2,6-bisphosphate concentration curve became sigmoidal and the activation constant increased. Between 15C and 37 'C, temperature did not influence the kinetic properties of Mg2 " -pyruvate kinase with respect to phosphoenolpyruvate. Key words: Pyruvate kinase; Trypanosoma hrucei; AIIosteric interaction: Hcterotropic and homotropic activators; Biwdent metal ions: pH
Introduction ('orrespomlence addre'ss. Dr. F.R. Opperdoes, ICP-TROP 7439, Av. Hippocrate 74, B-1200 Brussels, Belgium. Tel.: 02: 764 74 39; Fax: 02/762 68 53. Email: Opperd(a TROP.UCL.AC.BE. Abbreviations." Fru(2.6)Px, fructose 2,6-bisphosphate: Penolpyruvate, phosphoenolpyruvate" Fru(l,6)P2, fructose 1,6bisphosphate: Glc(I,6)P2, glucose 1,6-bisphosphate: Ru(I,5)P,, ribulose 1,5-bisphosphate; PRPP, 5-phosphorylribose 1-pyrophosphate: k, turnover number of pyruvate kinasc; So,, substrate concentration at half-maximal velocity: K05, activator concentration at half-maximal velocity.
Pyruvate kinase from 7)'ypanosoma brucei, a haemoflagellate belonging to the order Kinetoplastida, is a homotetrameric enzyme with a subunit size of 54500 [1]. The enzyme is allosterically regulated and exhibits sigmoidal kinetics with respect to P-enolpyruvate and the activating metal ions Mg 2~, Mn 2+, Co 2 N H 4 ÷ and K ~. In the presence of heterotropic activators the sigmoidal kinetics for Penolpyruvate are transformed into hyperbolic
236 kinetics with a significantly decreased S0.5 for the substrate [2 5]. While for all the other allosterically regulated pyruvate kinases studied thus far, Fru(l,6)Pe is the most potent activator [6], this is not the case ['or the enzymes from the kinetoplast-containing organisms Trypam, soma hrucei [3], Trypanosoma cru:i [7], Leishmania mqjor [3,8], CrithMia luciliae [3] and Truumoldasma horelli [9]. The latter group of enzymes is activated by Fru(2,6)P2, a well-known activator of phosphotYuctokinase 1, at concentrations below 1 laM, rather than by Fru(l,6)P2 and GIc(1,6)P2 which activate the enzyme only in the millimolar range. Although we have previously reported [5] that Mg e~ ions can be replaced by Mn 2' or Co 2~ , the characterisation of the T. hrucei enzyme and the effect of its actiwttors and inhibitors was only studied with Mg 2~ as activating metal ion, at pH 7.2 and 25 C. For a better understanding of the role of the enzyme in both the vertebrate and insect stages of the parasite, we have extended our kinetic studies of the T. hrza'ei enzyme to its allosteric behaviour when actiwlted either by Mg 2' Mn 2~ or Co 2+ We have also analysed the influence of pH and temperature on its allosteric properties. Furthermore we found that the enzyme, in addition to Fru(2,6)P2, is activated by Ru(I,5)P2 and PRPP, at concentrations in the micromolar range.
Materials and Methods
Materials. Rabbit muscle lactate dehydrogenase, NADH, ADP, P-enolpyruvate, Fru(1,6)P: and GIc(l,6)P: were obtained from Boehringer Mannheim. All other biochemicals were purchased from Sigma. The concentration of PRPP was measured enzymatically [10]. Enzyme assay and kinetic studies. The purification of T. hrucei pyruvate kinase and the assay procedure were the same as described previously [5]. The purified enzyme migrated as a single protein band on SDS-PAGE corresponding to a subunit M,. of 57 000. The
specific activity of the enzyme was 417 units mg ' [5]. In the standard assay the activity was measured at 2 5 C in a volume of 1 ml containing 50 mM triethanolamine buffer, pH 7.2/2.5 mM P-enolpyruvate/2 mM ADP/6 mM MgSO4/50 mM KC1/0.42 mM N A D H / 6.25 l~g lactate dehydrogenase. When the effect of pH was studied triethanolamine buffer was replaced by 50 mM barbital/K-acetate buffer, in the pH range 6 8. The ionic strength of the buffers was adjusted to 0.1 M with KC1. When hyperbolic kinetics were obtained the initial rates were fitted to a Michaelis-Menten equation. When sigmoidal kinetics were obtained, the concentration giving half-maximal velocity values (S0.s) and the Hill coefficient (h) were calculated from Hill plots of the kinetic data using linear regression analysis. The activation constants were calculated from v vo versus actiwttor concentration plots. All kinetic experiments were perl`ormed at least 3 times and the standard deviations were less than 10%.
Results
Interaction between P-emd/u'ruvatc, bivalent metal io,s and heterolropic acHvalors. In this study we have analysed the kinetics of pyruvate kinase with respect to Peno/pyruvate (Table !), bivalent metal ions (Table II) and heterotropic activators (Table III). The experimental conditions were chosen such that at all times the effect of one substrate or co-factor was studied at saturating concentrations of the other substrate and co-factors. The kinetic parameters of Mg e - p y r u v a t e kinase, Co2'-pyruvate kmase and Mn 2~ pyruvate kinase for P-emdpyruvate in the presence and the absence of activators are compared in Table I. Both turnover number of the enzyme and apparent affinity for Penolpyruvate depended on the nature of the activating metal ion. In the presence of Mn-" " ions, pyruvate kinase had the highest apparent affinity for P-emdpyruvate. more than 5 times higher than with Mg e ~ and more than 10 times higher than with Co e ~, but. Mne~-pyruvate
237
kinase had the lowest turnover number. The reaction rate with respect to P-enolpyruvate was no longer allosteric when pyruvate kinase was activated by Co 2 ~. In the presence of all
the heterotropic activators tested the sigmoidal kinetics with respect to P-enolpyruvate became more hyperbolic and the S0.5 for Penolpyruvate decreased simultaneously. With
TABLE I Kinetic p a r a m e t e r s of p y r u v a t c kinase for P-eno/pyruvate m
Activator
None Ru (I,5) P, PRPP Fru(2,6)P2 Fru(1,6)P2 GIc( 1,6)P2
Mg 2 ' -PK
Co 2 +- P K
&~ ~ (mM)
/,(min
1.48 0.40 0.75 0.15 0.46 0.44
33.6 32.6 29.11 28.6 31.0 33.6
h I)×(10
s) 2.4 1.5 2.0 1.0 1.4 1.3
M n 2 ' -PK
&~.5 (mM)
k (min
3.89 0.23 0.57 1.07 0.76 0.37
18.5 6.82 7.32 10.8 17.4 17.8
h I)×(10
~) 1.2 0.95 0.95 1.0 1.0 1.0
So 5 (mM)
k (rain
0.28 0.07 0.13 0.07 0.38 0. I 1
15.4 111.2 I 1.5 12.5 15.4 11.6
h I)x(10
~) 2.0 (I.99 1.0 0.99 1.1 1.1 I
T h e e x p e r i m e n t s were p e r f o r m e d at 2 5 C in 50 m M t r i e t h a n o l a m i n e buffer pH 7.2 c o n t a i n i n g 2 m M A D P / 5 0 m M KCI variable c o n c e n t r a t i o n s o f P-enolpyruwtte. T h e reactant c o n c e n t r a t i o n s were for: M g 2 + - p K , 6 m M MgSO4, 0.1 9 Ru(I,5)P2 or I m M P R P P or 0.5 itM Fru(_,6)P, or 0.5 m M F r u ( I , 6 ) P , or 1 m M GIc(I,6)P,; C o - ~ - P K . I m M C o C h , 0.1 Ru(1,5)P2 or I m M P R P P or I itM Fru(2,6)Pe-or 5 m M Fru(1,6)P2 or 5 m M GIc(I,6)Pe:-Mn- ' -PK, I m M MnCI~, 0.1 Ru(I,5)P2 or 1 m M P R P P or 2 ItM Fru(2,6)P2 or 5 m M Fru(1,6)P2 or 2 m M GIc(I,6)P2.
and mM mM mM
T A B L E I1 Kinetic p a r a m e t e r s o f p y r u v a t c kinasc for the activating bivalent metal ions Actiwttor
None Rn (1,5)P2 PRPP Fru(2,6)P2 Fru(I,6)P2 GIc( 1,6)P~
Mg: -PK
M n 2 +-PK
Co 2 +-PK
So s (raM)
k (min
1.24 0.36 0.70 0.20 0.51 0.49
33.4 31.4 31.4 34.3 35.0 36.7
h ')x(10
3) 2.4 1.9 2.1 1.0 1.1 1.2
S0.5 (mM)
k (min
0.34 0.08 0.25 0.02 0.18 0.20
10.2 8.0 8.46 9.26 12.3 I 1.7
h I)x(10
31 2.4 1.6 1.6 1.0 I.I 1.0
&~.5
(raM)
k (min
0.07 0.02 1).04 0.03 0.06 0.07
14.5 13.3 14.5 14.9 17.2 16.4
[1
I)x(10
3) 1.4 1.8 1.5 I.I I.I I.I
T h e experimental conditions were 5 m M P-enolpyruvate, 2 m M A D P , 50 m M KCI a n d 0.1 m M Ru(I,5)Pe or I m M P R P P or 1 itM Fru(2,6)P2 or 5 m M Fru(1,6)P2 or 1 m M Glc(1,6)P2 respectively a n d wtrious a m o u n t s o f bivalent metal ions in 50 m M t r i e t h a n o l a m i n c buffer pH 7.2 at 25 C. T A B L E I11 Kinetic p a r a m e t e r s ot" p y r u v a t e kinase for actiwUors Activator
Ru (1,5) P_~ PRPP Fru(2,6)P2 Fru(1,6)P2 GIc(I,6)P2
M g 2 ~ -PK
Co 2 ' -PK
K0.5 (laM)
k (rain
12 113 0.116 121 169
29.0 17.4 27.4 23.2 28.8
/7 I)x(10
3) 0.94 0.98 1.0 1.2 1.2
M n 2 ~-PK
KII ~ (btM)
k (rain
3.3 8.7 0.02 620 2620
5.4 4.5 6.0 10.6 19.2
h t)x(10
3) 1.0 2.3 1.0 1.0 1.1
Ko 5 (btM)
k (rain
0.02 20 130
8.4 7.9 6.2
h I)x(10
~)
1.0 1.0 1.0
T h e e x p e r i m e n t s were p e r f o r m e d at 25 C in 50 m M t r i e t h a n o l a m i n e buffer pH 7.2 c o n t a i n i n g 2 m M A D P , 50 m M KCI and w m a b l e c o n c e n t r a t i o n s o f respective actwator. T h e reactant c o n c e n t r a t i o n s were tor: M g - -PK, 6 m M MgSO4 a n d 0.7 m M P-enolpyruvate; Co 2 ~-PK, 1 m M COC12 a n d 1 m M P-enolpyruvate: M n 2 ~-PK, I m M MnC12 and 0.1 m M P-enolpyruvate.
238 M g 2~ o r M n 2~ a s activating metal ions, addition of Fru(2,6)P2 resulted in the lowest So.5 for P-enolpyruvate and the maximal activities of both Mg2~-pyruvate kinase and Mn 2 ~-pyruvate kinase were not significantly changed. A quite different effect of heterotropic activators on pyruvate kinase was observed when Mg 2~ or Mn 2 ~ were replaced by Co 2~. Co2+-pyruvate kinase exhibited its lowest So.5 for P-enolpyruvate in the presence of Ru(I,5)P> while activation by Fru(2,6)P2 led only to a 3-fold decrease in S0.5. Moreover in the presence of Fru(2,6)P> Ru(l,5)P2 and PRPP the maximal activity of Co2 ~-pyruvate kinase was reduced by more than 50%. The heterotropic activators not only influenced the kinetic parameters for Penolpyruvate but also modified the kinetic constants for the bivalent metal ions (Table ||). Both the Hill coefficient and the S0.5 value tk)r the bivalent metal ions were diminished. The highest apparent affinity of pyruvate kinase for Mg 2~ and Co x' was obtained in the presence of Fru(2,6)P2. The So.5 ik~r Mn 2 was the lowest of the three metal ions and still decreased in the presence of Fru(2,6)P2, Ru(1,5)P2 and PRPP. In the absence of heterotropic activators the experiment was not performed at P-enolpyruvate saturation since the 5'{}.5 of Co2 ~-pyruwtte kinase for Pemdpyruvate is 4 mM (Table I). In the presence of the activators, however, the P-emdpyruvate concentration was at least 5 times the S0.5 value of the activated Co 2 ~-pyruwtte kinase for P-enolpyruvate. Thus, the lower rate in the presence of the heterotropic activators has to be explained as an inhibition of Co: ~-pyruvate kinase by them. Neither Mne~-pyruvate kinase nor Mg2 ~-pyruvate kinase was inhibited by the heterotropic activators. Table Ill summarises the kinetic parameters for the heterotropic activators of Mg 2+pyruvate kinase, C o 2 - p y r u v a t e kinase and Mn2 '-pyruvate kinase at sub-saturating concentrations of P-enolpyruvate. The kinetics were ahnost hyperbolic, except the reaction rate of Co 2 +-pyruvate kinase as a function of PRPP. The K0.5 of pyruvate kinase for the activators depended on the metal ion. The
lowest values were obtained with Mn 2~ pyruvate kinase. The apparent affinity of Mg2~-pyruvate kinase for Fru(2,6)P2 is at least 3 orders of magnitude higher than for PRPP, Fru(l,6)P2 and GIc(I,6)P2. The concentration of Ru(1,5)P2 and PRPP required [k~r hall-maximal activity of Co n ~-pyruvate kinase were higher than for Fru(2,6)P2, but still 2 orders of magnitude lower than that required l\~r the classical activator Fru(l,6)P2. At subsaturating concentrations of P-em)lpyruvate, Mn2f-pyruvate kinase was not activated by Ru(1,5)P2 or PRPP, ahhough, from the kinetics for both the substrate and the metal ions it must be concluded that Mn2 ~-pyruvate kinase interacts with both Ru(1,5)P: and PRPP. Effb{'t of pH. The dependence of the allosteric kinetics of T. bruce| pyruvate kinase on pH was analysed over a pH range of 6 8. In this ptt range the enzyme remains stable for at least 5 minutes [5]. We first studied the effect of pH on Mg 2 +-pyruvate kinase in the absence of any heterotropic activator (Table IV). The cooperativity of the enzyme reaction rate with respect to P-emdpyruvate ahered with the pH. At pH 7.9, the saturation curve fitted a hyperbola and the Hill coefficient was equal to I. By decreasing the pH, the velocity curves changed gradually from hyperbolic to sigmoidal and the Hill coefficient increased to 2.5 at pH 5.8. The S0.5 for P-emdpyruwlte remained the same over the whole pH range. The T A B L E IV Kinetic p a r a m e t e r s o f M g 2 +-activated pyruvate kinasc for
P-emdpyruvate at different pH values pH
SII.¢ {raM)
5.8 6.2 6.9 7.2 ~' 7.4 7.9
1.20 1.05 1.02 I. 14 1.20 1.16
k{nlin
i)(lO
)
h
38.66
2.46
42.28
2.29
28.99 3O.20 21.74 17.40
2.13 2.2O 1.62 1.14
;'Standard bul]er. T h e experimental conditions wcrc 2 m M A D P , 6 m M M g S 0 4 , 50 m M KCI and various anlounts o f Penolpyruvate in 50 m M barbital-K-acetate buffer.
239 TABLE V Kinetic parameters of Mg 2 ' -activated pyruvate kinase for Fru(2,6)P2 at different pH values pH
K, 5(/~M)
k(min
5.8 6.3 6.9 7.2" 7.4 7.8
0.157 0.065 0.034 0.056 0.025 0.039
30.20 31.89 30.44 27.53 20.54 16.67
i)(10
~)
h 1.95 1.45 1.05 1.03 1.13 1.16
~'Standard buffer. Reactant conditions were 50 mM barbital-K-acetate buffer, 1-0.1 M, 0.7 mM P-enolpyruvate, 2 mM ADP, 6 mM MgSO4, 50 mM KCI and various amounts of Fru(2,6)P2.
optimal activity was obtained at pH values around 6. The effect of pH on the interaction of Fru(2,6)P2 at subsaturating concentrations of P-enolpyruvate was subsequently studied and the results are shown in Table V. At pH values below 6.9 the reaction rate with respect to Fru(2,6)P2 was allosteric, but at pH values above 7, positive cooperativity was no longer observed. Maximal activation by Fru(2,6)P2 was achieved at pH values below 7. The activation constant for Fru(2.6)P2 did not significantly change, between pH 7 and 8, but at lower pH the Ko5 value increased.
Discussion
This study demonstrates that the allosteric regulation of T. brucei pyruvate kinase is not limited to the heterotropic activation by Fru(2,6)P:, which converts the sigmoidal kinetics of Mg 2 ~-pyruvate kinase with respect to P-enolpyruvate into normal MichaelisMenten kinetics. The allosteric behaviour of the T. brucei enzyme is much more complex and depends on a variety of factors, such as the nature of the activating metal ion, pH, and the nature of the heterotropic activator. Comparison of T. brucei pyruvate kinase with homologous enzymes from mammalian origin is essential for the identification of differences between parasite and host enzyme. Unique features of the enzyme may then be
exploited for the design of trypanosomespecific drugs. However, a comparison of the T. brucei enzyme with that of the vertebrates is complicated by the fact that there exist 4 isoenzymes (M1, M2, L and R-type). Their distribution depends on tissue and metabolic function, M1 being mainly found in skeletal muscle, heart and brain, L in liver, R in erythrocytes and M2 in fetal tissue, but the latter persists also widely in adult tissues. Of the 4 iso-enzymes of rat, only the M l-type displays hyperbolic kinetics with regard to Penolpyruvate, while the three other types are all allosterically regulated. These kinetic properties are explained by the existence of different conformational states. A high affinity or Rstate which is stabilised by P-enolpyruvate, Fru(1,6)P2 and H ~ and a low affinity or Tstate which is stabilised by alkaline pH, ATP and certain amino acids [11 13]. Pyruvate kinase from T. hrucei differs in several respects from this generalisation and these differences will be discussed below. In the presence of Mg 2~ and Mn 2+ respectively, pyruvate kinase from T. hrucei exhibits sigmoidal kinetics with respect to the substrate P-enolpyruvate. Heterotropic activators, such as Fru(2,6)P2 change the saturation curves from sigmoidal to hyperbolic without changing V. This is however not the case with Co 2~ as activating metal ion. Velocity versus P-enolpyruvate concentration curves in the presence of Co 2~ and in the absence of an effector are already hyperbolic but the apparent affinity for P-enolpyruvate is weak (S0.5 = 4 raM). This may suggest that Co 2 ~-pyruvate kinase is not in the R-state, which is corroborated by the fact that Coe ~-pyruvate kinase is still sensitive to heterotropic activators that shift the enzyme into a conformational state with a high affinity for P-enolpyruvate. Fru(2,6)P2, which is the best activator for Mg 2 ~ and Mn: ~-pyruvate kinase, is much less effective in the case of Co 2 ~-pyruvate kinase. The S0.s for P-enolpyruvate decreases only 3 times, as compared to 10 times in the presence of Mg 2÷, and the turnover number of Co 2 pyruvate kinase is even lowered by Fru(2,6)P2. No such behaviour has ever been documen-
240
ted for isoenzymes of mammalian origin. For the M1 isotype it is known that Mg 2 ~ can be replaced by Co 2 ~ resulting in an enzyme with about the same catalytic efficiency [14]. For the L-isotype from rats, the kinetic data for Penolpyruvate with either Mg 2' or Mn 2 ~ as activating metal ion are comparable with our data for Mg 2 ÷ or Mn 2 ~-pyruvate kinase from T. brucei. The kinetics for P-enolpyruvate in the absence of a heterotropic activator are sigmoidal and become hyperbolic in the presence of Fru(1,6)P2. M n 2 ' - p y r u v a t e kinase has a lower S0.5 value for Penolpyruvate than Mg2 ' -pyruvate kinase and the apparent affinity of the Mn 2 ~-enzyme for P-enolpyruvate does not change in the presence of Fru(l,6)P2, in contrast to the situation with Mg2t-pyruvate kinase [12,15]. Our kinetic data for P-enolpyruvate in the presence of Mg 2~ or Mn 2~ are also comparable with those described for the enzymes from E. coli [16] and Bacillus lk'hen(formis [17]. Pyruvate kinase from Concholepas concholepas, however, shows sigmoidal kinetics in the presence of Mg 2+ and hyperbolic kinetics in the presence of Mn 2+ The latter resembles T. hrucei pyruvate kinase activated by Co 2~ except that Concholepas-Mn: ~-pyruvate ki2 nase is insensitive to Fru(l,6)P2 [18,19] whereas the affinity of Co2 ~-pyruvate kinase from T. brucei for P-enolpyruvate still increases. A strong influence of the bivalent metal ions on the S0.5 values for P-enolpyruvate is not unexpected since these metal ions are bound to the active site. The enzyme-bound bivalent metal ion is ligated to P-enolpyruvate and its function is to correctly orientate the phosphate group of P-enolpyruvate. The bivalent metal ion ligated to A D P is also involved in the correct orientation of the overlapping phosphates at the active site [20]. Comparison of the kinetics for the bivalent metal ions obtained at 2.5 mM P-enolpyruvate [5] with the present data, obtained at 5 mM Penolpyruvate (Table II), indicates modifications with the concentration of P-enolpyruvate. A higher P-enolpyruvate concentration partially abolished the allostericity and decreased the S0.s values for the metal ions.
In the presence of a heterotropic activator, the homotropic activation of the metal ion was almost abolished. A similar phenomenon has been described for pyruvate kinase from Phycomyces blukesh, eunus [21] and from E. coli [16]. Addition of Fru(l,6)P2 abolished the sigmoidal kinetics. In the case of rat-liver pyruvate kinase the binding of bivalent metal ions is already hyperbolic also in the absence of heterotropic activators [15]. It is, however, unexpected that the So.s value for the metal ions also depends on the nature of the heterotropic activators (and vice versa). According to Muirhead and coworkers [13,20], based on 3-dimensional structure of cat muscle pyruvate kinase, the binding site of the effectors is supposed to be located between domains A and C whereas the active site is located between domains A and B. This suggests that direct contacts between effector and metal ion are excluded, and, therefore, the interaction between metal ion and effector probably should be explained in terms of indirect allosteric conformational changes. A similar effect has been described in the case of phenylalanine, an allosteric inhibitor of MItype pyruvate kinase, that affects Mg 2~ pyruvate kinase and M n 2 - p y r u v a t e kinase differently [22]. The kinetics for the activators were almost hyperbolic under the conditions used. This is in agreement with the observations by Flynn and Bowman [23] who also reported the absence of cooperativity in the reaction rate of Mg 2~ pyruvate kinase from T. brucei with respect to Fru(1,6)P2 and at various concentrations of Penolpyruvate. In the case of E. coli pyruvate kinase, however, the velocity versus Fru(1,6)P2 concentration curves are sigmoidal at low concentrations of P-enolpyruvate while they change to hyperbolic at 2 mM P-enolpyruvate [16]. For the L-isotype from rat liver the binding of Fru(l,6)P2 is cooperative with a Hill coefficient of 2 at 0.5 mM P-enolpyruvate which still increases at higher P-enolpyruvate concentrations (4 raM) [24~15]. A comparison of the potency of the heterotropic activators of trypanosomal pyruvate kinase shows that Fru(2,6)P2 is by far the
241
most effective one, followed by Ru(1,5)P2, P R P P and Fru(1,6)P2 and/or GIc(I,6)P2. Although these heterotropic activators can be considered as structural analogues of Fru(2,6)P2 having the 2 phosphate groups at roughly the same distance, their degree of effectiveness is remarkably different. The apparent high affinity of pyruvate kinase for Ru(1,5)P2 and P R P P and their reduced capacity to stimulate Co2~-pyruvate kinase to maximal activity suggest that the l a t t e r 2 effectors structurally resemble more Fru(2,6)P2 than Fru(1,6)P?_ or Glc(I,6)P2. A similar conclusion has recently been drawn in the case of the pyruvate kinase from E. coli [25]. This enzyme was stimulated by Fru(1,6)P2 which was 100 times more active than Fru(2,6)P2. Ru(I,5)P2 and P R P P also activated the enzyme but the latter two behaved as weak activators that resembled Fru(2,6)P2 in their action, rather than Fru(1,6)P2. Detailed information about the structural requirements for the binding of the heterotropic activators is not yet available. Therefore it is still an open question as to why Fru(2,6)P2, Ru(1,5)P2 and PRPP are so much better as activators of the trypanosome pyruvate kinase than Fru(l,6)P2 and why the situation is the opposite for all other regulated pyruvate kinases [6,25]. Based on a comparison of amino acid sequences of different isoenzymes and their modelling into the 3dimensional structure of cat muscle pyruvate kinase some predictions have been made about a possible binding pocket for a heterotropic activator [13,20,26]. Such a binding site would be located between domains A and C of the subunit structure, while intersubunit contacts also seem to play a role in the allosteric behaviour of the enzymes. Recently, Speranza et al. [27] have identified a peptide of the E. coli enzyme that could be involved in the binding of Fru(1,6)P2. However, a definite answer about the nature of the binding pocket for the heterotropic activator should await the successful crystallization of the activatorenzyme complex or should come from sitedirected mutagenesis of the amino acids involved in the binding of the activator.
Like many other pyruvate kinases, activity and allosteric regulation of the T. brucei enzyme strongly depend on pH. The optimal pH for catalysis is situated at pH values below 7, while the activity sharply decreased at higher pH. This is in agreement with the results published by Flynn and Bowman [23]. A similar pH-dependency has also been reported for pyruvate kinase of human erythrocytes, which exhibits its optimal activity at pH 6.6 with a rapid decline in activity between pH 7 and pH 7.6. By contrast, rabbit-muscle pyruvate kinase has a pH optimum above pH 7.2 with almost no variation in activity between pH 7 and pH 7.6 [28]. With partially purified pyruvate kinase from T. hrucei the Hill coefficient did not change between pH 5.7 and pH 7.8 but the apparent affinity for Penolpyruvate was pH dependent [23]. Our purified pyruvate kinase behaves differently. Variation in pH from 6 to 8 changes the cooperativity in the reaction rate of Mg 2~ pyruvate kinase with respect to P-enolpyruvate without changing the S0.5 value for Penolpyruvate. The kinetics of P-enolpyruvate were sigmoidal up to 7.4, but at pH 8.0 the cooperativity disappeared. In this respect the T. brucei enzyme resembles the Ml-type pyruvate kinase from rabbit muscle and the enzyme from yeast, where the &~.5 lbr Penolpyruvate is the same at pH 6.5 and at pH 8.5, while the Hill coefficient increases with decreasing pH [29]. The reverse has been published for pyruvate kinase from liver [30,24], M-type pyruvate kinases from rat liver [31], erythrocytes [28] and P. blakesleeanus pyruvate kinase [32], where in all cases protons act as allosteric activator, converting the kinetics with respect to P-enolpyruvate from sigmoidal to hyperbolic and shifting the enzyme into a conformational state with a high affinity for Penolpyruvate. The effect of pH on the interaction of T. brucei pyruvate kinase with Fru(2,6)P2 resembles that for P-enolpyruvate, except that the apparent affinity for Fru(2,6)P2 decreases at pH values below pH 7.0. This partially resembles the kinetic behaviour of yeast
242
pyruvate kinase. At pH values above 7.5 the binding of the activator, Fru(l,6)P2, is no longer allosteric while the affinity decreases [29]. For rabbit-muscle pyruvate kinases, however, both the S0.s and the Hill coefficient remain unchanged [29]. Pyruvate kinases from liver [30], erythrocytes [28] and P. blakesleeanus [32] were no longer sensitive to heterotropic activation at acidic pH. In conclusion, the kinetic behaviour of the trypanosomal pyruvate kinase depends remarkably on its interaction with Penolpyruvate, the heterotropic activators and the activating bivalent metal ions. In addition to its unique stimulation by Fru(2,6)P2 the enzyme differs from its mammalian counterparts by the effect of pH on the allosteric regulation of the enzyme.
Acknowledgements We thank Mr. Joris Van Roy for the supply of the trypanosomes, Drs. E. Van Schaflingen and M.-F. Vincent for the assistance with some of the PRPP measurements, and Ms Franqoise Van de Calseyde-Mylle for secretarial assistance. This research received the financial support of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases; The Belgian State Prime Minister's Office Science Policy Programming. References 1 Allert, S., Ernest, 1., Poliszczak, A., Opperdoes, F.R. and Michels, P.A.M. (1991) Molecular cloning and analysis of two tandemly linked genes for pyruvate kinase of Trypanosoma brucei. Eur. J. Biochem., 200, 19 27. 2 Elynn, I.W. and Bowman+ I.B.R. (1980) Purification and characterization of pyruvate kinase from Trypanosoma brucei. Arch. Biochem. Biophys. 200, 401 409. 3 Van Schaftingen, E., Opperdoes, E.R. and Hers, H.-G. (1985) Stimulation of TI3"panosoma brucei pyruvate kinase by fructose 2,6-bisphosphate. Eur. J. Biochem. 153,403~406. 4 Barnard, J.P. and Pedersen, P.L. (1988) Purification in a single step and kinetic characterization of the pyruvate kinase of Trypanosoma hrucei. Mol. Biochem. Parasitol. 31, 141 147.
5 Callens, M., Kuntz, D.A. and Opperdoes, E.R. (1991) Characterization of pyruvate kinase of Trypanosoma hrucei and its role in the regulation of carbohydrate metabolism. Mol. Biochem. Parasitot. 47+ 19 29. 6 Kayne, F.J. (1973) Pyruvate kinase. In: The Enzymes, 3rd edn. (Boyer, P.D., ed.), Vol. 8, pp. 353 382. Academic Press, New York and London. 7 Cazzulo, J.J+, Cazzulo Franke, M.C. and Eranke de Cazzulo, B.M. (1989) On the regulatory properties of tile pyruvate kinase from Trypanosoma cruzi epimastigotes. FEMS Microbiol. Lett. 50, 259 263. 8 Etges, R. and Mukkada, A.J. (1988) Purification and characterization of a metabolite-regulated pyruvate kinase from Leishmania mq/or promastigotes. Mol. Biochem. Parasitol. 27, 281 289. 9 0 p p e r d o e s , F.R., Nohynkova, E., Van Schaftingen, E., Lambeir, A.-M., Veenhuis, M. and Van Roy, J. (1988) Demonstration of glycosomes (microbodies) in the Bodonid flagellate Trypanoplasma horelli (protozoa. kinetoplastida). Mol. Biochem. Parasitol. 30, 155 164. 10 Kornberg, A., Lieberman, I. and Simms, E.S. (1955) Enzymatic synthesis and properties of 5-phosphoribosylpyrophosphate. J. Biol. Chem. 215, 389 401. 11 Kahn, A. and Marie, J. (1982) Pyruvate kinasc from human erythrocytes and liver. Methods Enzymol. 90. 131 140. 12 hnamura, K. and Tanaka, T. (1982) Pyruvate kinase isozymes from rat. Methods Enzymol. 90, 150 165. 13 Muirhead, H. (1990) Isoenzymes of pyruvate kinase. Biochcm. Soc. Trans. 18, 193 196. 14 Baek, Y.H. and Nowak, T. (1982) Kinetic evidence for a dual cation role for muscle pyruvate kinase. Arch. Biochem. Biophys. 217, 491 497. 15 Blair, J.B. and Walker, R.G. (1984) Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6bisphosphate binding. Arch. Biochem. Biophys. 232, 202 213. 16 Waygood, E.B. and Sanwal, B.D. (1974) The control ot pyruwtte kinases of k5"cherichia coli, I. Physico-chemical and regulatory properties of the enzymes activated by fructose 1,6-diphosphate. J. Biol. Chem. 249, 265 274. 17 Tuominen, F.W. and Bernlohr, R.W+ (1971) Pyruvate kinase of spore-forming bacterium Bacillus lichen(fi~rmis 11. Kinetic properties. J. Biol. Chem 246, 1746 1755. 18 Gonzalez+ R., Carvajal, N. and Moran, A. (1984) Differences between magnesium-activated and manganese-activated pyruvate kinase from the muscle of Concholepas concholepas. Comp. Biochem. Physiol. 78B, 389 392. 19 Carwtjal, N., Gonzalez, R., Moran, A. and Oyarce. A.M. (1985) Comparative kinetic studies of Mn :+actiwlted and fructose-l,6-P-modified Mg 2+ -activated pyruvate kinase from Concholepas concholepas. Comp. Biochem. Physiol. 82B, 63 65. 20 Muirhead, H., Clayden, D.A., Barford, D., Lorimer, C.G., FothergilI-Gilmore, L.A., Schlitz, E. and Schmitt, W. (1986) The structure of cat muscle pyruwtte kinase. EMBO J. 5, 475 481. 21 del Valle, P., de Arriaga, D., Busto, E. and Soler+ J. (1986) A study of the allosteric kinetics of Phycomyces pyruvate kinase as judged by the effect of L-alanine and fructose 1,6-bisphosphate. Biochcm, Biophys. Acta 874, 193 204. 22 Kayne, F.J. and Price, N.C. (1972) Conl\)rmational changes in the allosteric inhibition of muscle pyruvatc
243 kinase by phenylalanine. Biochemistry 11, 4415 4420. 23 Flynn, [.W. and Bowman, I.B.R. (1981) Some kinetic properties of pyruvate kinase from Trypanosoma brucei: influence of pH and fructose 1,6-diphosphate. Mol. Biochem. Parasitol. 4, 95 106. 24 EI-Maghrabi, M R . , Claus. T.H., McGrane, M.M. and Pilkis, S.J. (1982) Influence of phosphorylation on the interaction of effiectors with rat liver pyruvate kinase. J. Biol. Chem. 257. 233 240. 25 Speranza, M.L., Valentini, G. and Malcovati, M. (1990) Fructose-l,6-bisphosphate-activated pyruvate kinase from E. coli. Eur. J. Biochem. 191, 701 704. 26 McNally, T. and Fothergill-Gilmore, L.A. (1990) Sitedirected mutagenesis as a tool for the study of the allosteric control of pyruvate kinase. Biochem. Soc. Trans. 18, 258. 27 Speranza, M.L., Valentini, G., ladarola, P., Stoppini, M., Malcovati, M. and Ferri, G. (1989) Primary structure of three peptides at the catalytic and allosteric sites of the fructose-l,6-bisphosphate-activated pyruvate kinase from Escherichia coli. Biol. Chem. HoppeSeyler 370, 211 216.
28 Black, J.A. and Henderson, M.H. (1972) Activation and inhibition of human erythrocyte pyruvate kinase by organic phosphates, amino acids, dipeptides and anions. Biochim. Biophys. Acta 284, 115 127. 29 Brown, C.E., Taylor, J.M. and Chan, L.-M. (1985) The effect of pH on the interaction of substrates and el'lEctor to yeast and rabbit muscle pyruvate kinasc. Biochem. Biophys. Acta 829, 342 347. 30 Rozengurt, E., Jimdnez de Asua, L. and Carminatti, H. (1969) Some kinetic properties of liver pyruvate kinase (type L). Effect o f p H on its allosteric behaviour. J. Biol. Chem. 244, 3142 3147. 31 Van Berkel, T.J.C., Koster, J.F. and HOlsmann, W.C. (1973) Some kinetic properties of the allosteric M-type pyruvate kinase from rat liver; influence of pH and the nature of amino acid inhibition. Biochim. Biophys. Acta 321, 171 180. 32 de Arriaga, D.~ Busto, F., del Valle, P. and Soler, J. (1989) A kinetic study of the pH effect on the allosteric properties of pyruvate kinase from Phycomvces hlakesleeanus. Biochim. Biophys. Acta 998, 221 230.
Moh, cular and Biochemical Parasitology. 50 (1992) 235 244 ¢~' 1992 Elsevier Science Publishers B.V. All rights reserved.
O166-6851/92/$05.00
MOLBIO 01658
Some kinetic properties of pyruvate kinase from Trypanosoma brucei Mia Callens and Fred R. Opperdoes International Institute O/('el/ular and Moh'cular Pathology. Research Unit./or Tropical Diseases. Brussel.s. Belgium (Received 10 June 1991; accepted 28 August 1991)
We have studied the kinetics of the allosteric interactions of pyruvate kinase from I)'ypanosoma hrucei. The kinetics ['or phosphoenolpyruvate depended strongly on the nature of the bivalent metal ions. Pyruvate kinase activated by Mg 2 ' had the highest catalytic activity, but also the highest So 5 for phosphoenolpyruvate, while the opposite was true for pyruvate kinase activated by Mn 2 ' . The reaction rates of Mg 2 " -pyruvate kinase and Mn e +-pyruwtte kinase were clearly allosteric with respect to phosphoenolpyruvatc, while the kinetics with Co 2 -pyruvate kinase were hyperbolic. However, Co 2 ~-pyruvate kinase was still sensitive to heterotropie activation. Trypanosomal pyruvate kinase is unique in that the best activator was fructose 2,6-bisphosphate. Ribulose 1,5-bisphosphate and 5-phosphorylribose I-pyrophosphate were also strong heterotropic activators, which were much more effective than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. In the presence of the heterotropic activators, the sigmoidal kinetics with respect to phosphoenolpyruw~te and the bivalent metal ions were modified as were the concentrations of phosphoenolpyruvate and the bivalent metal ions needed to attain the maximal activity. Maximal activities were not significantly changed with Mg 2 ' and Mn 2 t as the activating metal ions. Moreover, with Co -~' and fructose 2,6-bisphosphate or ribulose 1,5-bisphosphate or 5-phosphorylribose 1-pyrophosphate, the maximal activity was significantly reduced. Ribulose 1,5-bisphosphate and 5-phosphorylribose I-pyrophosphate resembled fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate in their action in that the Ko5 wdues l`or the former 3 compounds increased when Mg :~ was replaced by C o 2 ' , while the Ko~ for fructose 1,6-bisphosphate and glucose 1,6-bisphosphate increased. The effect of pH on the cooperativity of the reaction rate of pyruwttc kinasc from T. hrucei towards phosphoenolpyruwtte and fructose 2,6-bisphosphate was unique. At alkaline pH the positive cooperativity in the conversion of phosphoenolpyruvatc was abolished while the apparent affinity for phosphoenolpyruwtte was not altered and the enzyme was still sensitive to heterotropic actiwttion. Optimal catalysis occurred at pH 6.0. At acidic pH the velocity versus fructose 2,6-bisphosphate concentration curve became sigmoidal and the activation constant increased. Between 15C and 37 'C, temperature did not influence the kinetic properties of Mg2 " -pyruvate kinase with respect to phosphoenolpyruvate. Key words: Pyruvate kinase; Trypanosoma hrucei; AIIosteric interaction: Hcterotropic and homotropic activators; Biwdent metal ions: pH
Introduction ('orrespomlence addre'ss. Dr. F.R. Opperdoes, ICP-TROP 7439, Av. Hippocrate 74, B-1200 Brussels, Belgium. Tel.: 02: 764 74 39; Fax: 02/762 68 53. Email: Opperd(a TROP.UCL.AC.BE. Abbreviations." Fru(2.6)Px, fructose 2,6-bisphosphate: Penolpyruvate, phosphoenolpyruvate" Fru(l,6)P2, fructose 1,6bisphosphate: Glc(I,6)P2, glucose 1,6-bisphosphate: Ru(I,5)P,, ribulose 1,5-bisphosphate; PRPP, 5-phosphorylribose 1-pyrophosphate: k, turnover number of pyruvate kinasc; So,, substrate concentration at half-maximal velocity: K05, activator concentration at half-maximal velocity.
Pyruvate kinase from 7)'ypanosoma brucei, a haemoflagellate belonging to the order Kinetoplastida, is a homotetrameric enzyme with a subunit size of 54500 [1]. The enzyme is allosterically regulated and exhibits sigmoidal kinetics with respect to P-enolpyruvate and the activating metal ions Mg 2~, Mn 2+, Co 2 N H 4 ÷ and K ~. In the presence of heterotropic activators the sigmoidal kinetics for Penolpyruvate are transformed into hyperbolic
236 kinetics with a significantly decreased S0.5 for the substrate [2 5]. While for all the other allosterically regulated pyruvate kinases studied thus far, Fru(l,6)Pe is the most potent activator [6], this is not the case ['or the enzymes from the kinetoplast-containing organisms Trypam, soma hrucei [3], Trypanosoma cru:i [7], Leishmania mqjor [3,8], CrithMia luciliae [3] and Truumoldasma horelli [9]. The latter group of enzymes is activated by Fru(2,6)P2, a well-known activator of phosphotYuctokinase 1, at concentrations below 1 laM, rather than by Fru(l,6)P2 and GIc(1,6)P2 which activate the enzyme only in the millimolar range. Although we have previously reported [5] that Mg e~ ions can be replaced by Mn 2' or Co 2~ , the characterisation of the T. hrucei enzyme and the effect of its actiwttors and inhibitors was only studied with Mg 2~ as activating metal ion, at pH 7.2 and 25 C. For a better understanding of the role of the enzyme in both the vertebrate and insect stages of the parasite, we have extended our kinetic studies of the T. hrza'ei enzyme to its allosteric behaviour when actiwlted either by Mg 2' Mn 2~ or Co 2+ We have also analysed the influence of pH and temperature on its allosteric properties. Furthermore we found that the enzyme, in addition to Fru(2,6)P2, is activated by Ru(I,5)P2 and PRPP, at concentrations in the micromolar range.
Materials and Methods
Materials. Rabbit muscle lactate dehydrogenase, NADH, ADP, P-enolpyruvate, Fru(1,6)P: and GIc(l,6)P: were obtained from Boehringer Mannheim. All other biochemicals were purchased from Sigma. The concentration of PRPP was measured enzymatically [10]. Enzyme assay and kinetic studies. The purification of T. hrucei pyruvate kinase and the assay procedure were the same as described previously [5]. The purified enzyme migrated as a single protein band on SDS-PAGE corresponding to a subunit M,. of 57 000. The
specific activity of the enzyme was 417 units mg ' [5]. In the standard assay the activity was measured at 2 5 C in a volume of 1 ml containing 50 mM triethanolamine buffer, pH 7.2/2.5 mM P-enolpyruvate/2 mM ADP/6 mM MgSO4/50 mM KC1/0.42 mM N A D H / 6.25 l~g lactate dehydrogenase. When the effect of pH was studied triethanolamine buffer was replaced by 50 mM barbital/K-acetate buffer, in the pH range 6 8. The ionic strength of the buffers was adjusted to 0.1 M with KC1. When hyperbolic kinetics were obtained the initial rates were fitted to a Michaelis-Menten equation. When sigmoidal kinetics were obtained, the concentration giving half-maximal velocity values (S0.s) and the Hill coefficient (h) were calculated from Hill plots of the kinetic data using linear regression analysis. The activation constants were calculated from v vo versus actiwttor concentration plots. All kinetic experiments were perl`ormed at least 3 times and the standard deviations were less than 10%.
Results
Interaction between P-emd/u'ruvatc, bivalent metal io,s and heterolropic acHvalors. In this study we have analysed the kinetics of pyruvate kinase with respect to Peno/pyruvate (Table !), bivalent metal ions (Table II) and heterotropic activators (Table III). The experimental conditions were chosen such that at all times the effect of one substrate or co-factor was studied at saturating concentrations of the other substrate and co-factors. The kinetic parameters of Mg e - p y r u v a t e kinase, Co2'-pyruvate kmase and Mn 2~ pyruvate kinase for P-emdpyruvate in the presence and the absence of activators are compared in Table I. Both turnover number of the enzyme and apparent affinity for Penolpyruvate depended on the nature of the activating metal ion. In the presence of Mn-" " ions, pyruvate kinase had the highest apparent affinity for P-emdpyruvate. more than 5 times higher than with Mg e ~ and more than 10 times higher than with Co e ~, but. Mne~-pyruvate
237
kinase had the lowest turnover number. The reaction rate with respect to P-enolpyruvate was no longer allosteric when pyruvate kinase was activated by Co 2 ~. In the presence of all
the heterotropic activators tested the sigmoidal kinetics with respect to P-enolpyruvate became more hyperbolic and the S0.5 for Penolpyruvate decreased simultaneously. With
TABLE I Kinetic p a r a m e t e r s of p y r u v a t c kinase for P-eno/pyruvate m
Activator
None Ru (I,5) P, PRPP Fru(2,6)P2 Fru(1,6)P2 GIc( 1,6)P2
Mg 2 ' -PK
Co 2 +- P K
&~ ~ (mM)
/,(min
1.48 0.40 0.75 0.15 0.46 0.44
33.6 32.6 29.11 28.6 31.0 33.6
h I)×(10
s) 2.4 1.5 2.0 1.0 1.4 1.3
M n 2 ' -PK
&~.5 (mM)
k (min
3.89 0.23 0.57 1.07 0.76 0.37
18.5 6.82 7.32 10.8 17.4 17.8
h I)×(10
~) 1.2 0.95 0.95 1.0 1.0 1.0
So 5 (mM)
k (rain
0.28 0.07 0.13 0.07 0.38 0. I 1
15.4 111.2 I 1.5 12.5 15.4 11.6
h I)x(10
~) 2.0 (I.99 1.0 0.99 1.1 1.1 I
T h e e x p e r i m e n t s were p e r f o r m e d at 2 5 C in 50 m M t r i e t h a n o l a m i n e buffer pH 7.2 c o n t a i n i n g 2 m M A D P / 5 0 m M KCI variable c o n c e n t r a t i o n s o f P-enolpyruwtte. T h e reactant c o n c e n t r a t i o n s were for: M g 2 + - p K , 6 m M MgSO4, 0.1 9 Ru(I,5)P2 or I m M P R P P or 0.5 itM Fru(_,6)P, or 0.5 m M F r u ( I , 6 ) P , or 1 m M GIc(I,6)P,; C o - ~ - P K . I m M C o C h , 0.1 Ru(1,5)P2 or I m M P R P P or I itM Fru(2,6)Pe-or 5 m M Fru(1,6)P2 or 5 m M GIc(I,6)Pe:-Mn- ' -PK, I m M MnCI~, 0.1 Ru(I,5)P2 or 1 m M P R P P or 2 ItM Fru(2,6)P2 or 5 m M Fru(1,6)P2 or 2 m M GIc(I,6)P2.
and mM mM mM
T A B L E I1 Kinetic p a r a m e t e r s o f p y r u v a t c kinasc for the activating bivalent metal ions Actiwttor
None Rn (1,5)P2 PRPP Fru(2,6)P2 Fru(I,6)P2 GIc( 1,6)P~
Mg: -PK
M n 2 +-PK
Co 2 +-PK
So s (raM)
k (min
1.24 0.36 0.70 0.20 0.51 0.49
33.4 31.4 31.4 34.3 35.0 36.7
h ')x(10
3) 2.4 1.9 2.1 1.0 1.1 1.2
S0.5 (mM)
k (min
0.34 0.08 0.25 0.02 0.18 0.20
10.2 8.0 8.46 9.26 12.3 I 1.7
h I)x(10
31 2.4 1.6 1.6 1.0 I.I 1.0
&~.5
(raM)
k (min
0.07 0.02 1).04 0.03 0.06 0.07
14.5 13.3 14.5 14.9 17.2 16.4
[1
I)x(10
3) 1.4 1.8 1.5 I.I I.I I.I
T h e experimental conditions were 5 m M P-enolpyruvate, 2 m M A D P , 50 m M KCI a n d 0.1 m M Ru(I,5)Pe or I m M P R P P or 1 itM Fru(2,6)P2 or 5 m M Fru(1,6)P2 or 1 m M Glc(1,6)P2 respectively a n d wtrious a m o u n t s o f bivalent metal ions in 50 m M t r i e t h a n o l a m i n c buffer pH 7.2 at 25 C. T A B L E I11 Kinetic p a r a m e t e r s ot" p y r u v a t e kinase for actiwUors Activator
Ru (1,5) P_~ PRPP Fru(2,6)P2 Fru(1,6)P2 GIc(I,6)P2
M g 2 ~ -PK
Co 2 ' -PK
K0.5 (laM)
k (rain
12 113 0.116 121 169
29.0 17.4 27.4 23.2 28.8
/7 I)x(10
3) 0.94 0.98 1.0 1.2 1.2
M n 2 ~-PK
KII ~ (btM)
k (rain
3.3 8.7 0.02 620 2620
5.4 4.5 6.0 10.6 19.2
h t)x(10
3) 1.0 2.3 1.0 1.0 1.1
Ko 5 (btM)
k (rain
0.02 20 130
8.4 7.9 6.2
h I)x(10
~)
1.0 1.0 1.0
T h e e x p e r i m e n t s were p e r f o r m e d at 25 C in 50 m M t r i e t h a n o l a m i n e buffer pH 7.2 c o n t a i n i n g 2 m M A D P , 50 m M KCI and w m a b l e c o n c e n t r a t i o n s o f respective actwator. T h e reactant c o n c e n t r a t i o n s were tor: M g - -PK, 6 m M MgSO4 a n d 0.7 m M P-enolpyruvate; Co 2 ~-PK, 1 m M COC12 a n d 1 m M P-enolpyruvate: M n 2 ~-PK, I m M MnC12 and 0.1 m M P-enolpyruvate.
238 M g 2~ o r M n 2~ a s activating metal ions, addition of Fru(2,6)P2 resulted in the lowest So.5 for P-enolpyruvate and the maximal activities of both Mg2~-pyruvate kinase and Mn 2 ~-pyruvate kinase were not significantly changed. A quite different effect of heterotropic activators on pyruvate kinase was observed when Mg 2~ or Mn 2 ~ were replaced by Co 2~. Co2+-pyruvate kinase exhibited its lowest So.5 for P-enolpyruvate in the presence of Ru(I,5)P> while activation by Fru(2,6)P2 led only to a 3-fold decrease in S0.5. Moreover in the presence of Fru(2,6)P> Ru(l,5)P2 and PRPP the maximal activity of Co2 ~-pyruvate kinase was reduced by more than 50%. The heterotropic activators not only influenced the kinetic parameters for Penolpyruvate but also modified the kinetic constants for the bivalent metal ions (Table ||). Both the Hill coefficient and the S0.5 value tk)r the bivalent metal ions were diminished. The highest apparent affinity of pyruvate kinase for Mg 2~ and Co x' was obtained in the presence of Fru(2,6)P2. The So.5 ik~r Mn 2 was the lowest of the three metal ions and still decreased in the presence of Fru(2,6)P2, Ru(1,5)P2 and PRPP. In the absence of heterotropic activators the experiment was not performed at P-enolpyruvate saturation since the 5'{}.5 of Co2 ~-pyruwtte kinase for Pemdpyruvate is 4 mM (Table I). In the presence of the activators, however, the P-emdpyruvate concentration was at least 5 times the S0.5 value of the activated Co 2 ~-pyruwtte kinase for P-enolpyruvate. Thus, the lower rate in the presence of the heterotropic activators has to be explained as an inhibition of Co: ~-pyruvate kinase by them. Neither Mne~-pyruvate kinase nor Mg2 ~-pyruvate kinase was inhibited by the heterotropic activators. Table Ill summarises the kinetic parameters for the heterotropic activators of Mg 2+pyruvate kinase, C o 2 - p y r u v a t e kinase and Mn2 '-pyruvate kinase at sub-saturating concentrations of P-enolpyruvate. The kinetics were ahnost hyperbolic, except the reaction rate of Co 2 +-pyruvate kinase as a function of PRPP. The K0.5 of pyruvate kinase for the activators depended on the metal ion. The
lowest values were obtained with Mn 2~ pyruvate kinase. The apparent affinity of Mg2~-pyruvate kinase for Fru(2,6)P2 is at least 3 orders of magnitude higher than for PRPP, Fru(l,6)P2 and GIc(I,6)P2. The concentration of Ru(1,5)P2 and PRPP required [k~r hall-maximal activity of Co n ~-pyruvate kinase were higher than for Fru(2,6)P2, but still 2 orders of magnitude lower than that required l\~r the classical activator Fru(l,6)P2. At subsaturating concentrations of P-em)lpyruvate, Mn2f-pyruvate kinase was not activated by Ru(1,5)P2 or PRPP, ahhough, from the kinetics for both the substrate and the metal ions it must be concluded that Mn2 ~-pyruvate kinase interacts with both Ru(1,5)P: and PRPP. Effb{'t of pH. The dependence of the allosteric kinetics of T. bruce| pyruvate kinase on pH was analysed over a pH range of 6 8. In this ptt range the enzyme remains stable for at least 5 minutes [5]. We first studied the effect of pH on Mg 2 +-pyruvate kinase in the absence of any heterotropic activator (Table IV). The cooperativity of the enzyme reaction rate with respect to P-emdpyruvate ahered with the pH. At pH 7.9, the saturation curve fitted a hyperbola and the Hill coefficient was equal to I. By decreasing the pH, the velocity curves changed gradually from hyperbolic to sigmoidal and the Hill coefficient increased to 2.5 at pH 5.8. The S0.5 for P-emdpyruwlte remained the same over the whole pH range. The T A B L E IV Kinetic p a r a m e t e r s o f M g 2 +-activated pyruvate kinasc for
P-emdpyruvate at different pH values pH
SII.¢ {raM)
5.8 6.2 6.9 7.2 ~' 7.4 7.9
1.20 1.05 1.02 I. 14 1.20 1.16
k{nlin
i)(lO
)
h
38.66
2.46
42.28
2.29
28.99 3O.20 21.74 17.40
2.13 2.2O 1.62 1.14
;'Standard bul]er. T h e experimental conditions wcrc 2 m M A D P , 6 m M M g S 0 4 , 50 m M KCI and various anlounts o f Penolpyruvate in 50 m M barbital-K-acetate buffer.
239 TABLE V Kinetic parameters of Mg 2 ' -activated pyruvate kinase for Fru(2,6)P2 at different pH values pH
K, 5(/~M)
k(min
5.8 6.3 6.9 7.2" 7.4 7.8
0.157 0.065 0.034 0.056 0.025 0.039
30.20 31.89 30.44 27.53 20.54 16.67
i)(10
~)
h 1.95 1.45 1.05 1.03 1.13 1.16
~'Standard buffer. Reactant conditions were 50 mM barbital-K-acetate buffer, 1-0.1 M, 0.7 mM P-enolpyruvate, 2 mM ADP, 6 mM MgSO4, 50 mM KCI and various amounts of Fru(2,6)P2.
optimal activity was obtained at pH values around 6. The effect of pH on the interaction of Fru(2,6)P2 at subsaturating concentrations of P-enolpyruvate was subsequently studied and the results are shown in Table V. At pH values below 6.9 the reaction rate with respect to Fru(2,6)P2 was allosteric, but at pH values above 7, positive cooperativity was no longer observed. Maximal activation by Fru(2,6)P2 was achieved at pH values below 7. The activation constant for Fru(2.6)P2 did not significantly change, between pH 7 and 8, but at lower pH the Ko5 value increased.
Discussion
This study demonstrates that the allosteric regulation of T. brucei pyruvate kinase is not limited to the heterotropic activation by Fru(2,6)P:, which converts the sigmoidal kinetics of Mg 2 ~-pyruvate kinase with respect to P-enolpyruvate into normal MichaelisMenten kinetics. The allosteric behaviour of the T. brucei enzyme is much more complex and depends on a variety of factors, such as the nature of the activating metal ion, pH, and the nature of the heterotropic activator. Comparison of T. brucei pyruvate kinase with homologous enzymes from mammalian origin is essential for the identification of differences between parasite and host enzyme. Unique features of the enzyme may then be
exploited for the design of trypanosomespecific drugs. However, a comparison of the T. brucei enzyme with that of the vertebrates is complicated by the fact that there exist 4 isoenzymes (M1, M2, L and R-type). Their distribution depends on tissue and metabolic function, M1 being mainly found in skeletal muscle, heart and brain, L in liver, R in erythrocytes and M2 in fetal tissue, but the latter persists also widely in adult tissues. Of the 4 iso-enzymes of rat, only the M l-type displays hyperbolic kinetics with regard to Penolpyruvate, while the three other types are all allosterically regulated. These kinetic properties are explained by the existence of different conformational states. A high affinity or Rstate which is stabilised by P-enolpyruvate, Fru(1,6)P2 and H ~ and a low affinity or Tstate which is stabilised by alkaline pH, ATP and certain amino acids [11 13]. Pyruvate kinase from T. hrucei differs in several respects from this generalisation and these differences will be discussed below. In the presence of Mg 2~ and Mn 2+ respectively, pyruvate kinase from T. hrucei exhibits sigmoidal kinetics with respect to the substrate P-enolpyruvate. Heterotropic activators, such as Fru(2,6)P2 change the saturation curves from sigmoidal to hyperbolic without changing V. This is however not the case with Co 2~ as activating metal ion. Velocity versus P-enolpyruvate concentration curves in the presence of Co 2~ and in the absence of an effector are already hyperbolic but the apparent affinity for P-enolpyruvate is weak (S0.5 = 4 raM). This may suggest that Co 2 ~-pyruvate kinase is not in the R-state, which is corroborated by the fact that Coe ~-pyruvate kinase is still sensitive to heterotropic activators that shift the enzyme into a conformational state with a high affinity for P-enolpyruvate. Fru(2,6)P2, which is the best activator for Mg 2 ~ and Mn: ~-pyruvate kinase, is much less effective in the case of Co 2 ~-pyruvate kinase. The S0.s for P-enolpyruvate decreases only 3 times, as compared to 10 times in the presence of Mg 2÷, and the turnover number of Co 2 pyruvate kinase is even lowered by Fru(2,6)P2. No such behaviour has ever been documen-
240
ted for isoenzymes of mammalian origin. For the M1 isotype it is known that Mg 2 ~ can be replaced by Co 2 ~ resulting in an enzyme with about the same catalytic efficiency [14]. For the L-isotype from rats, the kinetic data for Penolpyruvate with either Mg 2' or Mn 2 ~ as activating metal ion are comparable with our data for Mg 2 ÷ or Mn 2 ~-pyruvate kinase from T. brucei. The kinetics for P-enolpyruvate in the absence of a heterotropic activator are sigmoidal and become hyperbolic in the presence of Fru(1,6)P2. M n 2 ' - p y r u v a t e kinase has a lower S0.5 value for Penolpyruvate than Mg2 ' -pyruvate kinase and the apparent affinity of the Mn 2 ~-enzyme for P-enolpyruvate does not change in the presence of Fru(l,6)P2, in contrast to the situation with Mg2t-pyruvate kinase [12,15]. Our kinetic data for P-enolpyruvate in the presence of Mg 2~ or Mn 2~ are also comparable with those described for the enzymes from E. coli [16] and Bacillus lk'hen(formis [17]. Pyruvate kinase from Concholepas concholepas, however, shows sigmoidal kinetics in the presence of Mg 2+ and hyperbolic kinetics in the presence of Mn 2+ The latter resembles T. hrucei pyruvate kinase activated by Co 2~ except that Concholepas-Mn: ~-pyruvate ki2 nase is insensitive to Fru(l,6)P2 [18,19] whereas the affinity of Co2 ~-pyruvate kinase from T. brucei for P-enolpyruvate still increases. A strong influence of the bivalent metal ions on the S0.5 values for P-enolpyruvate is not unexpected since these metal ions are bound to the active site. The enzyme-bound bivalent metal ion is ligated to P-enolpyruvate and its function is to correctly orientate the phosphate group of P-enolpyruvate. The bivalent metal ion ligated to A D P is also involved in the correct orientation of the overlapping phosphates at the active site [20]. Comparison of the kinetics for the bivalent metal ions obtained at 2.5 mM P-enolpyruvate [5] with the present data, obtained at 5 mM Penolpyruvate (Table II), indicates modifications with the concentration of P-enolpyruvate. A higher P-enolpyruvate concentration partially abolished the allostericity and decreased the S0.s values for the metal ions.
In the presence of a heterotropic activator, the homotropic activation of the metal ion was almost abolished. A similar phenomenon has been described for pyruvate kinase from Phycomyces blukesh, eunus [21] and from E. coli [16]. Addition of Fru(l,6)P2 abolished the sigmoidal kinetics. In the case of rat-liver pyruvate kinase the binding of bivalent metal ions is already hyperbolic also in the absence of heterotropic activators [15]. It is, however, unexpected that the So.s value for the metal ions also depends on the nature of the heterotropic activators (and vice versa). According to Muirhead and coworkers [13,20], based on 3-dimensional structure of cat muscle pyruvate kinase, the binding site of the effectors is supposed to be located between domains A and C whereas the active site is located between domains A and B. This suggests that direct contacts between effector and metal ion are excluded, and, therefore, the interaction between metal ion and effector probably should be explained in terms of indirect allosteric conformational changes. A similar effect has been described in the case of phenylalanine, an allosteric inhibitor of MItype pyruvate kinase, that affects Mg 2~ pyruvate kinase and M n 2 - p y r u v a t e kinase differently [22]. The kinetics for the activators were almost hyperbolic under the conditions used. This is in agreement with the observations by Flynn and Bowman [23] who also reported the absence of cooperativity in the reaction rate of Mg 2~ pyruvate kinase from T. brucei with respect to Fru(1,6)P2 and at various concentrations of Penolpyruvate. In the case of E. coli pyruvate kinase, however, the velocity versus Fru(1,6)P2 concentration curves are sigmoidal at low concentrations of P-enolpyruvate while they change to hyperbolic at 2 mM P-enolpyruvate [16]. For the L-isotype from rat liver the binding of Fru(l,6)P2 is cooperative with a Hill coefficient of 2 at 0.5 mM P-enolpyruvate which still increases at higher P-enolpyruvate concentrations (4 raM) [24~15]. A comparison of the potency of the heterotropic activators of trypanosomal pyruvate kinase shows that Fru(2,6)P2 is by far the
241
most effective one, followed by Ru(1,5)P2, P R P P and Fru(1,6)P2 and/or GIc(I,6)P2. Although these heterotropic activators can be considered as structural analogues of Fru(2,6)P2 having the 2 phosphate groups at roughly the same distance, their degree of effectiveness is remarkably different. The apparent high affinity of pyruvate kinase for Ru(1,5)P2 and P R P P and their reduced capacity to stimulate Co2~-pyruvate kinase to maximal activity suggest that the l a t t e r 2 effectors structurally resemble more Fru(2,6)P2 than Fru(1,6)P?_ or Glc(I,6)P2. A similar conclusion has recently been drawn in the case of the pyruvate kinase from E. coli [25]. This enzyme was stimulated by Fru(1,6)P2 which was 100 times more active than Fru(2,6)P2. Ru(I,5)P2 and P R P P also activated the enzyme but the latter two behaved as weak activators that resembled Fru(2,6)P2 in their action, rather than Fru(1,6)P2. Detailed information about the structural requirements for the binding of the heterotropic activators is not yet available. Therefore it is still an open question as to why Fru(2,6)P2, Ru(1,5)P2 and PRPP are so much better as activators of the trypanosome pyruvate kinase than Fru(l,6)P2 and why the situation is the opposite for all other regulated pyruvate kinases [6,25]. Based on a comparison of amino acid sequences of different isoenzymes and their modelling into the 3dimensional structure of cat muscle pyruvate kinase some predictions have been made about a possible binding pocket for a heterotropic activator [13,20,26]. Such a binding site would be located between domains A and C of the subunit structure, while intersubunit contacts also seem to play a role in the allosteric behaviour of the enzymes. Recently, Speranza et al. [27] have identified a peptide of the E. coli enzyme that could be involved in the binding of Fru(1,6)P2. However, a definite answer about the nature of the binding pocket for the heterotropic activator should await the successful crystallization of the activatorenzyme complex or should come from sitedirected mutagenesis of the amino acids involved in the binding of the activator.
Like many other pyruvate kinases, activity and allosteric regulation of the T. brucei enzyme strongly depend on pH. The optimal pH for catalysis is situated at pH values below 7, while the activity sharply decreased at higher pH. This is in agreement with the results published by Flynn and Bowman [23]. A similar pH-dependency has also been reported for pyruvate kinase of human erythrocytes, which exhibits its optimal activity at pH 6.6 with a rapid decline in activity between pH 7 and pH 7.6. By contrast, rabbit-muscle pyruvate kinase has a pH optimum above pH 7.2 with almost no variation in activity between pH 7 and pH 7.6 [28]. With partially purified pyruvate kinase from T. hrucei the Hill coefficient did not change between pH 5.7 and pH 7.8 but the apparent affinity for Penolpyruvate was pH dependent [23]. Our purified pyruvate kinase behaves differently. Variation in pH from 6 to 8 changes the cooperativity in the reaction rate of Mg 2~ pyruvate kinase with respect to P-enolpyruvate without changing the S0.5 value for Penolpyruvate. The kinetics of P-enolpyruvate were sigmoidal up to 7.4, but at pH 8.0 the cooperativity disappeared. In this respect the T. brucei enzyme resembles the Ml-type pyruvate kinase from rabbit muscle and the enzyme from yeast, where the &~.5 lbr Penolpyruvate is the same at pH 6.5 and at pH 8.5, while the Hill coefficient increases with decreasing pH [29]. The reverse has been published for pyruvate kinase from liver [30,24], M-type pyruvate kinases from rat liver [31], erythrocytes [28] and P. blakesleeanus pyruvate kinase [32], where in all cases protons act as allosteric activator, converting the kinetics with respect to P-enolpyruvate from sigmoidal to hyperbolic and shifting the enzyme into a conformational state with a high affinity for Penolpyruvate. The effect of pH on the interaction of T. brucei pyruvate kinase with Fru(2,6)P2 resembles that for P-enolpyruvate, except that the apparent affinity for Fru(2,6)P2 decreases at pH values below pH 7.0. This partially resembles the kinetic behaviour of yeast
242
pyruvate kinase. At pH values above 7.5 the binding of the activator, Fru(l,6)P2, is no longer allosteric while the affinity decreases [29]. For rabbit-muscle pyruvate kinases, however, both the S0.s and the Hill coefficient remain unchanged [29]. Pyruvate kinases from liver [30], erythrocytes [28] and P. blakesleeanus [32] were no longer sensitive to heterotropic activation at acidic pH. In conclusion, the kinetic behaviour of the trypanosomal pyruvate kinase depends remarkably on its interaction with Penolpyruvate, the heterotropic activators and the activating bivalent metal ions. In addition to its unique stimulation by Fru(2,6)P2 the enzyme differs from its mammalian counterparts by the effect of pH on the allosteric regulation of the enzyme.
Acknowledgements We thank Mr. Joris Van Roy for the supply of the trypanosomes, Drs. E. Van Schaflingen and M.-F. Vincent for the assistance with some of the PRPP measurements, and Ms Franqoise Van de Calseyde-Mylle for secretarial assistance. This research received the financial support of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases; The Belgian State Prime Minister's Office Science Policy Programming. References 1 Allert, S., Ernest, 1., Poliszczak, A., Opperdoes, F.R. and Michels, P.A.M. (1991) Molecular cloning and analysis of two tandemly linked genes for pyruvate kinase of Trypanosoma brucei. Eur. J. Biochem., 200, 19 27. 2 Elynn, I.W. and Bowman+ I.B.R. (1980) Purification and characterization of pyruvate kinase from Trypanosoma brucei. Arch. Biochem. Biophys. 200, 401 409. 3 Van Schaftingen, E., Opperdoes, E.R. and Hers, H.-G. (1985) Stimulation of TI3"panosoma brucei pyruvate kinase by fructose 2,6-bisphosphate. Eur. J. Biochem. 153,403~406. 4 Barnard, J.P. and Pedersen, P.L. (1988) Purification in a single step and kinetic characterization of the pyruvate kinase of Trypanosoma hrucei. Mol. Biochem. Parasitol. 31, 141 147.
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243 kinase by phenylalanine. Biochemistry 11, 4415 4420. 23 Flynn, [.W. and Bowman, I.B.R. (1981) Some kinetic properties of pyruvate kinase from Trypanosoma brucei: influence of pH and fructose 1,6-diphosphate. Mol. Biochem. Parasitol. 4, 95 106. 24 EI-Maghrabi, M R . , Claus. T.H., McGrane, M.M. and Pilkis, S.J. (1982) Influence of phosphorylation on the interaction of effiectors with rat liver pyruvate kinase. J. Biol. Chem. 257. 233 240. 25 Speranza, M.L., Valentini, G. and Malcovati, M. (1990) Fructose-l,6-bisphosphate-activated pyruvate kinase from E. coli. Eur. J. Biochem. 191, 701 704. 26 McNally, T. and Fothergill-Gilmore, L.A. (1990) Sitedirected mutagenesis as a tool for the study of the allosteric control of pyruvate kinase. Biochem. Soc. Trans. 18, 258. 27 Speranza, M.L., Valentini, G., ladarola, P., Stoppini, M., Malcovati, M. and Ferri, G. (1989) Primary structure of three peptides at the catalytic and allosteric sites of the fructose-l,6-bisphosphate-activated pyruvate kinase from Escherichia coli. Biol. Chem. HoppeSeyler 370, 211 216.
28 Black, J.A. and Henderson, M.H. (1972) Activation and inhibition of human erythrocyte pyruvate kinase by organic phosphates, amino acids, dipeptides and anions. Biochim. Biophys. Acta 284, 115 127. 29 Brown, C.E., Taylor, J.M. and Chan, L.-M. (1985) The effect of pH on the interaction of substrates and el'lEctor to yeast and rabbit muscle pyruvate kinasc. Biochem. Biophys. Acta 829, 342 347. 30 Rozengurt, E., Jimdnez de Asua, L. and Carminatti, H. (1969) Some kinetic properties of liver pyruvate kinase (type L). Effect o f p H on its allosteric behaviour. J. Biol. Chem. 244, 3142 3147. 31 Van Berkel, T.J.C., Koster, J.F. and HOlsmann, W.C. (1973) Some kinetic properties of the allosteric M-type pyruvate kinase from rat liver; influence of pH and the nature of amino acid inhibition. Biochim. Biophys. Acta 321, 171 180. 32 de Arriaga, D.~ Busto, F., del Valle, P. and Soler, J. (1989) A kinetic study of the pH effect on the allosteric properties of pyruvate kinase from Phycomvces hlakesleeanus. Biochim. Biophys. Acta 998, 221 230.
