Phosphoenolpyruvate carboxykinase from Fasciola hepatica

PHOSPH~ENOLPYRUVATE ~ARBOXYKINASE FROM FASCIOLA HEPA TICA and C. BRYANT

CAROLYN A. BEHM

Department of Zoology, Australian National University, P.O. Box 4, Canberra, A.C.T. 2600, Australia (Received 24 Jr& 198I) Abstract-Bmn.f C. A. and BRYANT C. 1982. Phosphoenoipyruvate carboxykinase from Fasciola hepatica. International Journal for Parasitology 12: 271-278. The kinetic properties of a partially purified preparation of phosphoenolpyruvate carboxykinase (PEPCK) from F. hepatica were examined. The pH optimum for the carboxyiat& reaction is 5;8-6.2. The enzyme is more active with Mn2+ than Mgz+ and the Mnz+ saturation curve was sigmoid. Apparent Km values for the substrates GDP, IDP, PEP and HCOr were determined and found to be in the same range as those reported for other helminths except that the enzyme is less sensitive to low PEP concentrations. GTP and ATP at 0.5 and 1.0 mtw inhibit the enzyme; the GTP inhibition was greater in the presence of Mgz+ than Mn2+ and was competitive with GDP. It was concluded that the activity of PEPCK from F. hepaticu is controlled by the concentration of reactants and the ambient pH, that the accumulation of GTP is a sensitive mechanism for inhibiting the carboxylation reaction and that PEPCK activity in the cytosol is likely to be favoured over that of pyruvate kinase except when pH is high and PEP concentration low.

INDEX KEY WORDS: Fasciolu he~atjcu; phosphoenolpyruvate INTRODUCTION PHOSPHOENOLPYRUVATE

carboxykinase (PEPCK; E.C.4.1.1.32) and pyruvate kinase (PK; E.C.2.7.1.40) play key roles in invertebrate energy metabolism because they direct the flow of carbon from phosphoenolpyruvate (PEP) into the end-products of anaerobic metabolism. The enzymes compete for the substrate, PEP, and their relative activities are thought to account for the relative amounts of endproducts formed by the PEP-lactate or acetate and PEP-succinate or propionate pathways. Pyruvate kinase activity is known to be under tight allosteric control (see Behm & Bryant, 1980), but PEPCK activity appears to be controlled primarily by the concentrations of both the enzyme itself and its substrates and products only. An additional feature of PEPCK is that its reaction is readily reversible, aliowing synthesis of PEP and gluconeogenesis in many organisms. In parasitic helminths, PEPCK has been shown to be very active in nearly all groups studied, but its properties have been examined in only a small number of species, including Hymenolepis diminuta (Moon, Mustafa, Hulbert, Podesta & Mettrick, 1977; Reynolds, 1980; Wilkes, Cornish & Mettrick, 1981), ~~ni~i~ expansu (Behm & Bryant, 1975b), Ascaris suum muscle (Van den Bossche, 1969; Wilkes, Cornish & Mettrick, 1982), Moniliformis dubius (Cornish, Wilkes & Mettrick, 1981a) and Fasciola hepatica (Prichard & Schofield, 1968; Prichard, 1976, 1980). In addition, the enzyme has recently been purified from several species (Cornish

carboxykinase; metabolic regulation.

et al., 1981a; Wilkes et al., 1981, 1982). As part of a continuing study of regulatory metabolism in the liver fluke, F. hepatica, we have examined the properties of a partially purified preparation of PEPCK, with the aim of elucidating its role at the PEP branchpoint in this species. Preliminary studies of the liver fluke enzyme have been made by Prichard and Schofield (1968) and Prichard (1976, 1980). With cytosol supernatants, Prichard (1976) found that NADH added to the reaction mixture greatly reduced the enzyme’s apparent .&, (PEP); he suggested that this effect could be significant in determining flow through the succinate-propionate pathway under changing conditions of oxygen tension. In a later study, Prichard (1980) calculated that the reaction catalysed by the enzyme was out of equilibrium under steadystate conditions in vivo, and found that, in cytosol supernatants, (a) it was more active with Mn2+ than Znz+ as the added cation, (b) the pH optimum with Mn2+ was between 5.7 and 6.7, and (c) that ATP inhibited activity by increasing the apparent K, for both substrates, PEP and IDP. This study suggested that the enzyme’s activity was regulated by endproduct inhibition, by availability of the substrate PEP and, particuiarly, by ATP inhibition. These conclusions are investigated in more detail below.

MATERIALS

AND METHODS

Adult F. heputicu were collected from the bile ducts of sheep at the Goulburn Abattoirs, NSW. They were

271

272

CAROLYN A. BEHM and C. BRYANT

maintained overnight at 39°C in H&don-Fleig solution containing 4 g/l glucose, 100 i.u./ml penicillin G, 100 pg/ml streptomycin sulphate and 2.5 ,ugcg/ml amphotericin B. Flukes that had disgorged their caecal contents were selected the next day for preparation of PEPCK. Enzymes, substrates and cofactors were purchased from Boehringer Mannheim (Australia). MES, PIPES, MOPS, and HEPES buffers were obtained from Calbiochem (Australia) and bovine serum albumin from Commonwealth Serum Laboratories, Melbourne. Sephacryl S-200 and Sephadex G25 were purchased from Pharmacia (South Seas). Analytical grade chemicals and glass-distilled water were used throughout. Preparation of PEPCK. Flukes were blotted, weighed and rinsed twice in a buffer containing 0.1 M MES-KOH, pH 6.0, 5 mM dithiothreitol and 1 mM EGTA. This buffer was employed throughout the preparation. They were homogenised 1:2 (w:v) at 0-2°C in the same buffer using an all-glass Dounce homogeniser. The homogenate was ce&ifuged at loo0 g, 4°C. for 10 min and the suuernatant centrifuged at 20,&O g, i”C, for 20 min. The sipernatant was brought to 50% saturation (at O’C) with solid (NH&SO, and allowed to stand on ice for 2 h. The suspension was centrifuged at 20,000 g, 4”C, for 20 min and the supernatant brought to 75% saturation with (NH,)$iO,. This suspension was allowed to stand overnight. The precipitate was recovered by centrifugation at 20,000 g, 4”C, for 20 min and redissolved in a volume of buffer equal to the original wet weight of flukes. The ammonium sulphate fractionation was repeated, with the fraction precipitating at 50-65% saturation being collected. The precipitate was redissolved in buffer at the rate of 5 ml/25 g wet weight of flukes, precipitated with 80% (NH&SO, and stored at 4°C until the next stage of purification. Approximately 20 units of the PEPCK preparation were recovered by centrifugation as above and redissolved in 5 ml buffer. This solution was centrifuged for 2 min at 14,OOOg, 4”C, and the supernatant applied, with a sample applicator, to a column, 65 x 1.6 cm, containing Sephacryl S-200 equilibrated with the same buffer. The preparation was eluted at a flow rate of 0.25 ml/min and 1 ml fractions were collected. Those fractions with the highest PEPCK activities were pooled and stored as a suspension at 4°C in 80% (NH&SO, in buffer. Under these conditions the preparation loses about 10% activity in 2 months. For assay, PEPCK was recovered by centrifugation at 20,000 g, 4”C, for 20 min. redissolved in 1 ml buffer and desalted in a column (0.9 x 13 cm) of Sephadex G25 equilibrated with the same buffer. The preparation, which was stable for 12-24 h, was used immediately. Assay of PEPCK. The enzyme was routinely assayed spectrophotometrically at 25°C in the carboxylation direction, using NADH and malate dehydrogenase (MDH, E.C.l.l.1.37) to follow the production of oxaloacetate (OAA). The reaction mixture giving the maximum activity was as given below and was used routinely. In a final volume of 2 ml the mixture contained: 0.1 M MES-KOH, pH 6.0; 10 rnM MnCI,; 0.14 mM NADH; 1.0 mM GDP; 12 units MDH; 20 mM KHCO,; 1.0 mM PEP; approx. 15 wg PEPCK preparation. All the components except KHCO, were brought to pH 6.0 with KOH before addition to the cuvettes. After preincubation of the enzyme at 25°C with all the other constituents, the reaction was initiated with PEP; the disappearance of NADH was followed at 334 nm, with a light path of 1 cm in an Eppendorf recording photometer, thermostatted at 25”C, for up to 4 min. No reaction occurred if GDP, or PEP, or HCOr was omitted from the

1.1.~. VOL. 12. 1982

mixture. All assays were performed in quintuplicate using disposable plastic cuvettes; reaction rates were calculated by the method of Henderson (1971); apparent values of Km and v,,, were computed by the method of Wilkinson (1961). Protem was determined by the method of Lowry, Rosebrough, Farr & Randall (1951) using bovine serum albumin as a standard. Other assuys. The following methods were used to test for other enzymes present in the PEPCK preparations: enolase (E.C.4.2.1.11), adenylate kinase (E.C.2.7.4.3), lactate dehydrogenase (LDH, E.C. 1.1.1.27), (Bergmeyer, Gawehn & Gras& 1974); malic enzyme (E.C.1.1.1.40) (Hsu 8.1 Lardy, 1969); malate dehydrogenase (E.C. 1.1.1.37) (Bergmeyer & Bernt, 1974). The concentrations of substrates in

prepared solutions were assayed by the following methods: PEP (Czok & Lamprecht, 1974); GDP, IDP (Grassl, 1974a); GTP (Gras& 1974b). It was necessary in the GTP inhibition studies to correct the added GDP concentration for the GDP content of GTP solutions. RESULTS

Partial purification

of PEPCK activity in the 20,000 g supernatant

PEPCK of homogenates was 0.33 units/mg protein. In the SO-65% (NH&SO, preparation the activity was 0.52 units/mg protein, with a yield of about 40%. After Sephacryl S-200 fractionation the pooled PEPCK samples had an activity of 1.5 units/mg protein, a five-fold purification with a final yield of 30-35%. Ammonium sulphate fractionation effectively separates PEPCK from pyruvate kinase. The 50-65% fraction contains high activities of PEPCK, plus substantial contamination by MDH, enolase, malic enzyme and minor amounts of LDH. The results of Sephacryl S-200 elution of PEPCK are given in Fig. 1. This method removes significant quantities of malic enzyme from the preparation, but MDH and enolase remain in the pooled PEPCK samples in considerable quantities. The relative composition of the pooled PEPCK preparation was PEPCK 1.0, malic enzyme 0.43, enolase 4.68, MDH 11.39. The MDH activity was stable upon storage, but malic enzyme and enolase activities decreased by approx. 40% in a week under the storage conditions employed. Since MDH is utilised in the coupled assay system for PEPCK, its presence is unlikely to affect the results. Malic enzyme had little activity with NAD+ compared with NADP+ ; it was therefore considered unlikely to interfere with the assays for PEPCK. Enolase catalyses a readily reversible reaction which may affect determination of the apparent K,,, (PEP); aged (2 or 4 weeks) preparations were therefore used for this determination, but the results were similar to those obtained using fresh preparations. The pooled S-200 PEPCK preparation was free from adenylate kinase activity. Attempts were made to separate MDH from PEPCK in the S-200 preparation in order to test directly for an effect of NADH on PEPCK activity. Affinity chromatography using GDP-agarose (Sigma Chemical Corp.) or GTP-agarose (Pharmacia) proved unsuccessful due to coelution of the two

273

PEP carboxykinase from Fasciola hepatica

I.J.P. VOL.12. 1982

45

50

55

60

65

70

75

60

65

90

ml eluted

FIG. 1. Elution of PEPCK and other enzymes by Sephacryl S-200. Column dimensions 65 cm optical density at 280 nM; -PEPCK flow rate 0.25 ml/min, sample volume 5 ml. ,,,,,N// malic enzyme activity;

I

II

enolase

enzymes under a variety of binding-elution conditions. It was not possible, therefore, to test the effect of NADH on this preparation. The NADH effect observed by Prichard (1976) is most likely an artefact due to end-product inhibition of the carboxylation reaction (which is readily reversible) by accumulation of oxaloacetate in the reaction system employed. Addition of NADH to that system would permit reduction of oxaloacetate to acid-stable malate catalysed by the high levels of MDH known to be present in F. hepatica cytosol preparations. The result would be an apparent increase in the rate of 2.0

.g

activity

x

10-t; IIIUILI MDH activity

x

X

1.6 cm, activity; 10-t.

reaction by preventing oxaloacetate accumulation. In the present work a coupled spectrophotometric assay system was employed to minimise the effects of endproduct accumulation on measured kinetic parameters. The partial purification miniiised interference from pyruvate kinase and other enzymes. pH Curves Figure 2 shows typical pH curves for PEPCK with MS+ and Mgz+ . Each curve has a single major peak, at pH 54-6.2 with MS+ and pH 5.8 with Mg2+. There is a steady decrease in activity to pH

1

(b)

1.5-

0.3

;; k

_

:

-

1 c 0

1 \

5 :

t.o\

-

z 5

0.2-

;

,:

c E

-

lo.,i;

0.5-

I

5.6

5.0

5.4

6.8 PH

7.2

7.6

6.0

5.6

6.0

6.4

6.6

7.2

7.6

6.0

PH

FIG. 2. Effects of pH on PEPCK activity (a) with Mn*+, (b) with Mg z+. A combined buffer, 0.05 M in each of MES, PIPES, MOPS, and HEPES, adjusted to the required pH with KOH, was employed across the whole pH range. The reaction mixture was as in Methods except that PEP concentration was 2.5 mM, GDP was 0.82 mM and Mn2+ was replaced by 12.5 mM Mgz+ for curve (b). The activities in the two curves are not directly comparable because the tests were performed on different days. Points are X f S.D.

274

CAROLYN

I.J.P. VOL. 12. 1982

A. BEHM and C. BRYANT

8.0, with the decline more pronounced in the case of Mg2+. With MS+, activity at pH 7.0 is 63% of that at pH 6.0; in the case of Mgz+ it is 18%. With Mgz+, therefore, the enzyme is probably not significantly active above pH 6.4: its activity is only 30% of the activity with MS+ at pH 6.4. All subsequent kinetic work with this enzyme was performed at pH 6.0. Effects of Mn2 + and Mg2 +

Typical saturation curves for MS+ and Mg2+ are shown in Fig. 3 as Lineweaver-Burk plots. For MS+ the relationship is sigmoid, with a Hill coefficient of 2.2. The reaction rate with MS+ decreased with time below 2.5 mM Mn2+; initial reaction rates were therefore determined in the first 30 s in individual cuvettes to obtain the curve illustrated. Mg2+ shows a hyperbolic relationship and the reaction rate was linear with time at all concentrations tested, after an initial lag of 1 min. The calculated values for apparent Km are 1.18 & 0.15 mM for Mn2+ and 0.83 + 0.03 mM for Mg2+. There is no inhibition at concentrations up to 15 mM for either cation. Tests for interaction between Mn2+ and Mg2+ showed only additive effects. Effects of PEP

Hyperbolic saturation curves were evident for PEP in the presence of Mn2+ or Mg2+. Table 1 gives the calculated values of apparent Km and V, for the determinations on one preparation. Relative affinities for PEP with Mn*+ and Mg2+ varied with different preparations, but a higher maximal velocity is attained when Mn*+ is the cation added. At 0.2 mM PEP, activity with Mn*+ is six times greater than with Mg2+. No inhibition is evident at high PEP concentrations with either cation. Effects of GDP and IDP

Both IDP and GDP showed hyperbolic saturation curves. Apparent values of K,,, for one preparation are recorded in Table 1. The enzyme is more sensitive to low concentrations of either nucleotide when Mg2+ is the cation present, but the maximal velocity is 4-5 times greater in the presence of Mn2+. GDP

0

6

cation.

rnM

Effects of HCO,

Figure 4 depicts typical saturation curves for HCO, with MS+ or Mg2+. In order to avoid bubble formation and pH changes at the higher HCO,- concentrations, the KHCO, was added to the preincubated reaction mixture 0.5 min before initiating the reaction with PEP. Each cuvette was read individually. The enzyme shows hyperbolic kinetics with either Mn2+ or Mg2+, and it is clear that the optimal HCOj concentration with Mg2+ is above 30 mM. Calculated values of apparent Km and V, are given in Table 1. The enzyme is much less sensitive to HCO, with Mg2+ as cation than with Mn2+; at 20 mM KHCO, activity with Mn2+ is approximately six times greater than with Mg2+. V,,, FOR PEPCK

FROM F. hepatica

vnl* + Mg2+

+ Mn2+

+ Mg2+

0.022 * 0.002

0.012 + 0.002

1.25 z!z0.02

0.29 ” 0.01

IDP t

0.083 + 0.013

0.058 ? 0.006

1.38 ? 0.06

0.38 ? 0.01

PEP $

0.238 f 0.014

0.614 f 0.082

1.65 f 0.03

0.44 2 0.03

1.32 2 0.03

0.37 ? 0.03

Q

4.18

-to.35

19.70

10

-1

showed slight inhibitory activity (about 20%) at 2.05 mM; the optimal GDP concentration for this assay system was approx. 1.0 mM. The enzyme was inactive with ADP.

GDP t

HCOj

8

)

FIG. 3. Lineweaver-Burk plot of saturation curves for Mn2+ or Mg*+. Assays were performed as in Methods, with 0.82 mM GDP, 2.5 rnM PEP at pH 6.0. The lines are drawn from intercepts calculated by the method of Wilkinson (1961). l with Mn2+ ; n with Mgz+.

&I* +Mn2+

4

2

1 diYale”t

VALUESOF K,,, AND

TABLE I-APPARENT

Substrate

> -2

% 2.62

*Calculated by the method of Wilkinson (1961). ~mole/min/mg protein (V,). TConcentrations tested 0+?08-2.05 mM (10 points). $Xoncentrations tested O+XI8-2~0 mM (10 points). 5Concentrations tested 0.50-60 IIIM(8 points).

The units are mM (K,)

and

I.J.P. VOL. 12. 1982

PEP carboxykinase from Fasciola hepatica

FIG. 4. Saturation curves for HCO, with Mn2+ or Mgz+. Assays were performed as in Methods, with 1.5 mM GDP and 10 rnM Mnz+ or Mg*+. Points are x f S.D. + with

275

same conditions. The GTP inhibition was significantly greater with Mg*+ than Mn2+ at the substrate concentrations employed. It is possible that some of this inhibition is caused by chelation of the divalent cation by the nucleotide triphosphate; however, the inhibition by ATP is likely to be a measure of the maximum chelation effect, and therefore the GTP inhibition is unlikely to be due to chelation alone. The effects of GTP on the apparent kinetic constants of PEPCK are shown in Table 2. The inhibition is mixed-competitive with PEP and competitive with GDP, with either Mn*+ or Mg*+ as cation. For HCO,, the effect is non-competitive with Mn*+ but with Mg*+ it is clearly competitive. L-Alanine at a concentration of 1 or 2.5 mM had no effect on PEPCK activity, whether tested at low or high concentrations of GDP or PEP, with Mn*+ or Mg*+. Similarly, it had no effect on the enzyme inhibited by GTP.

Mn*+; 0 with Mg*+. Effecters

of PEPCK

activity

DISCUSSION

ATP and GTP both inhibited PEPCK activity. At 1.0 mM, GTP reduced the activity by 34 and 94% with Mn2+ and Mg*+ respectively; ATP at the same concentration inhibited by 31 and 30% under the TABLE ~-EFFECTS OF GTP

The results from the present study are summarised and compared, in Table 3, with those obtained by other workers with PEPCK from other parasitic helminths.

ON KOJETNZ PARAMETERS OF PEPCK

Mn*+ No GTP

FROMF. hepatica*

Mg*+ 1.0 rnM GTP

No GTP

0.5 mM GTP

PEP

K,,, “Ill

0.50 f 0.042 1.13 2 0.03

0.60 f 0.07 0.93 2 0.04

0.37 f 0.07 0.21 2 0.01

0.69 f 0.14 0.10 t 0.01

GDP

K, “Ill

0.01 f 040 0.81 f 0.01

0.38 f 0.06 0.89 f 0.05

0.02 f 0.01 0.20 f 0.01

0.18 2 0.06 0.19 2 0.03

HCOj K, “lIl

4.18 * 0.35 1.32 ? 0.03

4.39 f 0.15 1.19 f 0.01

19.70 f 2.62 0.37 f 0.03

86.42 f 44.59 0.26 ? 0.14

*GDP concentration employed was 1.5 mM; corrections were made for GDP and CTP content of GTP solutions. PEP concentration employed was 2.0 mM. K,,,‘s and V,,,‘s calculated by the method of Wilkinson (1961). All curves had 6 or 7 points. Units are mM (K,) or pmole/min/mg protein (V,). TABLE 3-PEPCK

Enzyme

source

FROMF. heparica; SUMMARYOF RESULTSAND COMPARISONS WITH OTHERPEPCKs

Reference

PH optimum

K,,, HCOj mM

K,

K,,, GDP mM

K,,, IDP mM

4.18 (Mn*+) 19.70 (Mg*+)

0.24 (Mnzf) 0.61 (Mg*+)

0.083 (Mnz+) 0.058 (Mg*+)

0.15 (Mn*+)

0.022 (Mn* +) 0.012 (Mg*+) -

-

0.09 (Mnz+) 0.10 (Mg*+) 0.08 (Mn* +)

0.15 (Mn*+) 0.03 (Mg* + ) 0.10 (Mnz+)

0.15 (Mn*+) 0.04 (Mg* +) 0.20 (Mnz+)

PEP mM

Fasciola hepatica

This paper

5.8-6.2

Fasciola hepatica

Prichard & Schofield (1968) Prichard (1980)

5.7-6.7

Moniezia expansa (cytosol) (mitochondria)

Behm & Bryant (1975b)

Hymenolepis

Wilkes et al. (1981)

5.6

3.3 (Mnz+)

0.039 (Mn*+)

0.02

I (Mn* + )

0.078 (Mn*+)

Moniliformis dubius

Cornish

5.5

4.3 (Mn*+)

0.069 (Mn*+)

0.002 (Mn* +)

0.017 (Mn*+)

Ascaris suum

Wilkes et al. (1982)

5.8

3.2 (Mn*+)

0.066 (Mn*+)

0.013 (Mnz+)

0.076 (Mnz+)

diminuta

6.6 6.6

et aL(1981a)

0.40 (Mn* +)

216

CAROLYN A. BEHM and C. BRYANT

All workers agree that the pH optima for the helminth enzymes are low, in comparison with the normally accepted ‘physiological’ pH. However, even PEPCKs from vertebrate sources have pH optima between 6 and 6.8 (see Utter & Kolenbrander, 1972). Since PEPCK requires CO, rather than HCO,as a substrate (Miller & Lane, 1968), pH curves obtained experimentally may be different for the forward and reverse reactions. The absolute concentration of CO, would be highest for a given concentration of added HCO, at pH values below 7 and it is possible that the steep negative slope of the pH curve fdr the carboxylation reaction above this pH is due to limiting concentrations of CO, available as a substrate. For all the studies in Table 3 the value of the apparent K,,, (HCO,-) is an overestimate of the K,,, (CO,) because actual concentrations of CO, present in the reaction mixtures are lower depending on pH, temperature, gas phase and ionic strength. In vivo, carbon dioxide concentrations are likely to be high and pH values likely to be below 7, so PEPCK in these organisms is probably able to operate under favourable conditions, at least with respect to the carboxylation reaction. The affinity of F. hepaticu PEPCK for CO, is similar to that found for other helminths, despite the fact that bile ducts and intestinal lumina probably have quite different ambient levels of CO,. It is doubtful whether Mg*+ is a direct activator of helminth PEPCK because the three helminth PEPCKs that have been purified, from ZZ. diminutu, M. dubius and A. suum, show no in vitro activity with Mg2+ alone, though the M. dubius and A. mum enzymes were active with Mg2+ at the ammonium sulphate stage of purification (R. A. Cornish and J. Wilkes, personal communication). Hence activity with Mg2+ could be due to interaction with another protein in the impure (but washed) preparations from F. hepatica and M. expansa. Zn vivo, of course, the enzyme is not pure, so Mg2+ activation may still play a physiological role, but the high apparent K,,, (HCOj-) with Mg2+ suggests that this is unlikely.

TABLE 4-INTERNAL

I.J.P.VOL.

The apparent Km (PEP) for F. hepatica PEPCK is 2-4 times higher than that measured for PEPCKs from other helminths (see Table 4). Measured internal PEP pools in F. hepatica are within the same range as those found in other helminths, so fluke PEPCK is possibly less saturated by PEP in vivo than is the case in other helminth species. Its activity, therefore, may be more likely to vary with changes in PEP levels than in other helminths. All the helminth PEPCKs so far studied have a higher affinity for GDP than IDP as ,a substrate, so GDP is the most likely candidate for that role in vivo. The GTP formed could be utilised directly in anabolic reactions such as synthesis of purines and nucleic acids, or transformed to ATP by nucleoside diphosphokinase which is known to be very active in F. hepatica cytosol (Barrett, 1975). Since GTP is an effective inhibitor of PEPCK in addition to being a reaction product, GDP/GTP ratios could influence the enzyme’s activity significantly. ATP, also, may play some part, but it is noteworthy that the three helminth enzymes purified to homogeneity are not affected by ATP although GTP is a potent inhibitor. ATP added to a reaction mixture containing GDP and nucleoside diphosphokinase (which is likely to contaminate impure PEPCK preparations) will form GTP; this could be the source of the apparent ATP inhibition of PEPCK reported by Prichard (1976, 1980). Since absolute concentrations of GTP (or possibly ITP) in liver fluke tissues are not known, it is difficult to predict whether this effect is likely to be important under physiological conditions. Table 5 lists the kinetic parameters for pyruvate kinase and PEPCK from F. hepatica at pH 6.4 and 6.0 respectively. Although pyruvate kinase regulation is more sensitive around pH 7, the pH at which the two enzymes are most likely to compete for PEP is probably around 6-6.5 because PEPCK activity is much reduced at higher pH values. The maximal activities of the two enzymes measured in supernatant preparations from F. hepaticu are 0.33 units for PEPCK and 0.012 units for pyruvate kinase,

CONCENTRATIONSOF PEP IN HELMINTHS

K,,, (PEP) for Species

F. hepatica M. expansa M. dubius

A. suum H. diminuta

[PEP],

mM*

Reference?

12. 1982

PEPCK,

mM

Reference+

0.11, 0.17

1, 2

0.24

3

0.06-O. 10

4

0.09

5

0.25-0.42 0.14 0.35

6 8 10

0.069 0.066 0.039

7 9 11

*Assuming even distribution throughout the body. 1. Cornish & Bryant (1976). 2. Prichard (1976). 3. This j-References: paper. 4. Behm & Bryant (1975a). 5. Behm & Bryant (1975b). 6. Cornish, Wilkes & Mettrick (1981b). 7. Cornish et al. (1981a). 8. Barrett & Beis (1973). 9. Wilkes et al. (1982). 10. Bueding & Saz (1968). 11. Wilkes et al. (1981).

I.J.P. VOL. 12. 1982

PEP carboxykinase TABLE ~-KINETIC

Enzyme PK? PEPCK

PH 6.4 6.0

*Units are mM +Behm & Bryant

217

from Fasciola hepatica

PARAMETERSFOR PK

AND

PEPCK

FROMF. hepatica

K,,, (PEP)* -FBP + FBP

K,,, -FBP

Mn2+

0.05

0.05

0.17

0.19

Mg2+

0.42

0.04

0.55

0.60

Mn2+ Mgz+

0.24 0.61

-

Cation

-

(ADP)*

K,,, (GDP)*

+ FBP

-

0.02 0.01

(1980).

giving a relative PEPCK:PK activity of 27.5: 1. Hence, under optimal conditions, PEPCK is likely to be the more active. However, PEPCK is much less sensitive to low levels of PEP than pyruvate kinase, and it may not be fully saturated with PEP at concentrations prevailing in fluke tissues. Inhibition of pyruvate kinase at high HCO, concentrations may also play a role if HCO, concentrations are particularly high. Both enzymes are inhibited by elevated levels of ATP, though PEPCK is more sensitive to GTP inhibition which is more likely to play a physiological role. The rate of utilisation of GTP may have a significant controlling effect on PEPCK activity in vivo. If pyruvate kinase and PEPCK are directly competing in liver fluke cytosol (they may, of course, reside in different tissues), pyruvate kinase probably has some advantage at high pH and low PEP levels but its overall competitive ability is limited by its considerably lower concentration in the cytosol. In conclusion, PEPCK activity in F. hepaticu appears to be controlled by the concentration of its substrates and products and by the ambient pH. At pH values around 6 the ambient CO, concentration may be sufficient to saturate the enzyme but above pH 7 this is probably not the case. Low in vivo levels of PEP are likely to limit the enzyme’s activity because of its relatively high apparent Km (PEP). Accumulation of the reaction product GTP, or its recruitment from ATP via nucleoside diphosphosensitive method for kinase, is a potentially inhibition of the carboxylation reaction; whether this operates in vivo remains to be demonstrated. Acknowledgements-This work was supported by a grant from the Wool Research Trust Fund on the recommendation of the Australian Wool Corporation.

REFERENCES BARRETT J. 1975. The occurrence and intracellular distribution of nucleoside diphosphokinase in parasitic helminths. Journal of Parasitology 61: 545-546. BARRETT J. & BEIS I. 1973. The redox state of the free nicotinamide-adenine dinucleotide couple in the cytoplasm and mitochondria of muscle tissue from Ascaris lumbricoides (Nematoda). Comparative Biochemistry and Physiology 44A: 331-340.

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