AbstractSheep liver pyruvate carboxylase was mixed with avidin at a molar ratio of 1:1 in the presence of various combinations of the components of the assay systems required for either the acetyl-CoA-dependent or the acetyl-CoA-independent activity and negatively stained samples were examined by electron microscopy. Significant numbers of chain-like polymers of enzyme-avidin complexes were evident only when acetyl-CoA or high levels of pyruvate were present in the media. Similar results were also obtained for chicken liver pyruvate carboxylase despite this enzyme's almost complete lack of acetyl-CoA-independent activity. Thus, although acetyl-CoA and high concentrations of pyruvate may induce pyruvate carboxylase to adopt a ‘tight’ tetrahedron-like conformation which can interact with avidin to form chains, this structural change alone does not result in an enzymic form that is maximally active. This suggests that the allosteric activation of pyruvate carboxylase by acetyl-CoA is attributable, at least in part to more subtle conformational changes, especially in the case of the chicken enzyme

Attwood, Paul V.

Mayer, Frank

Wallace, John C.

Elsevier - Publisher Connector 

Volume 203, number 2 FEBS 3859 July 1986 
Avidin as a probe of the conformational changes induced in 
pyruvate carboxylase by acetyl-CoA and pyruvate 
Paul V. Attwoodt, Frank Mayer” and John C. Wallace* 
*Department of Biochemistry, University of Adelaide, Adelaide, South Australia, 5001, Australia and “Institut fcr 
~~rob~o~ogie d r ~eorg-Aunt-~~iversit~t zu Giittingen, ~r~se~a~hstr~se 8,D-3@0 e~tti~ge~, FRG 
Received 2June 1986 
Sheep liver pyruvate carboxylase was mixed with avidin at a molar ratio f I:1 in the presence of various 
combinations of the components of the assay systems required for either the acetyi-CoA-dependent or the 
acetyl-CoA-independent activity and negatively stained samples were examined by electron microscopy. Sig- 
nificant numbers of chain-like polymers of enzyme-avidin complexes were evident only when acetyl-CoA 
or high levels of pyruvate were present in the media. Similar rest&s were atso obtained for chicken liver 
pyruvate carboxylase despite this enzyme’s almost complete lack of acetyl-CoA-independent activity. 
Thus, although acetyl-CoA and high concentrations of pyruvate may induce pyruvate carboxylase to adopt 
a ‘tight’ tetrahedron-like conformation which can interact with avidin to form chains, this structural change 
alone does not result in an enzymic form that is maximally active. This suggests that the allosteric activation 
of pyruvate carboxylase by acetyl-CoA is attributable, at least in part to more subtle conformational 
changes, especially in the case of the chicken enzyme. 
Pyruvate carboxylase Ailosteric activation Conformational change Acetyl-CoA Pyruvate Avidin 
1. INTRODUCTION 
Despite the dramatic effects which acetyl-CoA 
has on both the activity and the stability of 
pyruvate carboxylase purified from vertebrate 
liver, the readily discernable effects which the 
allosteric effector has directly on the enzyme’s 
structure are mostly subtle {review II]). 
Nevertheless, markedly enhanced preservation 
of pyruvate carboxylase’s tetrameric structure has 
been observed in electron microscopic studies of 
this enzyme purified from a thermophilic Bacillus 
121, vertebrate liver 131, Aspergiks ~~~~~~~s 141, 
Rhizopus arrhizus [5] and Saccharomyces 
cerevisiae [6]. In the presence of acetyl-CoA the 
pyruvate carboxyiases from vertebrate Iivers were 
* To whom correspondence should be addressed 
r Present address: Merrell Dow Research Institute, 
Strasbourg Research Centre, 16, Rue d’Ankara, 67084 
Strasbourg Cedex, France 
found to exhibit a higher proportion of compact, 
triangular images enclosing three readily visible in- 
tensity maxima. In the absence of acetyl-CoA, 
besides a higher percentage of particles having no 
consistent geometry, there was evidence also of a 
much higher proportion of ‘open’, predominantIy 
rhomboidal projections 131. 
in the course of electron microscopic studies of 
avidin-enzyme complexes [7] it was noted that only 
in the presence of acetyl-CoA did the characteristic 
linear, unbranched polymers form when the 
avidin:enzyme ratio was between 2: 1 and 1: 2. It 
appeared, therefore, as if avidin, with its biotin- 
binding sites arranged 2 nm apart in pairs on op- 
posite sides of the 4.5 nm diameter molecule [8,9], 
may represent a sensitive probe of pyruvate car- 
boxylase’s conformation in the presence of various 
substrates and metabolic effecters. 
Under suitable conditions (viz., in the presence 
of high concentrations of enzyme, of NHd, and of 
the substrates pyruvate and HCO?), sheep liver 
Published by Elsevier Science Publishers B. V. (Biomedical Division) 
00145793/86/%3SO 0 1986 Federation of European Biochemical Societies 191 
Volume 203, number 2 FEBS LETTERS July 1986 
pyruvate carboxylase can exhibit specific activities 
in the absence of acetyl-CoA up to 25% of that 
found when this ligand is present at saturating con- 
centrations [lo]. Chicken liver pyruvate carbox- 
ylase, however, does not exhibit a comparable level 
of acetyl-CoA-independent activity under these 
conditions [lo]. Therefore, it seemed possible that 
some component(s) of the acetyl-CoA-independent 
assay mix may induce sheep liver pyruvate carbox- 
ylase to adopt the compact tetrahedral structure 
found in the presence of acetyl-CoA, whereas this 
would not be the case for the chicken liver enzyme. 
This was investigated using electron microscopic 
observation of avidin-enzyme chain formation as a 
measure of enzyme conformation. Contrary to ex- 
pectations, however, it was found that similar 
changes in conformation were induced in both 
sheep and chicken liver pyruvate carboxylases by 
high concentrations of pyruvate (or 2-oxobutyrate) 
alone. 
2. MATERIALS AND METHODS 
Avidin and d-biotin were obtained from Sigma, 
while [14C]biotin and NaH14COs were obtained 
from Amersham (Australia). All other materials 
were high purity preparations as described earlier 
[7,111. 
2.1. Preparation of pyruvate carboxylase 
Pyruvate carboxylase was purified from sheep 
and chicken liver mitochondria according to Goss 
et al. [l l] except that DEAE-Sepharose replaced 
DEAE-Sephadex. The fractions containing en- 
zyme of the highest specific activities, 25-30 
units - mg-’ protein (where 1 unit enzyme catalyses 
the formation of I pmol oxaloacetate . min-’ at 
3O”C), were pooled and the enzyme precipitated by 
the addition of ammonium sulphate (27.7 
g/100 ml). This enzyme could be stored at - 80°C 
without loss of activity if dissolved in a buffer 
composed of 0.1 M N-ethylmorpholine acetate, 
pH 7.0, 0.04 M ammonium sulphate and 1.6 M 
sucrose. Before use, these salts and sucrose were 
removed from the enzyme by gel filtration on a 
TSK G-4000SW column (25 cm x 7.2 mm) 
equilibrated in 0.1 M Tris-Cl, pH 7.2, containing 
0.1 M KCl. 
192 
2.2. Measurement of biotin and biotin-binding 
sites 
The biotin content of the enzyme and the biotin- 
binding capacity of the avidin were determined as 
described by Johannssen et al. [7]. Where ratios of 
avidin: enzyme are quoted, these refer to [biotin- 
binding sites] : [biotin]. 
2.3. Preparation of enzyme-avidin complexes 
In all experiments the enzymes were diluted to 
-16 units-ml-’ in the following solutions, each of 
final volume 96 pl: (a) The complete acetyl-CoA- 
independent assay mix (viz. 100 mM Tris-Cl, pH 
8.4 [sheep] or pH 7.8 [chicken], 2.5 mM ATP, 
8 mM MgClz, 40 mM Na pyruvate, 40 mM 
NaHC03, 100 mM NHQ. (b) The acetyl-CoA- 
dependent assay mix (viz. 100 mM Tris-Cl, pH 8.4 
[sheep] or pH 7.8 [chicken], 2.5 mM ATP, 7 mM 
MgC12, 10 mM Na pyruvate, 20 mM NaHCO3) 
with or without 0.25 mM acetyl-CoA. (c) The 
acetyl-CoA-dependent assay mix containing in- 
stead 40 mM Na pyruvate or 40 mM NaHCO3 or 
8 mM MgC12 or 100 mM NH4Cl or 40 mM 
2-oxobutyrate but no acetyl-CoA. (d) 0.1 M Tris- 
Cl, pH 8.4 [sheep] or pH 7.8 [chicken], containing 
40 mM pyruvate. The solutions were incubated at 
30°C for 30 min before the addition of 4 ~1 avidin 
solution (4 mg . ml-‘, so that the ratio of 
avidin: enzyme was 1: l), and then for a further 
2 h at 30°C to ensure as complete avidin binding as 
possible. At the end of this period, 50~1 of each 
sample were added to 950 $1 of 0.1 M Tris-Cl at 
the appropriate pH to give a final enzyme concen- 
tration of -40 pug eml-’ (-0.8 unit-ml-‘). These 
solutions were incubated at 30°C for a further 
hour to allow a large degree of dissociation of 
unstabilized enzyme to take place. Controls were 
also prepared which contained no avidin and 
which were incubated in the acetyl-CoA-dependent 
assay mix containing 0.25 mM acetyl-CoA, with 
the final dilution to 40 pg -ml-’ being performed in 
0.1 M Tris-Cl, containing 0.25 mM acetyl-CoA. 
Samples were then prepared for and examined by 
electron microscopy as described 171. 
3. RESULTS AND DISCUSSION 
Fig.1 shows a control sample of sheep pyruvate 
carboxylase prepared in the absence of avidin but 
in the presence of acetyl-CoA. The enzyme 
Volume 203, number 2 FEBS LETTERS July 1986 
molecules appear to be well preserved and show 
the ‘tight’ configuration [3,7]. Fig.2a shows sheep 
enzyme that had been mixed with avidin in the 
acetyl-CoA-dependent assay mix including acetyl- 
CoA. Numerous long chain-like polymers are visi- 
ble, some of them showing backfolding. Fig.2b 
shows sheep enzyme that had been mixed with 
avidin in the acetyl-CoA-independent assay mix. 
The result is very similar to that in fig.2a. Fig.2c 
shows a contrast where the sheep enzyme and 
avidin were prepared in the same way as in fig.2a 
except that acetyl-CoA was omitted. Only a few 
typical avidin-enzyme chains are visible. The 
markedly reduced formation of these chains, the 
occurrence of distorted enzyme molecules and 
unspecific aggregates, and the presence of many 
small particles of different sizes (presumably 
subunits of broken enzyme molecules and free 
avidin) (see table 1) are indicative of a low degree 
of structural preservation of the sheep enzyme 
under these conditions. 
These results demonstrate that some component 
of the acetyl-CoA-independent assay mix can 
mimic the structure-preserving effect of acetyl- 
CoA on sheep liver pyruvate carboxylase and in- 
duce this enzyme to adopt the ‘tight’ tetrahedron- 
like configuration which interacts well with avidin 
to form chains of avidin-enzyme polymer. Fig.Zd 
demonstrates that pyruvate is the factor responsi- 
ble for this effect. This micrograph shows that 
chain formation occurred when a sheep enzyme 
sample was mixed with avidin in the presence of 
pyruvate (40 mM) alone. Similar results (not 
shown) were obtained with 40 mM 2-oxobutyrate. 
Table 1 
Summary of electron microscopic observations of the quaternary structure of pyruvate carboxylase-avidin complexes 
under various conditions 
Enzymes/additionsb Well-preserved 
tetramers (rJ/o)c 
Well-preserved 
tetramers in 
chains (070)~ 
Tetramers 
per chain’ 
Number of small 
particles per unit 
areaf 
SLPC + acetyl-CoA (E fig.1) 
SLPC + acetyl-CoA-dependent 
assay mix + avidin (= fig.2a) 
SLPC + acetyl-CoA-independent 
assay mix + avidin (” fig.2b) 
SLPC + acetyl-CoA-dependent 
assay mix minus acetyl-CoA + 
avidin (’ fig.2c) 
SLPC + pyruvate (40 mM) + avidin 
(‘fig.2d) 
CLPC + pyruvate (60 mM) + avidin 
(3 fig.3b) 
93.12 f 0.84 0 43 
95.9 88.4 3.53 f 0.09 (265) 19 
94.4 87.8 3.03 + 0.09 (235) 19 
n.d.g 25.2 2.42 f 0.08 (110) 119h 
95.8 91.4 3.67 + 0.13 (278) 19 
97.1 78.5 3.72 + 0.15 (194) 18 
a SLPC, sheep liver pyruvate carboxylase; CLPC, chicken liver pyruvate carboxylase 
b Details of the additions made to the enzyme solutions are given in the legend to the figure indicated in parentheses 
’ The number of well-preserved tetrameric enzyme molecules is given as a percentage of the total number of protein 
particles observed in 9 areas each 0.25 pm’ (‘unit area’) 
d The number of well-preserved tetrameric enzyme molecules present in the chain-like complexes with avtdm is given 
as a percentage as described in ’ 
e The values given are the means + SE for the number of chains indicated in parentheses 
f ‘Small particles’ are protein molecules (either avidin or broken enzyme molecules) smaller than intact tetrameric 
pyruvate carboxylase molecules; ‘unit area’ - as defined in ’ 
8 There were very few intact tetramers evident in the absence of acetyl-CoA, except hose stabilized by chain formation 
with avidin 
h Accompanied by a number (mean = 1Uunit area) of non-specific aggregates of broken enzyme and/or avidin 
molecules 
193 
Volume 203, number 2 FEBS LETTERS July 1986 
Volume 203, number 2 FEBS LETTERS July 1986 
Fig.1. Electron micrograph of sheep liver pyruvate carboxylase after negative staining. The enzyme was incubated at 
16 units.mi-’ in the presence of the components of the acetyl-CoA-dependent assay system and 0.25 mM acetyl-CoA 
for 2 h at 30°C. The sample was then diluted to a final enzyme concentration of -4O~(g*ml- (0.8 unit.ml-‘) using 
0.1 M Tris-Cl, pH 8.4, containing 0.25 mM acetyl-CoA and incubated for 1 h at 30°C before preparation for electron 
microscopy. 
Fig.2. Electron micrographs of preparations of sheep enzyme in the presence of avidin, after negative staining. In all 
the samples the avidin was present at a ratio of avidin : enzyme of 1: 1 ([biotin-binding site] : [biotin]) and final dilution 
to -4O/rg~ml- (0.8 unit-ml-‘) was performed using 0.1 M Tris-Cl, pH 8.4. These soiutions were incubated for 1 h 
at 30°C before samples were prepared for electron microscopy. The enzyme was incubated at 16 units.ml-’ before and 
during the reaction with avidin in the presence of the following: (a) the components of the acetyl-CoA-dependent assay 
system together with 0.25 mM acetyl-CoA; (b) the components of the acetyl-CoA-independent assay system; (c) as in 
(a) except that acetyl-CoA was omitted; (d) 40 mM pyruvate in 0.1 M Tris-Cl, pH 8.4. 
Fig.3. Electron micrographs of complexes of chicken liver pyruvate carboxylase and avidin which were present at a 
concentration ratio of 1: 1. The enzyme (16 unitsaml-‘f and avidin were incubated together in 0.1 M Tris-Cl buffer, 
pH 7.8, at 30°C in the presence of various concentrations of pyruvate, i.e. (a) 10 mM and (b) 60 mM. 
For figs 1-3, the bar is given in fig. 1; it represents 100 nm, and on the micrographs, the letters represent: A, non-specific 
protein aggregates; BC, backfolded chains; C, chains; P, small protein particles (broken enzyme and avidin); T, ‘tight’ 
tetrameric pyruvate carboxylase particles. 
Samples of the sheep liver enzyme taken prior to 
the addition of avidin were assayed for catalytic 
activity under conditions corresponding to figs 2a 
and b above. In the acetyl-CoA-independent assay 
system (fig.2b) the enzyme exhibited 10% of the 
activity which it yielded in the presence of 
saturating acetyl-CoA (fig.2a). However, when 
similar assays were conducted on the chicken liver 
enzyme, even with the pyruvate concentration as 
high as 80 mM the acetyl-CoA-independent activi- 
ty was found to be only 0.9% of that in the 
presence of acetyl-CoA. It was somewhat surpris- 
ing, therefore, to find, as illustrated in fig.3 that 
pyruvate alone, especially at 60 mM (see fig.3b), 
could induce chain formation between avidin and 
chicken liver pyruvate carboxylase. Such an obser- 
vation is consistent, however, with the finding of 
Mildvan et al. [ 121 that pyruvate enhances the rate 
of inhibition of chicken liver pyruvate carboxylase 
by avidin. 
From these observations, which are summarised 
in table 1, it would seem that pyruvate induces a 
generally similar effect on the quaternary structure 
of both enzymes. In view of the substantial dif- 
ferences in activity between these enzymes with ap- 
parently similar ‘tight’ tetrahedron-like conforma- 
tions, it would seem that this type of structural 
change in pyruvate carboxylase could account for 
only a small proportion of the stimulating effect 
that acetyl-CoA has on this enzyme’s activity. 
Thus, more subtle approaches (e.g. use of fluores- 
cent substrate analogues [13]) will be required to 
probe the structural basis of this allosteric ac- 
tivator’s several effects on the catalytic cycle of 
pyruvate carboxylase [ 10,141. 
ACKNOWLEDGEMENTS 
This investigation was supported by a grant 
(D284/15411) from the Australian Research 
Grants Scheme to J.C.W. We are indebted to the 
Research Sub-Committee of the University of 
Adelaide for financing F.M. as a Distinguished 
Visiting Scholar. It is a pleasure to acknowledge 
the skilled technical assistance of MS J. Brazier, 
and to thank Dr K. Bartusek for the Electron Op- 
tical Centre for the use of the Philips 300 electron 
microscope. 
REFERENCES 
[l] Wallace, J.C. and Easterbrook-Smith, S.B. (1985) 
in: Pyruvate Carboxylase (Keech, D.B. and 
Wallace, J.C. eds) Chapter 3, pp.65-108, CRC 
PI 
Press, Boca Raton. 
Libor, S., Sundaram, T.K., Warwick, R., 
Chapman, J.A. and Grundy, S.M.W. (1979) 
Biochemistry 18, 3647-3653. 
19s 
Volume 203, number 2 FEBS LETTERS July 1986 
[3] Mayer, F., Wallace, J.C. and Keech, D.B. (1980) 
Eur. J. Biochem. 112, 265-272. 
[4] Osmani, S.A., Mayer, F., Marston, F.A.O., 
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[S] Mayer, F., Osmani, S.A. and Scrutton, M.C. 
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[6] Rohde, M., Lim, F. and Wallace, J.C. (1986) Eur. 
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[7] Johannssen, W., Attwood, P.V., Wallace, J.C. 
and Keech, D.B. (1983) Eur. J. Biochem. 133, 
201-206. 
[S] Green, N.M., Konieczny, L., Toms, E.J. and 
Valentine, R.C. (1971) Biochem. J. 125, 781-791. 
[9] Safer, D., Hainfeld, J., Wall, J.S. and Reardon, 
J.F. (1982) Science 218, 290-291. 
[lo] Ashman, L.K., Keech, D.B., Wallace, J.C. and 
Nielsen, J. (1972) J. Biol. Chem. 247, 5818-5824. 
[ 1 l] Goss, N.H., Dyer, P.Y ., Keech, D.B. and Wallace, 
J.C. (1979) J. Biol. Chem. 254, 1734-1739. 
[12] Mildvan, A.S., Scrutton, M.C. and Utter, M.F. 
(1966) J. Biol. Chem. 241, 3488-3498. 
[13] Attwood, P.V., Coates, J.H. and Wallace, J.C. 
(1984) FEBS Lett. 175, 45-50. 
[14] Wallace, J.C., Phillips, N.B.F., Snoswell, M.A., 
Goodall, G.J., Attwood, P.V. and Keech, D.B. 
(1985) Ann. NY Acad. Sci. 447, 169-188. 
196 


Avidin as a probe of the conformational changes induced in pyruvate carboxylase by acetyl-CoA and pyruvate 

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Avidin as a probe of the conformational changes induced in pyruvate carboxylase by acetyl-CoA and pyruvate

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