Borders, C.L.

Zurcher, Jackie A.

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Volume 108, number 2 FEBS LETTERS December 1979 
RABBIT MUSCLE ENOLASE ALSO HAS ESSENTIAL ARCINY L RESIDUES 
C. L. BORDERS, jr and Jackie A. ZURCHER 
~~part~e~t of~e~~str~, College of ?Vooster, Wooster, OH 44691, USA 
Received 16 October 1979 
1. Introduction 
Recent studies have attempted to clarify the roles 
of various amino acid residues in the catalytic activity 
of yeast enolase. The yeast enzyme has a single essen- 
tial arginyl residue per subunit [l-3] which is likely 
involved’in binding the phosphate moiety of substrates 
to the active site [Z]. In addition, the enzyme can be 
inactivated by the modification of carboxyl [4,5] and 
histidyl [G-8] residues at the active site, but the roles 
played by these amino acid residues is less well defined. 
Inactivation of yeast enolase has also been corre- 
lated with the modification of me~ionyl residues 
[9,10] and cysteinyl residues under denaturing condi- 
tions [ 111, but in neither case are the modified resi- 
dues thought to be involved in the catalytic mecha- 
nism. Similarly, lysyl residues are not involved in sub- 
strate binding or catalysis [8]. 
Although there have been comparative studies on 
the structural and catalytic properties of yeast and 
rabbit muscle enolases [ 10,121, little information is 
available on catalytically functional amino acid resi- 
dues in the muscle enzyme. It has been shown that 
glycidol phosphate, a substrate analogue, inactivates 
rabbit muscle enolase by modification of a carboxyl 
group at the active site [ 13 ,141, while cysteinyl resi- 
dues have been shown to be non-essential [ 15 ,161. 
Here we report that rabbit muscle enolase, like the 
yeast enzyme [l-3], has a single essential arginyl 
residue per subunit which likely serves to bind the 
anionic substrates to the active site. This conclusion 
is based on chemical modification with butanedione 
in borate buffer, a system known to be highly selec- 
tive for the modification of arginyl residues in pro- 
teins [ 1,2,17-201. 
EisevierlNorth-Holland Biomedical Press 
2. Materials and methods 
2-Phosphoglycerate, 2,3-butanedione, Sephadex 
gels, and buffer salts were obtained from Sigma 
(St. Louis, MO). Commercial preparations of rabbit 
muscle enolase were obtained from Sigma, Calbio- 
them, and Boehringer-Mannheim. The preparation 
from Boehringer-Mannheim (lot 1078414) gave the 
highest specific activity (53 I*mol phosphoenolpyru- 
vate . min-’ . mg-‘) under the conditions of assay, and 
a single band on polyacrylamide gel electrophoresis 
1211, and was used for all further studies. Enzyme 
concentration was determined from the &se, using 
A (O.l%)= 0.89 [l(i]and mol. wt 82 000 [lO,lS] for 
the dimeric [22] enzyme. Enzyme activity was deter- 
mined at 25’C as in [23-251, with the exception that 
2-phosphoglycerate was 2 mM. Activity was deter- 
mined from the increase in Az4e as a function of 
time, using a molar absorption coefficient of 
13 10 M-l cm-’ for phosphoenolpyruvate under the 
assay conditions [26]. 
Modification with butanedione was carried out 
at 25*C under the conditions given in the figure and 
table legends. Modification of arginine was deter- 
mined by analysis on a Beckman 12OC amino acid 
analyzer after workup analogous to the procedures in 
[ 18,201. 
3. Results 
The time course for the inactivation of rabbit 
muscle enolase by butanedione in 50 mM borate 
(pH 8.3) depends on the concentration of reagent 
used (fig. I). Butanedione at 2.5 mM reduces the 
415 
Volume 108, number 2 FEBS LETTERS December 1979 
0 60 
Fig.1. Rabbit muscle enolase inactivation by butanedione. 
The enzyme (2 PM) in 50 mM borate, 1 mM magnesium 
acetate, 0.01 mM EDTA, pH 8.3, was modified by: (0) 
2.5 mM; (=) 5 mM; (A) 10 mM; or (T) 20 mM butanedione. 
The control retains full activity over this period of time. 
Table 1 
Correlation of rabbit muscle enolase inactivation by 
butanedione with arginine modification, and protection 
by substratesa 
Enzyme 
Control 
VI vc Arg per Arg modified 
x 100 subunit per subunit 
100 16.3 _ 
+ Butanedione 
5min 88 14.1 2.2 
20 min 57 14.0 2.3 
40 min 33 13.8 2.5 
75 min 15 13.3 3.0 
+ Butanedione 
+ 2-Phosphoglycerate 95 14.3 2.0 
a Modification of rabbit muscle enolase (18 PM) was carried 
out with 4 mM butanedione in 50 mM borate, 1 mM magne- 
sium acetate, 0.01 mM EDTA @H 8.3). Aliquots were with- 
drawn at the indicated times, gel filtered to remove excess 
butanedione, assayed for enzymatic activity, and subjected 
to amino acid analysis by the procedures in [ 18,201. For the 
sample modified in the presence of 2_phosphoglycerate, 
10 mM, an aliquot was subjected to gel filtration after 40 min 
modification and subsequently analyzed. The values of Arg/ 
subunit listed represent the average of duplicate analyses of 
single samples for each of the butanedione-treated samples, 
and an average of duplicate analyses of each of two separate 
control samples 
416 
activity to 50% of the control after 22 min, while 
only 14% activity remains after 1 h of modification. 
One feature of the modification of essential arginyl 
residues in enzymes by butanedione is that the inac- 
tivation is enhanced by borate [ 1,2,17-201. This 
is the case with muscle enolase, for under conditions 
where activity is reduced to 20% in the presence of 
50 mM borate, 80% activity remains if the borate 
is replaced by 50 mM NJ-bis-(2-hydroxyethyl)- 
glycine. This suggests that inactivation of muscle 
enolase by butanedione is due to the modification 
of essential arginyl residues. 
Protection against inactivation is provided by 
substrates of muscle enolase (data not shown). 
After 1 h of modification by 2.5 mM butanedione 
in 50 mM borate (pH 8.3), 75% of the control activity 
remains when an equilibrium mixture of substrates 
I-- 
Fig.2. Correlation of the inactivation of rabbit muscle enolase 
with arginine modification by butanedione. Details of the 
experiment are given in table 1. The solid line is theoretical, 
calculated on the assumption that complete inactivation cor- 
relates with the modification of the third arginyl residue per 
subunit. 
Volume 108, number 2 FEBS LETTERS December 1979 
plus Mg*‘(generated by adding 20 mM 2-phosphogly- involved in binding the phosphate moiety of substrates 
cerate plus 1 mM magnesium acetate before initiation to the active site [2]. It is quite likely that the 
of modification) is also present, while only 14% activ- essential arginyl residue of the muscle enzyme plays 
ity remains when modification is carried out in the a similar role, and experiments to ascertain this are in 
absence of substrates. progress. 
When inactivation by butanedione is correlated 
with the modification of arginyl residues, the data 
shown in table 1 are obtained. Muscle enolase has 
two very reactive arginyl residues per subunit which 
can be modified by butanedione without loss of 
activity. However, as shown in fig.2, activity is 
completely lost concomitant with the modification 
of a third arginyl residue per subunit. When modifi- 
cation is carried out in the presence of substrates, 
only two arginyl residues per subunit are modified 
and very little loss of enzymatic activity observed 
(table 1). 
Cardenas and Wold, comparing structural and 
catalytic properties of enolases from widely different 
sources, concluded that the chemical compositions of 
the active sites of different enolases are identical or 
very similar throughout the biological world [ 121. 
The yeast and muscle enzymes are now known to con- 
tain essential carboxyl [5,13,14] and arginyl residues. 
Further studies will reveal whether or not other 
types of amino acid residues which are known to be 
essential for the activity of the yeast enzyme also 
play a crucial role in the catalytic mechanism of rabbit 
muscle enolase. 
4. Discussion Acknowledgements 
Arginyl residues play a very general role in the 
binding of anionic substrates and cofactors to enzyme 
active sites [ 11. These data based on chemical modifi- 
cation with butanedione in borate buffer, suggest 
that rabbit muscle enolase has a single essential arginyl 
residue per subunit (fig.2). Since substrates protect 
against the inactivation of muscle enolase and against 
modification of the essential arginine (table l), it is 
very likely that arginine plays a similar role in this 
enzyme. 
Acknowledgement is made to the Donors of the 
Petroleum Research Fund, administered by the 
American Chemical Society, for support of this 
research. A grant from the Research Corporation for 
the purchase of the Beckman 12OC amino acid 
analyzer is gratefully acknowledged. This work was 
taken in part from the senior Independent Study 
thesis of J. A. Z., The College of Wooster, 1979. 
An unexpected feature of this study was that 
muscle enolase has two extremely reactive arginyl 
residues per subunit which can be modified by 
butanedione, either in the presence or absence of 
substrates, without loss of activity (table 1). It has 
been pointed out that quite often functional arginyl 
residues at enzyme active sites can be selectively modi- 
fied by arginine-specific reagents [18,27]. However, 
these data represent the first example of an arginyl 
enzyme which has hyper-reactive arginyl residues 
which can be modified more rapidly than the essential 
arginyl residue. 
References 
[ 1] Riordan, J. F., McElvany, K. D. and Borders, C. J. jr. 
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418 


Rabbit muscle enolase also has essential arginyl residues 

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Rabbit muscle enolase also has essential arginyl residues

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