OPTICAL PROPERTIES OF ACETOACETYL-X-COENZYME A AND ITS METAL CHELATES ...

13 Pages·2003·0.59 MB·English

OPTICAL PROPERTIES OF ACETOACETYL-X-COENZYME A
AND ITS METAL CHELATES*
BY JOSEPH R. STERN
(From the Department of Pharmacology, School of Medicine, Western Reserve
University, Cleveland, Ohio)
(Received for publication, November 7, 1955)
Acetoacetic thio esters of CoA’ (l-3) and possibly pantetheine (4, 5)
play an important role in fatty acid metabolism. Effective use has been
made of the optical properties of these thio esters in developing sensitive
assays (6) for the enzymes concerned in their metabolism.
Lynen and collaborators (7) were the first to discover that acetoacetic
thio esters, specifically synthetic S-acetoacetyl-N-acetyl-p-mercaptoethyl-
OH
I ii
(1) CH a- 81CH 2- !-SR + CH,-C=CH-C-SR G
CH 3- 8=,,_8,, + H+
amine, possessa strong absorption band between 280 and 320 rnp, with a
maximum around 303 mp. Since the intensity of this band was dependent
on pH, increasing with increasing pH, they attributed it to formation of
the enolate ion, as indicated by reaction (1). The pK’ of S-acetoacetyl-
N-acetyl-fl-mercaptoethylamine was determined to be 8.5. Acetoacetyl-
S-CoA prepared enzymatically (8,9) or synthetically (1) was found to have
a similar absorption band. Stern et al. (10) showed that the intensity of
this band was greatly increased in the presence of Mg++. These observa-
tions have been confirmed by Beinert (9) and extended to include the Ca
and Cs fi-ketoacyl-S-CoA compounds.
This paper deals with the optical properties of metal chelates2 of aceto-
* This investigation was aided by grant No. A739 from the National Institute of
Arthritis and Metabolic Diseases of the National Institutes of Health, United States
Public Health Service, and by a grant from the Williams-Waterman Fund of the
Research Corporation.
1 The following abbreviations are used: CoA (or CoA-SH), coenzyme A (reduced) ;
R-SH, thiol compound; AcAc, acetoacetate; M, metal; E, molecular extinction coeffi-
cient ; E, optical density; Am,,, wave-length at which E is maximum.
2 The term chelate is used in its strict chemical sense (cf. (11)). Although the
compounds described have not been isolated, their behavior in solution is completely
in accord with that of known metal chelates of p-diketones (11-13).
33
This is an Open Access article under the CC BY license.
34 OPTICAL PROPERTIES OF ACETOACETYL COA
acetyl-S-CoA and related thio esters. The significance of these properties
in the interpretation of optical assays will be discussed.
i IS,OOl
:
5
%2 12,001
a
8,001
4,00
f
WAVE LENGTH (ma)
FIG. 1. Effect of divalent ions on the absorption spectrum of acetoacetyl-S-CoA.
These are difference absorption spectra obtained by first measuring the enolate (or
enolate + chelate) spectrum at pH 8.1, then adding HCl (final concentration, 0.1 N)
to obtain the non-enolate absorption spectrum. The latter is subtracted from the
former. The difference spectrum between 260 and 280 rnp may be attributed in part
to changes in absorption of the adenosine moiety of the thio ester. The lowest curve
represents the acetoacetyl-S-CoA spectrum in absence of metal. Metal indicated
added as chloride salt, final concentration 5 X 1OP M. Acetoacetyl-S-CoA concen-
tration, 0.68 X 1OP M (total acetoacetic thio ester concentration, 0.76 X 1OP M).
Standard optical conditions.
EXPERIMENTAL
Absorption Xpectrum-Fig. 1 shows the difference absorption spectrum
at pH 8.1 of acetoacetyl-S-CoA in the range 260 to 340 mp and the effect
of various divalent metal ions (concentration, 5 X 1OP M) on it. It is
evident that Mg++, Mn++, and Ni++, in order of increasing effectiveness,
cause a great increase in the intensity of the enolate ion absorption as well
as some shift in X,,,. This increase in absorption may be attributed to
J. R. STERN 35
the formation of a 1: 1 metal chelates (reaction (2)) which possesseas greater
degree of conjugation than the chelating agent, i.e. acetoacetyl-S-CoA.
(2) M++ + acetoacetyl-S-CoA- F‘, M-acetoacetyl-S-CoA+
The X,,, of acetoacetyl-S-CoA and of its Mg chelate both occurred in
the range 300 to 303 mp. For the Co, Mn, and Ni chelates of acetoacetyl-
S-CoA, X,,, was 306 f 2 rnp, 308 rnp, and 309 to 310 rnp, respectively.
26.000 pH 10.0
+ +
24,000 n ------¤ .
vH 9.4
I I I I I I I I
O 5 IO 15 20 25 30 35 40
[MS++] M x IO-'
FIG. 2. Effect of Mg++ concentration and pI-1 on the extinction of acetoacetyl-s-
CoA solutions at wave-length 310 rnp.
This small but definite shift of X,,, toward the red indicates that the Co,
Mn, and Ni chelates may differ in structure from the Mg chelate.
3 In all experiments described the concentration of metal was high compared to
that of chelating agent, so negligible quantities of M(AcAcCoA)z were present (cf.
(14)). For example, consider the further reaction MAcAcCoA+ + AcAcCoA- =
M(AcAcCoA) 2 where (M++) N 5 X 10e3 M and (AcAcCoA-) N lo+ M. Even if IQ =
((M(AcAcCoA)z)/(MAcAcCoA+)(AcAcCoA-)) were approximately equal to IL
(= 2750), the ratio (M(AcAcCoA)~)/(MAcAcCoA-) would not exceed 2750 X lo+ N
0.028. Further evidence that a 1:l chelate is formed under the experimental condi-
tions is given in the text.
36 OPTICAL PROPERTIES OF ACETOACETYL COA
+
These changes in absorption spectra resemble those described for metal
chelates of a simpler ,%diketone, acetylacetone (12, 13), which possesses
an ultraviolet absorption band whose X,,, = 273 rnp in ethanol. How-
ever, in the case of the Co and Ni chelate compounds of acetylacetone there
is a more pronounced shift of X,,, to the red.
Effect of Mg++ Concentration on Extinction-The effect of Mg++ con-
centration on the extinction of acetoacetyl-S-CoA and acetoacetyl-X-
pH 10.0
pH 9.4
’ pH 9.0 ’
16,000 ;A’
<
t
Y
pH 8.05
g 12,000-
w
5
Et
a 8,000
15 20 25 30
[I&$+] MxlO“
FIG. 3. Effect of Mg++ concentration and pH on the extinction of acetoacetyl-s-
pantetheine solutions at wave-length 310 rnp.
pantetheine at X = 310 rnp is shown in Figs. 2 and 3 for a number of pH
values. It is seen that, at any given pH, the concentration of chelate in-
creases as the Mg++ concentration increases and that for a given concentra-
tion of Mg++ the extinction is greater the more alkaline the pH, as is the
case in the absence of Mg++.
Molecular Extinction Coeficient-The molecular extinction coefficient
(E) of the Mg-acetoacetyl-S-CoA chelate was determined by measuring its
extinction under conditions for which chelate formation may be considered
to be complete, i.e. at very alkaline pH or at less alkaline pH in the pres-
J. R. STERN 37
ence of a large excess of Mg++. By this method (cf. Figs. 2, 3, and 4) the
true for the Mg chelates of both acetoacetyl-S-CoA and acetoacetyl-X-
c31~
pantetheine was found to be 25,000. It is noteworthy that the e310o b-
tained by this method for the enolate ion form of the two thio esters is
also 25,000. At the X,,, indicated the values of e were as follows: e300‘v
32,000 for acetoacetyl-S-CoA and N 30,000 for its Mg chelate.
~303
E$ect of @-The effect of pH on the apparent molecular extinction
coefficient, c/,4 of acetoacetyl-S-CoA and acetoacetyl-S-pantetheine in the
absence and presence of various Mg* concentrations is presented in Fig.
70 8.0 9.0 10.0 I 1.0
PH
FIG. 4. The effect of pH on the extinction of acetoacetict hio esters. Acetoacetyl-
S-CoA, X, no metal; 0, 5.0 X 10V3 M MgCh; Cl, 20 X 10v3M MgCL; 0, 35 X 1OP M
MgCL. Acetoacetyl-S-pantetheine, n , no metal; A, 5 X 10W3MMgC12. a, points
common to upper four curves.
4. In each instance e’ increased continuously with pH. The pH at which
6’ has half the maximal value corresponds to the pK’ values of the aceto-
acetic thio esters or their metal chelates. The pK’ of acetoacetyl-S-CoA
is therefore 9.45. The value of pK’ decreasesw ith increasing Mg++ con-
centration, a factor of biological significance. Since Lynen (1) had re-
ported a pK’ of 8.50 for X-acetoacetyl-N-acetyl-@mercaptoethylamine,
the problem arose whether this difference of pK’ values was the result of
metal contamination or a property of the thiol moiety of the acetoacetic
thio ester. The effect of pH on the e’ of acetoacetyl-%pantetheine (4)
was therefore examined (Fig. 4). At any given pH the E’ of acetoacetyl-
4 2 is defined as log IO/I = E’cd where c is the molar concentration of total aceto-
acetic thio ester.
38 0PncA~ PROPERTIES 0s A~ETOMETYL COA
S-pantetheine was greater than that of acetoacetyl-S-CoA and the effect
of a given Mg++ concentration on E’ was less, since the pK’ of acetoacetyl-
X-pantetheine, 8.85, was less than that of acetoacetyl-S-CoA and also
greater than that of X-acetoacetyl-N-acetyl-p-mercaptoethylamine. These
results emphasize the influence of groups in the thiol moiety on the pK’
of acetoacetic thio esters.
Calculation of XtabiEity Constant-Let A, A-, and AM+ represent the
keto, enol, and chelate forms, respectively, of acetoacetyl-S-CoA and AT
the sum of the three forms. At any given pH, the dissociation constant
TABLE I
Effect of pH on Degree of Enolization and Chelation of Acetoacetyl-S-CoA
Conditions as in Fig. 2.
I No metal’ 5 X lo-, P Mg++
__
PH 7
Keto Enolt Keto Ed Chelate
_ _ -
per cenf per cent per cent per cent per cent
7.05 97.6 2.4 93.9 0.37 5.7 3080
7.55 95.1 4.9 83.3 1.05 15.6 2980
8.13 87.8 12.2 54.4 2.40 43.2 3460
8.35 84.4 15.6 47.1 3.34 49.6 2970
9.15 66.2 33.8 19.5 9.83 70.7 1450
9.40 50.4 49.6 7.51 6.70 85.8 2560
10.00 20.0 80.0 -0 -0 -100.0
10.25 12.7 87.3
-
Average.. . 2750
- - - -
* E not affected by addition of potassium versenate.
t Calculated from the equation per cent enol = 100~‘/25,000.
(Ka) of the reaction A G A- + Hf is given by the equation
Ka = (A-1 (H+)
(3)
(4
while at any given concentration of metal (&I++), the stability constant
(K,) of the reaction A- + &I++ e AM+ is given by the equation
(AM+)
(4)
Kc = (A-) (M++)
Also,
(5) (AT) = (A) + (A-) + (AM+)
Under the experimental conditions (MT) N (&I++) > (A) or (A-). Since
at XsIO eAM+ = CA- = 25,000, the sum ((AM+) -l- (A-)) can be determined
J. R. STERN 39
by measuring the extinction (at constant pH and (M*)), applying Beer’s
law (log IO/l = ecd), and solving for c. Also, (A *) can be measured en-
zymatically and (A) calculated from equation (6) as follows:
(6) (A) = (AT) - ((AM+) + (A-3)
(A-) can now be determined from equation (3), since pK’, pH, and (A)
are known. This then permits (AM+) to be computed, since ((AM+) +
(A-)) is known. Finally K, can be computed.
Table I gives (a) the proportion of keto and enol forms of acetoacetyl-
S-CoA as a function of pH in the absence of metal and (b) the proportions
TABLE II
Effect of pH on Degree of Enolization and Chelation of Acetoacetyl-S-pantetheine
Conditions as in Fig. 3.
No metal* 5X10-naMg++
PH KC
K&o Enolt Keto Ed Chelate
per cent per cent per c%%t per cent per cent
6.85 98.0 2.0 96.2 0.86 2.91 673
7.40 94.1 5.9 88.8 3.16 8.03 511
8.05 82.3 17.7 70.7 11.2 18.1 345
9.00 40.8 59.2 27.1 38.2 34.7 193
9.40 24.8 75.2 8.2 29.0 62.8 432
10.00 10.0 90.0 NO -0 -100.0
10.25 6.0 94.0
Average. . . . . . 431
* E not affected by addition of potassiumv ersenate.
t Calculated from the equation per cent enol = 100~‘/25,000.
of the keto, enol, and chelate forms in the presence of 5 X 1OP M Mg++,
computed as indicated above. It is seen that the values of K, are reason-
ably constant and independent of pH. Table II presents analogous data
for acetoacetyl-X-pantetheine. Here the values of K, show greater spread.
The standard free energy (AF”) of the formation of the 1: 1 Mg chelate
can be calculated from the equation AF” = - RT In Kc. For Mg-aceto-
acetyl-S-CoA AF” = -4670 calories per mole, while for Mg-acetoacetyl-
S-pantetheine AF” N -3580 calories per mole.
Stability of Metal Chelates-At alkaline pH acetoacetic thio esters are
unstable and slowly hydrolyze according to reaction (7).
(7) Acetoacetyl-SR- + Hz0 4 acetoacetate- + HSR
The time-course of spontaneous hydrolysis of acetoacetyl-S-CoA at pH
40 OPTICAL PROPERTIES OF ACETOACETYL COA
8.1 is illustrated in Fig. 5. The metal chelates of acetoacetyl-S-CoA also
hydrolyze spontaneously, at varying rates, at alkaline pH according to
reaction (8).
(8) M-acetoacetyl-SR+ f H20 + ill++ + acetoacetate- + HSR
The stability of various metal chelates at pH 8.1 and 25” is shown in Fig.
5. The Ni chelate was the most stable, followed by the Co, Mn, and Mg
?
E 1.0 ‘x,
0
-‘-r Ni 5x IO-‘M
z 0.9 -
‘\A
zQ8; ’
0
Gi Mn 5x10sJM
fj Q7- \ 0 A
n
Co 5xWM
< CGr
I
; 0.5t
: I&(
0.4 -Lj(
\ -x-
*-x-x Mg 5 x 10-3M
0.3 ;;.
0 IO 20 30 40 50 60
MINUTES
FIG. 5. Time-course of spontaneous hydrolysis of acetoacetyl-S-CoA and various
metal chelates. The thio ester was added at zero time to solutions containing the
indicated concentration of metal in 0.067 M Tris-HCl buffer, pH 8.1. Final acetoace-
tyl-S-CoA concentration, 0.68 X 1OP M (total acetoacetic thio ester 0.76 X 10-4~).
Volume 1.50 ml.; temperature 25”.
chelates which possesss imilar stability. The Cu and, particularly, the
Zn chelates were much more unstable and it was not practical to determine
their difference spectra or X,,, in the Beckman DU spectrophotometer.
Reaction of Hg+f with Acetoacetyl-X-CoA--It was found that addition
of Hg++ in concentrations higher than 10m6M to solutions of acetoacetyl-
S-CoA (1.6 X 10e4M ) or to solutions containing its Mg chelate (0.8 X
low4 M total thio ester) at pH 8.1 resulted instantaneously in a partial
decrease of the extinction of these compounds. 1 to 2 X 10W4M HgClz
completely abolished the enolate ion absorption band of acetoacetyl-S-CoA
S. R. STERX 41
or its Mg chelate. The decrease in extinction at 310 mp was proportional
to the amount of Hg* added and it was determined that, to achieve com-
plete “titration” of the &IO of these compounds, just more than 1 mole of
Hg++ was required per mole of acetoacetyl-S-CoA. This observation sug-
gested that the Hg chelate of acetoacetyl-S-CoA was extremely unstable
and hydrolyzed immediately. This interpretation was supported (a) by
the disappearance of the typical enolate ion (or chelate) absorption band
and (b) by the fact that, after “titration” of the I&I,, with Hg++ and addi-
tion of potassium versenate to bind any excess Hg++, enzymatic assay for
acetoacetyl-S-CoA with L( +)-P-hydroxybutyryl-S-CoA dehydrogenase was
negative. Since Hg++ also hydrolyzed solutions of the Mg chelate, it must
have a greater affinity for acetoacetyl-S-CoA than Mg++; i.e., the stability
constant of the Hg chelate (reaction (9)) is greater than that of the Mg
chelate. The fact that Hg++ does not act catalytically in hydrolyzing
acetoacetyl-S-CoA and other acetoacetic thio esters can be explained by
the binding of Hgti- by the liberated thiol according to the following reac-
tion sequence :
(9) Hg++ + ncetoacetyl-SR- e Hg-ncetoacetyl-SR+
(10) Hg-acetoacetyl-SR+ + Hz0 ---t Hg++ + acet,oacetate- + HSR
(11) Hg++ + HSR + 2Cl- $ HgSR+ + HCl + Cl-
Hg++ also hydrolyzed the Ni, Mn, and Co chelates of acetoacetyl-S-CoA.
DISCUSSION
The experiments presented above permit the conclusion that a 1: 1
chelate is formed between acetoacetic thio esters of CoA or pantetheine
and certain divalent ions. Possibly an “internal” chelate is formed in
which the ligand groups are the enol and carbonyl oxygen atoms of the
acyl moiety and the carbonyl oxygen atoms (or possibly nitrogen atoms)
of the two peptide bonds, as follows:
CH,
I
HOCH?--C-CHOH-C-N-CHzCH2-C=~~~
I
/ I
CHs \O
\Mg//o CH2
// \
CHs---C=CH-C-
This structure of the chelate would differ drastically from that of the enol
42
OPTICAL PROPERTIES OF ACETOACETYL COA
form and would not be possible with X-acetoacetyl-N-acetyl-fi-mercapto-
ethylamine.
Optical tests of enzymatic reactions of acetoacetyl-S-CoA or acetoacetyl-
X-pantetheine measure the change in concentration of the enol form or
(if a particular divalent metal ion is present) of the sum of the enol and
chelate forms. The possibility exists that the keto form may be the ac-
tual substrate, in which case one is measuring not the enzymatic reaction
but the resultant and instantaneous chemical change in the equilibrium
concentration of the enol (=t chelate) form. Since none of the enzymes
concerned, dehydrogenase, thiolase, and CoA transferase (acetoacetyl-
succinic thiophorase6), requires Mg++ or other divalent ion, the chelate is
not an obligatory substrate. Although the problem of distinguishing be-
TABLE III
Apparent Equilibrium Constant K’ of CoA Transjerase Reaction Succinyl-S-CoA +
Acetoacetate + Acetoacetyl-S-CoA + Succinate
Recalculated from data of Stern et al. (15), assuming that only the keto form of
acetoacetyl-S-CoA is a substrate of CoA transferase. Concentration of keto form
calculated by method given in the text.
K' X 108
PH
No metal 5 XlO-anaMg++
7.00 2.8 4.6
7.50 4.0 4.9
8.10 3.9 4.8
9.20 5.5 3.0
tween keto and enol substrates invites solution by isotope experiments,
some experiments already reported (15) with CoA transferase are of in-
terest in this regard. The apparent equilibrium constant (K’) of reaction
(12) was found to increase continuously over the range pH 7.0 to 9.2 when
(12) Succinyl-S-CoA- + acetoacetate- s acetoacetyl-S-CoA- + succinate- + H+
determined in the presence or absence of Mg++, and a plot of log K’ versus
pH suggested that the total concentration of reactants did not approxi-
mate the active concentrations. However, if one recalculates K’ on the
assumption that only the keto form participates in the equilibrium, then,
as shown in Table III, the values of K’ are reasonably constant and inde-
pendent of pH. This indicates that the keto form of acetoacetyl-S-CoA
6 This name has been proposed by a Committee on Nomenclature, 2nd Interna-
tional Conference on Biochemical Problems of Lipides, Ghent, Belgium, July, 1955.