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Catalysis of the peroxidase-mediated oxidation of aldehydes by enolphosphates
Biochimica et Biophysica Acta, 789 (1984) 57-62 Elsevier
57
BBA 31962
CATALYSIS OF T H E PEROXIDASE-MEDIATED OXIDATION OF ALDEHYDES BY ENOLPHOSPHATES H U G O G A L L A R D O ~, LIDIA A. G U I L L O a, NELSON D U R A N b and GIUSEPPE CILENTO a,,
lnstituto de Qu'tmica, Universidade de Sgto Paulo, C.P. 20.780, Sgw Paulo, and b Universidade Estadual de Campinas, C.P. 6154, Campinas, CEP 13.000, S.P. (Brazil) (Received January 17th, 1984)
Key words." Peroxidase," Aldehyde oxidation," Enolphosphate
The peroxidase-catalyzed aerobic oxidation of aldehydes, which normally occurs only in phosphate or arsenate buffer, is efficiently promoted in Tris buffer by enolphosphates of isobutanal. The effect is catalytic and is exerted upon the substrate. It is inferred that the aldehyde reacts in the enol form, the enzymatic oxidation of the latter competing very efficiently with ketonization.
Introduction The peroxidase-catalyzed aerobic oxidation of appropriate substrates generates a carbonyl product in the electronically excited triplet state [1-4]. Among the reactions which have been thoroughly investigated are the oxidation of isobutanal (I; R = R ' = C H 3 ) to triplet acetone [5-8] and of propanal (I; R = CH3, R' = H) to triplet acetaldehyde [9] (Eqn. 1; HRP, horseradish peroxidase). A tentative mechanism has been formulated [4,7]. R
/I R'
O
C--C H
3R
+02
\ H
~,
enzyme. In order to investigate this possibility we have studied the monoanilinium salts of isobutanal-enolphosphate (II) and its methylester 0 ! l ) as well as the corresponding dimethyl ester (IV): H3C,~
H
II
•o
H3C
--
/ \ / / H3C
O
O--P--O-
\ OCH 3
~-I~H3 I1
+ HCOOH
H
"c=¢ /
.o/',o _
R' *
C
H3C"
c=c /
"
e~-/qH 3 III
(1)
0
H3C"
\c
These reactions occur in phosphate buffer but not in Tris buffer [6,7]. One possibility is that phosphate catalyses the formation of the enol form of the substrate, which is then attacked by the
* To whom correspondence should be addressed. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.
~H
/
9
H3C
O--P--OCH 3
\
OCH 3 IV
These compounds are novel. The rationale for the choice is that a trapped enol is analogous to
58 the enol formate (V), the substrate in the bioluminescence of Latia, and to coelenterazine enol sulfate, the substrate in the bioluminescence of coelenterates [11,12]. Furthermore, a luciferase and a peroxidase may, under certain circumstances, replace each other [13,14].
phosphate buffer, pH 7.4, 0.1 ml (95.0 mM final) of isobutanal stock solution (2.2 M, freshly prepared by mixing 0.1 ml of the neat compound and 0.4 ml of absolute ethanol). Isobutanal must be distilled weekly under nitrogen at atmospheric pressure and kept at 0°C. The reaction was initiated by addition of 10 #1 (2.0/~M) of an aqueous stock solution of peroxidase (0.5 mM) at 37-40°C.
Dimethyl 2-methyl-l-propenyl phosphate (IV). Compound IV was obtained by the Perkow reaction [17-19] using a-chloro-2-methylpropanal prepared by the method of Stevens and Gilles [17]. v An additional reason for the choice of compounds I I - I V is that ionized phosphates are capable of transferring an electron to appropriate acceptors [15]. H3C H II--' ~C = C/
O
/
,p
>
\
/
H
c=c n 3C /
H3CN -
<
~
~XO.
ide (14 g, 0.12 mol) was added dropwise under nitrogen at room temperature. The mixture was then heated at 50°C for 16 h and distilled under reduced pressure; b.p. 140°C at 10 mmHg, 45% yield. 1H-NMR (C2HC13) 0.31 (s, 9H, SiCH3); 1.69 (m, 6H, CH3-C=C); 3.68 (d, 3H, JP-H = 11 Hz, C H 3 0 - P ) ; 6.21 (m, 1H, C=CH-OP).
Bis(trimethylsilyl)-2-methyl-l-propenyl phosphate. This compound was prepared analogously
OH3C \
Methyl trimethylsilyl 2-methyl-l-propenyl phosphate. To (IV) (22 g, 0.1 mol), trimethylsilyl chlor-
/ /c.-c N
H 3C
H
O
vI
by refluxing IV for 54 h with 0.22 mol trimethylsilyl chloride; b.p. 160°C at 10 mmHg, 65% yield. 1H-NMR (C2HC13) 0.29 (s, 18H, SiCH3); 1.55 (m, 6H, CH3-C=C); 6.10 (m, 1H, C=CH-OP).
Anilinium methyl 2-methyl-l-propenyl phosphate (liD. To a mixture of aniline (4.65 g, 0.05 mol)
Materials and Methods
and ethanol (25 ml) in diethyl ether (20 ml) was added the silyl enolphosphate of IV (15.1 g, 0.05 mol) in 15 ml diethyl ether at room temperature. The white precipitate was collected, washed with diethyl ether, and dried at reduced pressure: 66% yield, m.p. 100°C. 1H-NMR (CZH302H) 1.48 (s, 6H, CH3-C=C), 3.34 (d, 3H, J = 11 Hz, CH3-OP); 6.25 (m, 1H, C=CH-OP); 6.71-731 (m, 5H, N-C6Hs); 8.25 (br. s, 4H, H3N , POH).
All chemicals were analytical grade reagents. Horseradish peroxidase (Type VI) ((E403 = 1.02. 10 5 c m - a . M-1 [16]) and catalase were from Sigma Chem. Co., whereas EDTA, phosphate salts, Tris, isobutanal, propanal, phenylacetaldehyde and pivaldehyde were from Merck. To investigate the kinetics of the peroxidasecatalyzed oxidation of isobutanal, the standard reaction medium was as follows: 0.8 ml of 0.1 M pyrophosphate buffer (pH 7.4), 1.4 ml of 1.0 M
Compound II was prepared by same procedure as described for III, using 0.1 ml bis(silyl)-enolphosphate of IV in ethanol (35 ml)/diethyl ether (30 ml); 45% yield, m.p. 136°C. 1H-NMR (DMSO-d6) 1.6 (s, 6H, CH3-C=C); 6.2 (m, 1H, C=CH-OP); 7.4 (m, 5H, N-C6Hs) 9.03 (br. s, 4H, H3N, POH). Other procedures. The enolphosphate samples were prepared as 0.27 M stock solutions in ethanol/water (1:4). A new spectrophotometric
In this regard, radical VI is a postulated intermediate in the peroxidase-catalyzed aerobic oxidation of isobutanal to triplet acetone [7]. We report here that II and III greatly enhance the enzymic oxidation of aldehydes by catalyzing the formation of the enol form of the substrate.
Anilinium 2-methyl-l-propenyl phosphate (II).
59 assay was used for phosphate analysis [20]. Acetone formed in the peroxidase-catalyzed oxidation of isobutanal p r o m o t e d by l I in Tris buffer was identified as its 2,4-dinitrophenylhydrazone. Oxygen consumption was measured in a Yellow Springs Instruments Model 53 oxygen monitor and in a W a r b u r g apparatus. The chemiluminescence from the enzymatic reaction was measured, depending upon its intensity: in a H a m a m a t s u TV Photocounter C-747 or HTV-R-562; on a sensitive photometer (MitchellHastings Type) of in-house construction with a 1P28 photomultiplier; or in a Perkin Elmer M P F - 4 fluorescence spectrophotometer with a R446F photomultiplier. The Hastings-Weber [21] scintillation cocktail was used as a light standard. Results
Reinvestigation of the kinetics of the peroxidase-catalyzed oxidation of isobutanal confirmed that the rate of O 2 uptake exhibits a firstorder dependence with respect to concentration of phosphate and of isobutanal (Fig. 1). At high isobutanal concentrations, the rate starts leveling off; from a Lineweaver-Burk plot (Fig. 2), K m was found to be 30_+0.2 m M at 40°C. The linear c o n s u m p t i o n of oxygen [6,7] indicates a zero-order dependence with respect to the oxygen concentra-
40 rE LD
~ 20 E c
I
0
I
I
I
20 4-0 1 / [ ISOBUTANAL] (M -~)
Fig. 2. Lineweaver-Burkplot of the data for the peroxidase-catalyzed aerobic oxidation of isobutanal (presented in the inset).
tion. The rate increases with horseradish peroxidase concentration but levels off at approx. 2.2 /~M (Fig. 3); under o t h e r experimental conditions a m a x i m u m can be observed [6]. Integrated emission data parallel those obtained for 02 uptake. Thus, there is a first-order dependence on the isobutanal and phosphate concentrations (Fig. 1) and saturation is observed at high enzyme concentration (Fig. 3).
Oxidation of the enol phosphates H R P does not p r o m o t e the oxidation of the enol phosphates, even in presence of H202.
Effect of the enol phosphates on 02 uptake by the aldehyde /peroxidase systems in Tris buffer As shown by O 2 consumption (Fig. 4), I I promotes the peroxidase-catalyzed aerobic oxidation of isobutanal. Likewise, I I j promotes the reaction; in contrast, anilinium chloride, IV, glucose 6-phosphate and phosphoenolpyruvate had no effect
F2.5
~ 1.5
Q5 10
I 1.5 LOG X
[ 20
F i g . 1. P l o t o f l o g Y ( e m i s s i o n i n t e n s i t y o r i n i t i a l v e l o c i t y o f 0 2
uptake) vs. log x (isobutanal or phosphate concentration), zx, O2 uptake (/tl/min in Warburg) vs. phosphate, slope 1.08' A, emission intensity vs. phosphate, slope 1.00; O, O2 uptake (/xM/30 s in oxygraph) vs. isobutanal, slope 0.96; e, emission intensity vs. isobutanal, slope 0.98.
V
oF
i 100
!
Io 250 HRP (~M} Fig. 3. Effect of peroxidase (HPR) concentration on the rate of [
[
I
oxidation of 95 mM isobutanal followed by oxygen uptake (0 O) or by emission intensity (El D)
60 100 LU
E Z LJ
z
W
X 0
0
X 0
100
2O
f I
I
50
I
MINUTES
f
I
250
Fig. 4. Promotion by I1 of the peroxidase-catalyzed aerobic oxidation of isobutanal in 0.05 M Tris buffer (pH 7.4). Lower curve: 7 mM isobutanal/70 mM I1; upper curve 7 mM isobutanal/70 mM II/peroxidase. In the middle curve, the concentrations of isobutanal and II have been reversed (70 mM isobutanal/7 mM lI/peroxidase). No oxygen consumption is observed with the systems 70 mM isobutanal/7 mM II, 70 mM isobutanal/peroxidase and 70 mM lI/peroxidase. Warburg equipment, t = 37°C.
whatsoever. Oxygen c o n s u m p t i o n (Warburg) is det e r m i n e d by the a m o u n t of i s o b u t a n a l (Fig. 4). As expected, acetone is the p r o d u c t of the reaction. Some phosphate (10% after 3 days) is released from 7 m M II d u r i n g the p r o m o t e d enzymatic oxidation of 70 m M isobutanal; little or no phos100
,,,I
z
DJ 0 X 0
I
1
I
I
I
MINUTES
I
5
Fig. 6. Acceleration by 7 mM II of the aerobic oxidation of 70 mM phenylacetaldehyde in 0.~ M Tris buffer, pH 7.4, in the absence (middle curve) and presence (upper curve) of peroxidase. The lower curve refers to phenylacetaldehyde either alone or in the presence of peroxidase.
phate was f o u n d u p o n o m i t t i n g isobutanal, the substrate, or both. W h e n II was replaced by phosphate in c o n c e n t r a t i o n equivalent to that released u n d e r these conditions, no O 2 c o n s u m p t i o n occurred. The data in Fig. 5 show that the very slow a u t o x i d a t i o n of p r o p a n a l is not accelerated by the e n o l p h o s p h a t e or b y horseradish peroxidase alone; however, when both are present, O 2 is depleted very rapidly. Results with phenylacetaldehyde are presented in Fig. 6. The enolphosphate (II) greatly enhances the s p o n t a n e o u s oxidation of this aldehyde; if peroxidase is also present, the rate of 0 2 uptake is further increased, b u t only modestly. The enolphosphates do not p r o m o t e the oxidation of a,c~-dimethylpropanal (pivaldehyde), even in the presence of peroxidase.
Effect of the enolphc ,hates upon 0 2 uptake in phosphate buffer I
MINUTES
5
Fig. 5. Promotion by II of the peroxidase-catalyzed aerobic oxidation of propanal in 0.05 M Tris buffer (pH 7.4). Curve 1, 7 mM propanal/70 mM II; curve 2, 70 mM propanal, alone or in the presence of either 7 mM ll or of peroxidase; curve 3, 70 mM propanal/7 mM II/peroxidase; curve 4, 7 mM propanal/ 70 mM II/peroxidase. Oxygraph, t = 37°C.
The effect is still discernible, being most promin e n t in the case of p r o p a n a l (data not shown). As in Tris buffer, the enolphosphate also has a marked effect u p o n the a u t o x i d a t i o n of phenylacetaldehyde in phosphate buffer.
Emission in the presence of enolphosphates I n phosphate buffer, the i s o b u t a n a l / p e r o x i d -
61 a s e / 0 2 reaction exhibits acetone phosphorescence. In Tris, however, no emission whatsoever is observed, despite the fast rate of reaction when both peroxidase and enolphosphate are present. Triplet acetone is expected to be quenched by the free base form of Tris. Quenching by aniline would appear to be the reason that enzyme generated acetone phosphorescence is not observed in phosphate when II or III are present (pK a of the anilinium ion is 4.63).
the C a hydrogen which is removed from the substrate rather than the aldehydic hydrogen; (v) saturation kinetics at high enzyme concentration imply rate-limiting generation of an active precursor under these conditions. The enol content of isobutanal in water is indeed very small, the keto-enol equilibrium constant being reported to be 1.28.10 -4 [24]. Furthermore, since the rate of ketonization is not particularly rapid - the half-life being about 10 min - the rate of enolization should be quite slow. If the autoxidation of the enol and its peroxidase (HRP-I)-promoted oxidation are fast enough to compete with ketonization, a fast rate of oxygen uptake, controlled by that of the catalytic step, should be observed (Scheme I). In the case o f phenylacetaldehyde, the enol appears to be easily autoxidizable (Fig. 6) perhaps due to the tendency to form a highly resonance stabilized radical ion. Our explanation requires that when the disappearance of the enolic form of the substrate is very fast, the rate of O 2 uptake should depend only upon the product of the concentrations of the substrate and of the enolphosphate; the deviations observed (e.g., Figs. 4 and 5) may also be due to medium effects. The above scheme readily accounts also for the kinetic dependence reported in Figs. 1-3. Further work directed toward an understanding of why II and III are much more efficient than phosphate in catalyzing enolization, is currently in progress.
Discussion
The very fact that II and III are not oxidized by horseradish peroxidase I and undergo at most only very limited alteration when added to the enzymatic systems (as judged by Pi release) indicates that these enolphosphates act catalytically. Accordingly, while complete (enzymatic) oxidation of isobutanal is observed when [isobutanal] << [II], upon reversing the relative concentrations, 02 consumption proceeds to a greater extent. A catalytic effect must, of course, be exerted either at the level of the enzyme or the substrate. An effect on the enzyme alone is untenable, since in the case of phenylacetaldehyde the effect is observed even in the absence of enzyme. The most readily conceivable catalyzed process involving the substrate is enolization. This interpretation is in accord with the following facts: (i) catalytic inactivity of IV; (ii) non-reactivity of pivaldehyde; (iii) easily enolizable/substrates, e.g., methylacetylacetone [22] and a-formylphenylacetic acid [23], are enzymically oxidized even in Tris buffer; (iv) it is autoxidation 02 R1
\
O
//" C--C ~ /I \ R2 H H
Rl
\
/
OH
' C=C / \ R 2
H
R1
O / C--C c / \ \
R2 S c h e m e I.
H
Rl
t,O
\
J • , C--C / \ R2
H
62
Acknowledgements The authors wish to express their gratitude to Prof. frank H. Quina for a critical examination of the manuscript. This work was supported by grants from the Financiadora de Estudos e Projetos (FINEP), the Volkswagenwerk and the Organization of American States Program (OAS). H.G. was a visiting professor through the Chile (CONYCIT)-Brazil (CNPq) bilateral program; L.G. is a predoctoral student of the.Fundaqao de Amparo Pesquisa do Estado de Sao Paulo (FAPESP). N.D. thanks the Guggenheim Foundation for a fellowship.
References 1 Cilento, G. (1980) Photochem. Photobiol. Rev. 5, 199-228 2 Cilento, G. (1980) Acc. Chem. Res. 13, 225-230 3 Cilento, G. (1982) in Chemical and Biological Generation of Excited States (Adam, W. and Cilento, G., eds.), pp. 277-307, Academic Press, New York 4 Adam, W. and Cilento, G. (1983) Angew. Chem. Int. Ed. Engl. 22 (7), 529-542 5 Durfin, N., Faria-Oliveira, O.M.M., Haun, M. and Cilento, G. (1977) J. Chem. Soc. Chem. Commun. 13, 442-443 6 Faria Oliveira, O.M.M., Haun, M., Durfin, N., O'Brien, P.J., O'Brien, C.R., Bechara, E.J.H. and Cilento, G. (1978) J. Biol. Chem. 253, 4707-4712 7 Bechara, E.J.H., Faria Oliveira, O.M.M., Durfin, N., De Baptista, R.C. and Cilento, G. (1979) Photochem. Photobiol. 30, 101-110
8 Durhn, N. and Cilento, G. (1980) Photochem. Photobiol. 32, 113-116 9 Haun, M., Durhn, N., Augusto, O. and Cilento, G. (1980) Arch. Biochem. Biophys. 200, 245-252 10 Shimomura, O. and Johnson, F.H. (1968) Biochemistry 7, 1734-1738 11 Inoue, S., Kakoi, H., Murata, M. and Goto, T. (1977) Tetrahedron Lett. 2685-2688 12 Shimomura, O. and Johnson, F.H. (1979) Comp. Biochem. Physiol. 64B, 105-107 13 Henry, S.P., Isambert, R.F. and Michelson, A.M. (1973) Biochemie 55, 83-93 14 Puget, K., Michelson, A.M. and Avrameas, S. (1977) Anal. Biochem. 79, 447-456 15 Goncharova, N.V. (1981) Biochemistry (USSR) 46, 12-16 16 Schonbaum, G.R. and Lo, S. (1972) J. Biol. Chem. 247, 3353-3360 17 Stevens, C.L. and Gillis, B.T. (1957) J. Am. Chem. Soc. 79, 3448-3451 18 Arbuzov, B.A. and Vinogradova, V.S. (1956) Dokl. Akad. Nauk, SSSR 106, 263-266 19 De Selms, R.C. and Lin, T.W. (1967) Tetrahedron 23, 1479-1488 20 Bencini, D.A., Wild, J.-R. and O'Donovan, G.A. (1983) Anal. Biochem. 132, 353-361 21 Hastings, J.W. and Weber, G. (1963) J. Opt. Soc. Am, 53, 1410-1415 22 Bechara, E.J.H. and Medeiros, M.H.G. (1984) in Oxygen Radicals in Chemistry and Biology (Bors, W., ed.), pp. 539 542, Walter de Gruyter, Berlin 23 Adam, A., Arias Encarnaci6n, L.A., Cilento, G., Haun, M. and Nassi, L. (1984) Photochem. Photobiol., in the press 24 Chiang, Y., Kresge, A.J. and Walsh, P.A. (1982) J. Am. Chem. Soc. 104, 6122-6123
57
BBA 31962
CATALYSIS OF T H E PEROXIDASE-MEDIATED OXIDATION OF ALDEHYDES BY ENOLPHOSPHATES H U G O G A L L A R D O ~, LIDIA A. G U I L L O a, NELSON D U R A N b and GIUSEPPE CILENTO a,,
lnstituto de Qu'tmica, Universidade de Sgto Paulo, C.P. 20.780, Sgw Paulo, and b Universidade Estadual de Campinas, C.P. 6154, Campinas, CEP 13.000, S.P. (Brazil) (Received January 17th, 1984)
Key words." Peroxidase," Aldehyde oxidation," Enolphosphate
The peroxidase-catalyzed aerobic oxidation of aldehydes, which normally occurs only in phosphate or arsenate buffer, is efficiently promoted in Tris buffer by enolphosphates of isobutanal. The effect is catalytic and is exerted upon the substrate. It is inferred that the aldehyde reacts in the enol form, the enzymatic oxidation of the latter competing very efficiently with ketonization.
Introduction The peroxidase-catalyzed aerobic oxidation of appropriate substrates generates a carbonyl product in the electronically excited triplet state [1-4]. Among the reactions which have been thoroughly investigated are the oxidation of isobutanal (I; R = R ' = C H 3 ) to triplet acetone [5-8] and of propanal (I; R = CH3, R' = H) to triplet acetaldehyde [9] (Eqn. 1; HRP, horseradish peroxidase). A tentative mechanism has been formulated [4,7]. R
/I R'
O
C--C H
3R
+02
\ H
~,
enzyme. In order to investigate this possibility we have studied the monoanilinium salts of isobutanal-enolphosphate (II) and its methylester 0 ! l ) as well as the corresponding dimethyl ester (IV): H3C,~
H
II
•o
H3C
--
/ \ / / H3C
O
O--P--O-
\ OCH 3
~-I~H3 I1
+ HCOOH
H
"c=¢ /
.o/',o _
R' *
C
H3C"
c=c /
"
e~-/qH 3 III
(1)
0
H3C"
\c
These reactions occur in phosphate buffer but not in Tris buffer [6,7]. One possibility is that phosphate catalyses the formation of the enol form of the substrate, which is then attacked by the
* To whom correspondence should be addressed. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.
~H
/
9
H3C
O--P--OCH 3
\
OCH 3 IV
These compounds are novel. The rationale for the choice is that a trapped enol is analogous to
58 the enol formate (V), the substrate in the bioluminescence of Latia, and to coelenterazine enol sulfate, the substrate in the bioluminescence of coelenterates [11,12]. Furthermore, a luciferase and a peroxidase may, under certain circumstances, replace each other [13,14].
phosphate buffer, pH 7.4, 0.1 ml (95.0 mM final) of isobutanal stock solution (2.2 M, freshly prepared by mixing 0.1 ml of the neat compound and 0.4 ml of absolute ethanol). Isobutanal must be distilled weekly under nitrogen at atmospheric pressure and kept at 0°C. The reaction was initiated by addition of 10 #1 (2.0/~M) of an aqueous stock solution of peroxidase (0.5 mM) at 37-40°C.
Dimethyl 2-methyl-l-propenyl phosphate (IV). Compound IV was obtained by the Perkow reaction [17-19] using a-chloro-2-methylpropanal prepared by the method of Stevens and Gilles [17]. v An additional reason for the choice of compounds I I - I V is that ionized phosphates are capable of transferring an electron to appropriate acceptors [15]. H3C H II--' ~C = C/
O
/
,p
>
\
/
H
c=c n 3C /
H3CN -
<
~
~XO.
ide (14 g, 0.12 mol) was added dropwise under nitrogen at room temperature. The mixture was then heated at 50°C for 16 h and distilled under reduced pressure; b.p. 140°C at 10 mmHg, 45% yield. 1H-NMR (C2HC13) 0.31 (s, 9H, SiCH3); 1.69 (m, 6H, CH3-C=C); 3.68 (d, 3H, JP-H = 11 Hz, C H 3 0 - P ) ; 6.21 (m, 1H, C=CH-OP).
Bis(trimethylsilyl)-2-methyl-l-propenyl phosphate. This compound was prepared analogously
OH3C \
Methyl trimethylsilyl 2-methyl-l-propenyl phosphate. To (IV) (22 g, 0.1 mol), trimethylsilyl chlor-
/ /c.-c N
H 3C
H
O
vI
by refluxing IV for 54 h with 0.22 mol trimethylsilyl chloride; b.p. 160°C at 10 mmHg, 65% yield. 1H-NMR (C2HC13) 0.29 (s, 18H, SiCH3); 1.55 (m, 6H, CH3-C=C); 6.10 (m, 1H, C=CH-OP).
Anilinium methyl 2-methyl-l-propenyl phosphate (liD. To a mixture of aniline (4.65 g, 0.05 mol)
Materials and Methods
and ethanol (25 ml) in diethyl ether (20 ml) was added the silyl enolphosphate of IV (15.1 g, 0.05 mol) in 15 ml diethyl ether at room temperature. The white precipitate was collected, washed with diethyl ether, and dried at reduced pressure: 66% yield, m.p. 100°C. 1H-NMR (CZH302H) 1.48 (s, 6H, CH3-C=C), 3.34 (d, 3H, J = 11 Hz, CH3-OP); 6.25 (m, 1H, C=CH-OP); 6.71-731 (m, 5H, N-C6Hs); 8.25 (br. s, 4H, H3N , POH).
All chemicals were analytical grade reagents. Horseradish peroxidase (Type VI) ((E403 = 1.02. 10 5 c m - a . M-1 [16]) and catalase were from Sigma Chem. Co., whereas EDTA, phosphate salts, Tris, isobutanal, propanal, phenylacetaldehyde and pivaldehyde were from Merck. To investigate the kinetics of the peroxidasecatalyzed oxidation of isobutanal, the standard reaction medium was as follows: 0.8 ml of 0.1 M pyrophosphate buffer (pH 7.4), 1.4 ml of 1.0 M
Compound II was prepared by same procedure as described for III, using 0.1 ml bis(silyl)-enolphosphate of IV in ethanol (35 ml)/diethyl ether (30 ml); 45% yield, m.p. 136°C. 1H-NMR (DMSO-d6) 1.6 (s, 6H, CH3-C=C); 6.2 (m, 1H, C=CH-OP); 7.4 (m, 5H, N-C6Hs) 9.03 (br. s, 4H, H3N, POH). Other procedures. The enolphosphate samples were prepared as 0.27 M stock solutions in ethanol/water (1:4). A new spectrophotometric
In this regard, radical VI is a postulated intermediate in the peroxidase-catalyzed aerobic oxidation of isobutanal to triplet acetone [7]. We report here that II and III greatly enhance the enzymic oxidation of aldehydes by catalyzing the formation of the enol form of the substrate.
Anilinium 2-methyl-l-propenyl phosphate (II).
59 assay was used for phosphate analysis [20]. Acetone formed in the peroxidase-catalyzed oxidation of isobutanal p r o m o t e d by l I in Tris buffer was identified as its 2,4-dinitrophenylhydrazone. Oxygen consumption was measured in a Yellow Springs Instruments Model 53 oxygen monitor and in a W a r b u r g apparatus. The chemiluminescence from the enzymatic reaction was measured, depending upon its intensity: in a H a m a m a t s u TV Photocounter C-747 or HTV-R-562; on a sensitive photometer (MitchellHastings Type) of in-house construction with a 1P28 photomultiplier; or in a Perkin Elmer M P F - 4 fluorescence spectrophotometer with a R446F photomultiplier. The Hastings-Weber [21] scintillation cocktail was used as a light standard. Results
Reinvestigation of the kinetics of the peroxidase-catalyzed oxidation of isobutanal confirmed that the rate of O 2 uptake exhibits a firstorder dependence with respect to concentration of phosphate and of isobutanal (Fig. 1). At high isobutanal concentrations, the rate starts leveling off; from a Lineweaver-Burk plot (Fig. 2), K m was found to be 30_+0.2 m M at 40°C. The linear c o n s u m p t i o n of oxygen [6,7] indicates a zero-order dependence with respect to the oxygen concentra-
40 rE LD
~ 20 E c
I
0
I
I
I
20 4-0 1 / [ ISOBUTANAL] (M -~)
Fig. 2. Lineweaver-Burkplot of the data for the peroxidase-catalyzed aerobic oxidation of isobutanal (presented in the inset).
tion. The rate increases with horseradish peroxidase concentration but levels off at approx. 2.2 /~M (Fig. 3); under o t h e r experimental conditions a m a x i m u m can be observed [6]. Integrated emission data parallel those obtained for 02 uptake. Thus, there is a first-order dependence on the isobutanal and phosphate concentrations (Fig. 1) and saturation is observed at high enzyme concentration (Fig. 3).
Oxidation of the enol phosphates H R P does not p r o m o t e the oxidation of the enol phosphates, even in presence of H202.
Effect of the enol phosphates on 02 uptake by the aldehyde /peroxidase systems in Tris buffer As shown by O 2 consumption (Fig. 4), I I promotes the peroxidase-catalyzed aerobic oxidation of isobutanal. Likewise, I I j promotes the reaction; in contrast, anilinium chloride, IV, glucose 6-phosphate and phosphoenolpyruvate had no effect
F2.5
~ 1.5
Q5 10
I 1.5 LOG X
[ 20
F i g . 1. P l o t o f l o g Y ( e m i s s i o n i n t e n s i t y o r i n i t i a l v e l o c i t y o f 0 2
uptake) vs. log x (isobutanal or phosphate concentration), zx, O2 uptake (/tl/min in Warburg) vs. phosphate, slope 1.08' A, emission intensity vs. phosphate, slope 1.00; O, O2 uptake (/xM/30 s in oxygraph) vs. isobutanal, slope 0.96; e, emission intensity vs. isobutanal, slope 0.98.
V
oF
i 100
!
Io 250 HRP (~M} Fig. 3. Effect of peroxidase (HPR) concentration on the rate of [
[
I
oxidation of 95 mM isobutanal followed by oxygen uptake (0 O) or by emission intensity (El D)
60 100 LU
E Z LJ
z
W
X 0
0
X 0
100
2O
f I
I
50
I
MINUTES
f
I
250
Fig. 4. Promotion by I1 of the peroxidase-catalyzed aerobic oxidation of isobutanal in 0.05 M Tris buffer (pH 7.4). Lower curve: 7 mM isobutanal/70 mM I1; upper curve 7 mM isobutanal/70 mM II/peroxidase. In the middle curve, the concentrations of isobutanal and II have been reversed (70 mM isobutanal/7 mM lI/peroxidase). No oxygen consumption is observed with the systems 70 mM isobutanal/7 mM II, 70 mM isobutanal/peroxidase and 70 mM lI/peroxidase. Warburg equipment, t = 37°C.
whatsoever. Oxygen c o n s u m p t i o n (Warburg) is det e r m i n e d by the a m o u n t of i s o b u t a n a l (Fig. 4). As expected, acetone is the p r o d u c t of the reaction. Some phosphate (10% after 3 days) is released from 7 m M II d u r i n g the p r o m o t e d enzymatic oxidation of 70 m M isobutanal; little or no phos100
,,,I
z
DJ 0 X 0
I
1
I
I
I
MINUTES
I
5
Fig. 6. Acceleration by 7 mM II of the aerobic oxidation of 70 mM phenylacetaldehyde in 0.~ M Tris buffer, pH 7.4, in the absence (middle curve) and presence (upper curve) of peroxidase. The lower curve refers to phenylacetaldehyde either alone or in the presence of peroxidase.
phate was f o u n d u p o n o m i t t i n g isobutanal, the substrate, or both. W h e n II was replaced by phosphate in c o n c e n t r a t i o n equivalent to that released u n d e r these conditions, no O 2 c o n s u m p t i o n occurred. The data in Fig. 5 show that the very slow a u t o x i d a t i o n of p r o p a n a l is not accelerated by the e n o l p h o s p h a t e or b y horseradish peroxidase alone; however, when both are present, O 2 is depleted very rapidly. Results with phenylacetaldehyde are presented in Fig. 6. The enolphosphate (II) greatly enhances the s p o n t a n e o u s oxidation of this aldehyde; if peroxidase is also present, the rate of 0 2 uptake is further increased, b u t only modestly. The enolphosphates do not p r o m o t e the oxidation of a,c~-dimethylpropanal (pivaldehyde), even in the presence of peroxidase.
Effect of the enolphc ,hates upon 0 2 uptake in phosphate buffer I
MINUTES
5
Fig. 5. Promotion by II of the peroxidase-catalyzed aerobic oxidation of propanal in 0.05 M Tris buffer (pH 7.4). Curve 1, 7 mM propanal/70 mM II; curve 2, 70 mM propanal, alone or in the presence of either 7 mM ll or of peroxidase; curve 3, 70 mM propanal/7 mM II/peroxidase; curve 4, 7 mM propanal/ 70 mM II/peroxidase. Oxygraph, t = 37°C.
The effect is still discernible, being most promin e n t in the case of p r o p a n a l (data not shown). As in Tris buffer, the enolphosphate also has a marked effect u p o n the a u t o x i d a t i o n of phenylacetaldehyde in phosphate buffer.
Emission in the presence of enolphosphates I n phosphate buffer, the i s o b u t a n a l / p e r o x i d -
61 a s e / 0 2 reaction exhibits acetone phosphorescence. In Tris, however, no emission whatsoever is observed, despite the fast rate of reaction when both peroxidase and enolphosphate are present. Triplet acetone is expected to be quenched by the free base form of Tris. Quenching by aniline would appear to be the reason that enzyme generated acetone phosphorescence is not observed in phosphate when II or III are present (pK a of the anilinium ion is 4.63).
the C a hydrogen which is removed from the substrate rather than the aldehydic hydrogen; (v) saturation kinetics at high enzyme concentration imply rate-limiting generation of an active precursor under these conditions. The enol content of isobutanal in water is indeed very small, the keto-enol equilibrium constant being reported to be 1.28.10 -4 [24]. Furthermore, since the rate of ketonization is not particularly rapid - the half-life being about 10 min - the rate of enolization should be quite slow. If the autoxidation of the enol and its peroxidase (HRP-I)-promoted oxidation are fast enough to compete with ketonization, a fast rate of oxygen uptake, controlled by that of the catalytic step, should be observed (Scheme I). In the case o f phenylacetaldehyde, the enol appears to be easily autoxidizable (Fig. 6) perhaps due to the tendency to form a highly resonance stabilized radical ion. Our explanation requires that when the disappearance of the enolic form of the substrate is very fast, the rate of O 2 uptake should depend only upon the product of the concentrations of the substrate and of the enolphosphate; the deviations observed (e.g., Figs. 4 and 5) may also be due to medium effects. The above scheme readily accounts also for the kinetic dependence reported in Figs. 1-3. Further work directed toward an understanding of why II and III are much more efficient than phosphate in catalyzing enolization, is currently in progress.
Discussion
The very fact that II and III are not oxidized by horseradish peroxidase I and undergo at most only very limited alteration when added to the enzymatic systems (as judged by Pi release) indicates that these enolphosphates act catalytically. Accordingly, while complete (enzymatic) oxidation of isobutanal is observed when [isobutanal] << [II], upon reversing the relative concentrations, 02 consumption proceeds to a greater extent. A catalytic effect must, of course, be exerted either at the level of the enzyme or the substrate. An effect on the enzyme alone is untenable, since in the case of phenylacetaldehyde the effect is observed even in the absence of enzyme. The most readily conceivable catalyzed process involving the substrate is enolization. This interpretation is in accord with the following facts: (i) catalytic inactivity of IV; (ii) non-reactivity of pivaldehyde; (iii) easily enolizable/substrates, e.g., methylacetylacetone [22] and a-formylphenylacetic acid [23], are enzymically oxidized even in Tris buffer; (iv) it is autoxidation 02 R1
\
O
//" C--C ~ /I \ R2 H H
Rl
\
/
OH
' C=C / \ R 2
H
R1
O / C--C c / \ \
R2 S c h e m e I.
H
Rl
t,O
\
J • , C--C / \ R2
H
62
Acknowledgements The authors wish to express their gratitude to Prof. frank H. Quina for a critical examination of the manuscript. This work was supported by grants from the Financiadora de Estudos e Projetos (FINEP), the Volkswagenwerk and the Organization of American States Program (OAS). H.G. was a visiting professor through the Chile (CONYCIT)-Brazil (CNPq) bilateral program; L.G. is a predoctoral student of the.Fundaqao de Amparo Pesquisa do Estado de Sao Paulo (FAPESP). N.D. thanks the Guggenheim Foundation for a fellowship.
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8 Durhn, N. and Cilento, G. (1980) Photochem. Photobiol. 32, 113-116 9 Haun, M., Durhn, N., Augusto, O. and Cilento, G. (1980) Arch. Biochem. Biophys. 200, 245-252 10 Shimomura, O. and Johnson, F.H. (1968) Biochemistry 7, 1734-1738 11 Inoue, S., Kakoi, H., Murata, M. and Goto, T. (1977) Tetrahedron Lett. 2685-2688 12 Shimomura, O. and Johnson, F.H. (1979) Comp. Biochem. Physiol. 64B, 105-107 13 Henry, S.P., Isambert, R.F. and Michelson, A.M. (1973) Biochemie 55, 83-93 14 Puget, K., Michelson, A.M. and Avrameas, S. (1977) Anal. Biochem. 79, 447-456 15 Goncharova, N.V. (1981) Biochemistry (USSR) 46, 12-16 16 Schonbaum, G.R. and Lo, S. (1972) J. Biol. Chem. 247, 3353-3360 17 Stevens, C.L. and Gillis, B.T. (1957) J. Am. Chem. Soc. 79, 3448-3451 18 Arbuzov, B.A. and Vinogradova, V.S. (1956) Dokl. Akad. Nauk, SSSR 106, 263-266 19 De Selms, R.C. and Lin, T.W. (1967) Tetrahedron 23, 1479-1488 20 Bencini, D.A., Wild, J.-R. and O'Donovan, G.A. (1983) Anal. Biochem. 132, 353-361 21 Hastings, J.W. and Weber, G. (1963) J. Opt. Soc. Am, 53, 1410-1415 22 Bechara, E.J.H. and Medeiros, M.H.G. (1984) in Oxygen Radicals in Chemistry and Biology (Bors, W., ed.), pp. 539 542, Walter de Gruyter, Berlin 23 Adam, A., Arias Encarnaci6n, L.A., Cilento, G., Haun, M. and Nassi, L. (1984) Photochem. Photobiol., in the press 24 Chiang, Y., Kresge, A.J. and Walsh, P.A. (1982) J. Am. Chem. Soc. 104, 6122-6123