~ ~ A L PE. H SCH.4CH.4T,
722
ERNEST I. BECKFRA N D A. D. MCLAREN
for under these conditions one is working in the approximately linear portion of the isotherm where dr/dAf is approximately constant. (c) The lower the value of interaction constants, lil and nl, the greater will be the possible variation in the total concentration of the fast-moving component before dr/dAf will diverge greatly from its value a t very low free anion concentrations. (d) The difference in mobilities of the two components should be a t least compatible with the attainment of actual separation of the descending peaks, in order t o promote resolution of the boundary. The results emphasize the fact that, in systems composed of mixtures the components of which interact in solution, the mere presence of a shoulder
Vol. 56
or inflection point in the Tiselius pattern, no matter how reproducible it may be, is not in itself sufficient evidence for postulating the existence of an additional component nor is it necessarily evidence for the existence of more than one type of site at which interaction can occur. It, appears that great caution must be exercised in the interpretation of such patterns. Acknowledgments.-The authors wish to thank Dr. R. F. Smith, for his kindness in supplying part of the data from which Fig. 4 was constructed. Our thanks are given to the Saskatchewan Agricultural Research Foundation for a fellowship awarded to one of us (J. R. C.) during the early part of this study. '
ENZYME MODELS. 11. THE KINETICS OF AN ARTIFICIAL CAKBOSYLBSE CATALYSIS1 BYRALPH E. SCHACHAT, ERNEST I. BECKER~" A N D A. D. MCLAREN*~ Department of Chemistry, Polytechnic Institute of Bvooklyn, Brooklyn, New Y o r k Received August 7, 1861
The thermal, catalyzed decarboxylation of phenylglyoxylic acid by 3-amino-~u-naphthoxindolea t 70' has been studied ,by measurement of the rate of evolution of carbon dioxide. A mechanism for the reaction has been proposed and the resulting rate equations give a satisfactory fit of the theoretical rates with the measured rate.
The kinetics and mechanism of the catalytic action of model substances having enzyme-like activity may be of considerable interest if eventually the information acquired can be applied to the mode of action of natural enzymes. A previous paper3 dealt with the preparation of some artificial carboxylase models. The present work is concerned with a partial elucidation of the action of these catalysts. The decarboxylation of a keto acid (e.g., pyruvic acid) by a carboxylase model (e.g., 3-aminooxindole) may be considered to take place by the following scheme (cf. Lange~ibeck~) H -C-NH:!
11
iJ
I
H
+ CHsCOCOOH
r
CH3
- H,U -+
rcarrangcmcn t
(1) For detailed iiwer (or extended version or inaterial sui>pleiiicntary to this article) order Uocuiiient 3484 from diiierican Docuinentation Institute, 1718 N Street, N.W., Waahington 6, D. C., reniitting 81.00 for iiiicrofilin (images 1 inch high on standard 35 nini. motion iiiuture f i l i i i ) or $1.60 f o r photocopies (6 X 8 inches) readable without ui>tiesl aid. Taken from the dissertation submitted by R. E. Schachat in vartial fulfillinent of the requirements for the Ph.D. degree a t the Polytechnic Institute of Brooklyn, June, 1950. Fellow of the National Institute of Health 1949-1950. ( 2 ) (a) To whom inquiries ehould be sent. (b) Department of Agricultme, University of California, Berkeley, Calif. (3) R. E. Schachat, E. I. Becker and A . D. hIcLaren, J . Ore. Chem., 16, 1348 (1951). (4) W. Langenbeck, "Die organisclion Katalysatoren," 2nd ed., Springer Verlag, Berlin, 1948.
The formed Schiff base is then ready to react with additional pyruvic acid, regenerating the Schiff base of the acid and repeating the cycle
+
CHaCOCOOH
Inasmuch as the react,ion stops far short of completion, there is, in addition, a termination step which will be described be lo^.^ Figure 1 is a photograph of a model of the Schiff base of pyruvic acid and 3aminooxindole (“PC’’ in reaction 2, below). Note the proximity between the carbonyl oxygen in the 2-carbon and the carboxyl hydrogen, making hydrogen bonding possible. As proposed in the above scheme, hydrogen bonding between the oxygen and hydrogen atoms probably precedes decarboxylation.
(‘o
ii P Po PC
0
= initial concentration of catalyst = k;/k-s = phenylglyoxylic acid concentration (sullstratc) = initial concentration of phenylglyoxylic acid = concentration of CaH5C(=NR)COOH
= concentration of benzaldehyde Qo = initial concentration of benzaldehyde added to EUIJstrate, if any QC = concentration of CeHsC(=NR)H X = moles of COZevolved per 1000 ml. of phenol
(b)
(e) (d)
(e)
Reaction 1 goes forward only as water produced by the reaction is carried off by a stream of hot dry nitrogen (water is known to inhibit the reaction3). One mole of benzaldehyde is produced per mole of carbon dioxide ( L e . , Q = X ) . The cat>alyst in its active forms reacts irreversibly with benzaldehyde to form inactive products and that each of the active forms (C, PC and QC) react with it at ampproximatelythe same rate. I t is furt,her assumed that this bimolecular reaction is the only terminating step operating. IVe assume that, 99% of the catalyst is destroyed in 140 minutes (see Fig. 2) by reaction 4.
Mathematical Treatment.-Data froni Table I are plotted in Fig. 2. The concentration of substrate TABLE I
RATE O F
REACTION O F 3 - ~ M l N O - a - N . ~ P H T H O X I N D O L ~ON
PHENYLGLYOXYLIC ACID
Fig. l.-hIodel of 3-amino-a-naphthosindole’ showing necessary configuration of molecule for decarboxylation by method described in text of article.
A discussion of the kinetics of these steps with phenylglyoxylic acid as a substrate and 3-amino-anaphthoxindole as catalyst follows : Proposed Mechanism. (a).-The decarboxylation of phenylglyoxylic acid takes place by the following four reaction steps with no other appreciable side reactions. Ileactioii 1 (1nitiat.ion) P. kl 1’C ILNH:! CoHSCOCOOH +CsH,jC(=Nll)COOH HzO Rc:iclion 2 (Decarboxylntion) PC b, QC s CaHjC(=NR)COOH +CeHsC(=NR)H COL Reaction 3 (Regeneration) QC P k3 CsHj(=NR.)H CsHsCOCOOH JJ k- 3 Q 1’ c CsHjCHO CsHjC(=NR)COOlX Reaction 4 (Temiination)
c:
+
+
Elapsed time, min.
Interval mg. Cor collected per 3.27 ml. phenol
5.0 1o:o 17.5 25.0 ‘32.5 40.0 4i.5 55.0
0.548 2.816 5.852 5 . GO6 4.838 4.020 3.369 2.842
Interval Elapsed time, min.
tiig.
COz collected
per 3.27 tn1. phenol
65.0 75.0 85.0
2150
2,786 2.005 1.510 1.785 0,657 ,268 ,271
275.0
,002
105,O
125,O 155,O
was Po = 1.18 molar, and the catalyst concentration was Co = 2.77 X molar. 6.0,
I
I
I
I
I
I
I
+
+
+
Q
GH:,CHO
+ A +Inactive products k4
The symbols are (all coucentrations are in grain moles per liter of solvent, ie., phenol) -4 C
concentration of all active forms of catalyst, C.V., A=C+PC+QC = catalyst in uncombined form, L e . , concentration of RNH? =
( 5 ) A marked resemblance can be found between this scheme and the 111rcI1anisin proposed b y Baddar for a quite different reaction, the Strcckcr tlcgradittion (F.G . Beddar, J . Ckem. SOC.(Supp. No. 1) 5109 [ 19.19)).
10
t , time in minutes.
Fig. 2.-Plot of rate of evolution of CO? 2jersus time. (Catalyst, 2.77 X 10-3 molar 3-amino-a-naphthosindolc; suktratc, 1.18 molar phenylglyosylic acid; temperaturc, 70 ; -.-e-, observed data; calculntcd data.)
-n-o-,
724
K
RALPHE. SCHACHAT, ERNEST I. BECKEH AND A. D. MCLAREN
From reaction 2 and the equilibrium constant, = ka/k-r = (PC)(&)/(&C)(P)we have dX/dt
k:(PC)
Vol. 50
equal to C,,, X is virtually zero, and equation l b may be written as
(la)
and d S / d L = k&(A
(la)
V-) --('- C) /(Po - X ) + Ii
Equation l b is the general rate equation, where ks and K are constants. A may be evaluated from -dA/A
= k,Qdl =
ka(X)tll
(2a)
as
By assumption (e) and equation 2b, IC4 Fig. 2). C may be evaluated from -dC/tl/
=
kl(P)(C) = h ( P ,
=
- X)(C)
(Ra)
A solution of equation l b requires a knowledge of lG2 and K . To evaluate K and IC2 we use a special case of equation l b ; namely, when t is large C is small (for a justification of this see below).
For examples, with t = 60 minutes, dX/dt E 19.4 X X/(Po - X ) = 0.226 and A = 8.70 X with t = 120 minutes, dX/dt = 1.50 X X/(Po - X ) = 0.294 and A = 0.693 X Solving the two examples in equation ICsimultaneously K = 2.00 and lez = 2.48. With these figures it is also possible to determiue kl and hence C. Thus, when t is small, A is nearly I
I
I
I
I
I
(dzX/dt2)t+o = ( k l k ~ ) P o C= o G . 3 X lo-'
I
(le)
and kl = 0.193/ks = 0.0778. Making use of this approximate evaluation of ICl, equation 3b may be solved for C (cf. Fig. 4). At t = 60 minutes, C = 1.91 X and at t = 120 minutes, C = 2.52 X lo-'. Summary results are presented in Table 11.
0.169 (cf.
via
.30(
From initid slope of Fig. 2 uiid equation Id by differentiation
TABLE I1 S U M M AOF R YCALCULATED DATA (dS/dt) x A x 10' c x 10'
Time Min.
X 104
(Po-X)
60
19.4
0.226
8.7
120
1.5
0.294
0.GW
(A
x
- C) 104
8.51
0.19 0.003
0.G90
Equation l b may now be solved simultaneously at 60 and 120 minutes to give better values for the constants; namely, K , 1.17; kz, 2.72; k l , 0.0709. With these constants the rate of C02 evolution was calculated from equation l b to give the dotted line, Fig. 2. It should be observed that theoretical and observed curves are in reasonable agreement.
t
1
t
i
o.8
I
0.4
o.2
t I
0
L
I 20
I
40
I 60
I
I
80
100
1
120
time in minutes. Fig. 4.-Plot of unreacted substrat#eversus time. (Catalyst, 2.77 X 10-3 molar 3-amino-a-naphthoxindole; suhstrate, 1.18 molar phenylglyoxylic acid; temperature, 70 ".) t,
;6-
.05
0
20
40
60
80
I00
120
140
160
t , time in minutes.
Fig. 3.-Plot of moles of COXper 1000 ml. solvent versus time (temperature, 70"): 0 , catalyst, 2.77 X 10-3 molar 3-amino-a-naphthoxindole; substrate, 1.18 molar phenylglyoxylic acid; solvent, phenol; A , catalyst] 2.42 X molar benzal-3-amino-a-naphthoxindole; substrate, 1.18 molar phenylglyoxylic acid; solvent, phenol; 0, catalyst, 2.60 X low3molar 3-amino-a-naphthoxindole; substrate, 1.20 molar phenylglyoxylic acid; solvent, water.
Scveral experiments were carried out to tcst the validity of the steps in the proposcd mechanism. Water inhibits the reaction (see Fig. 3) as ~vould be expected in a condensation in which water is formed (reaction 1). That the benzal Schiff base of 3-amino-a-naphthoxindole also behaves as a catalyst (see Fig. 3) for the decarboxylation is in agreement with the formulation of reactions 2 and 3. It would be predicted from reactions 3 and 4 that benzaldehyde would slow the reaction by decreasing the concentration of P C and by inactivation of the catalysts, A . It was found by Lan-
*
KINETICSOF
June, 1952
AN
725
ARTIFICIAL CARBOXYLASE CATALYSIS
genbeck6 that benzaldehyde does markedly inhibit the reaction. Inhibition bj- benzaldehyde is calculable. Using a modified form of equation l b , namely dX/dl = k2K(.4 - C)/Q,/Po K From the constants obtained in the present work, and the quantities given by Langenbeck, it was calculated that 1.21 X 10-3 mole of COs/liter of phenol should be evolved in 15 minutes (see Fig. 7). Langenbeck found experimentally 1.69 X which is somewhat larger but not too far in disagreement. Benzoin does not inhibit the decarboxylation (Fig. 6), although Langenbeck states that it does inhibit the aniline-catalyzed decarboxylation of phenylglyoxylic acid. When a new portion of catalyst was added after the reaction had essentially stopped, the rate picked up quickly and then fell again as in the initial phase of the experiment (see Fig. 5). This shows that the catalyst is consumed by a termination step, feasibly as in reaction 4.
+
t GO
0
40
20
t , times in minutes. Fig, 6.-Plot of rate of evolution of Cot per mole of catalyst versus time, showing effect of introduction of benzoin. (-@-@-, with benzoin added; catalyst, 8.25 X mole 3amino-or-naphthoxindole plus 2.16 X 10+ mole benzoin; -o-n-,without benzoin; catalyst, 9.05 X mole 3aminonaphthoxindole; both cases, substrate, 5.1 ml. of 1.18 molar phenylglyoxylic acid; temperature, 70 ”.) 1
.
2
,
1
,
,
I
I
1
1
,
1
In
0
20
40
€0
80
100
120
140
1GO 180 200 220
t , time in minutes.
Fig. (i.-Plot of rate of evolution of COn tersus time, showing effect of introduction of fresh catalyst after 135 minutes, (Initial catalyst, 8.48 X 10-e mole; supplementary catalyst added a t 135 min., 1.96 X mole; substrate, 1.18 molar phenylglyoxylic acid; temperature, 70 ”.) A . I . = activity index.
Finally, the curves of Figs. 2 and 3 are strikingly like those observed for the action of yeast carboxylase on pyruvic acid as observed by Akamatsu,’ Hagglund and Rosenquist,s Albers and Schneiderg and others. The percentage of substrate decomposed in such experiments as the rate approached zero was about 0.1%. An attempt was made to prepare synthetic polymers containing carboxylase model moieties as functional groups in the hope of studying the effect of colloidal size of catalyst particles on catalyst activity. The polymers used were a copolymer of (6) W. Langenbeok, “Die organisohen Katalysatoren,” 2nd ed., Springer Verlrtg, Berlin, 1949, p. 91. (7) 9. Akamstsu, Biochsm. Z.,157, 369 (1923). (8) E. Hagglund and T. Rosenquist, ibid., 181, 296 (1927). (9) H. Albers and A. Scbneider, Naturwiasonschaftsn, 14, 794
(1936).
1
1
1
0
2
4
1 6
1 8
1 10
1
1
1
1
1
l
12
14
16
18
20
22
t , time in minutes. Fig. 7.-Plot of theoretical rate of COe evolution versus time in the presence of benzaldehyde. (Assumptions: catalyst, 1.128 X 10-3 molar 3-amino-~naphthoxindole; substrate, 0.525 molar phenxlglyoxylic acid; benzaldehyde, 1.12 molar; temperature, 70 ; rate constants, see text.)
styrene and maleic anhydride (SYHM resin) and a copolymer of 2-vinylpyridine and methacrylyl chloride. No clear-cut positive results were obtained as an examination of the polymers by absorption spectrophotometry indicated that the model moiety was altered chemically during the coupling of it to the polymer. Experimental The apparatus used for the carbon dioxide determination is illustrated in Fig. 8. The various parts are lettered and may be described as follows: A is a pressure regulator adapted from one developed by Furter and Steyermarklo (10)
RI, F.
Furter and A. Steyerniark, Anal. Chsm.. PO, 257 (1948).
720
--
RALPHE. S C H A C H A T , ERNEST I. BECKER A N D A. D. MCLAREX
~701.
56
PVRF N/ZROS€N
Fig. 8.-Apparatus
for measurement of micro amounts of carbon dioxide.
for use in micro-combustion analysis. The fluid used is mercury in order to have a sufficient head of nitrogen without an overly large regulator. The mercury level and hence the nitrogen pressure can be adjusted by raising or lowering the leveling bulb. B, G and K are drying tubes. C is a combination bubble counter and carbon dioxide absorber. D is a capillary tube having an air-flow resistance of about 5 or 6 times the resistance of any of the changeable absor tion tubes ( I and J). E is the reaction vessel containing ti; substrate and is kept immersed in a thermostat maintained a t the desired temperature. The enzyme model is weighed into a micro-porcelain boat which is placed at the start of a determination at the lower end of movable arm F. Both H and 0 are two-way stopcocks, while I and J are standard micro-absorption tubes charged wibh a carbon dioxide absorbent (such as Ascarite) plus some drying agent. Inasmuch as these tubes are t o be weighed to 10-6 gram, they must be carefully prepared and handled. See, for example, the recommendations of Pregl.11 M is a flowmeter calibrated directly in liters per hour. The fluid used is colored water. L is a capillary tube of such a resistance that when the gas is flowing at its maximum rate, a reading will be obtained on the scale about three-fourths of the way up. N is a trap. P is a two-liter Mariotte bottle containing water. Q is a two-liter beaker which may be replaced by a graduated cylinder when calibrating flowmeter M. The procedure is as follows: six micro-absorption tubes are wiped and weighed in the standard micro-analytical manner and two of these tubes are connected to the apparatus ( I and J in Fig. 8). The substrate is weighed into vessel E. The usual substrate consists of a mixture of 4.5 g. of phenol, 0.5 g. of phenylglyoxylic acid and 0.1 g. of potassium phenylglyoxylate. About 10-6 mole of catalyst is weighed (11) J. Grant, "Quantitative Organic Microanalysis," 4th ed., The Blakiston Co., Philadelphia, Penna., 1945,p. 50.
on a micro-balance into a micro-porcelain boat which is inserted into the lower end of holder F. The apparatus is then assembled as in Fig. 8 and the flow of nitrogen started. While the apparatus is coming to equilibrium, the flow rate is adjusted to 2.00 =!= 0.05 liters per hour using the coarse adjustment leveling bulb on A and the fine adjustment side arm on P. Usually, about 5 minutes is allowed for the apparatus to come to equilibrium, after which arm F is tipped, causing the boat containing the catalyst to slide from the holder and fall into the substrate. A stopwatch is started and after a predetermined time interval (usually 7.5 min.) drying tube K is disconnected from J and hooked to I and stopcock H turned to permit the gas to flow through tube I. Tube J is wiped and weighed and a new tube inserted in its place. After another time interval, the gas is allowed to pass through the new tube and the cycle repeated until a number of tubes have been used. Then tube No. 1 can be re-used, etc., until the desired number of points have been taken. In practice, due to the fact that it takes approximately 20 minutes to wipe and weigh a tube, it has been found that the optimum number of tubes is 6, and the number of points taken varies between 6 and 12, depending upon the activity of the catalyst. A second stopwatch is used in order to ensure that the tube wiping and weighing follows a definite schedule. Inasmuch as some of the tcbe weight increments were very small (less than 1 mg.), it, was necessary to take a zero reading or rest point after each weighing and make the necessary correction. Aftcr such a series of readings had been taken, a plot was made of the moles of carbon dioxide evolved per minute per liter of solvent versus total elapsed time to the middle of the interval. Characteristic curves were thus obtained (Fig. 2 and Table I).
Acknowledgment.-The authors wish to extend their thanks to Dr. H. Morawetz for many helpful suggestions in connection with this paper.
