March 5 , 1961
transfer to give Tl(II1) and a new intermediate, perhaps NOz. If the first alternative is operative, Tl(I1) must react rapidly with Ce(IV) to give Tl(II1) and Ce(1V). If the second alternative is operative, Ce(1V) reacts rapidly with the new intermediate to give Ce(II1) and the compound from which the first intermediate was formed, perhaps NO3-. The second term in the empirical rate law implies a bimolecular reaction between Tl(1) and Ce(1V) which must be either a one electron transfer to give Tl(I1) or a two electron reaction, for example Ce(NOs),‘-*
+ Tl(1)
1253
COMMUNICATIONS TO THE EDITOR
=
Ce(NOl)tT?
+ T10+ + NOZ
Several mechanisms in agreement with results of experiments in the presence of Ce(II1) are possible within the above framework. Experiments are continuing to determine which mechanism is correct. Our results for low concentrations of reactants are in agreement with a mechanism proposed by HalpernZa on the basis of unpublished data. Now, if TI(I1) is involved in both the Ce(1V) reaction and the exchange reaction between Tl(1) and Tl(III), the Ce(1V) reaction in the presence of Tl(II1) could be no slower than the exchange reaction because the exchange could feed TI(I1) to the Ce(1V) reaction. However, the exchange is much more rapid than the Ce(1V) reaction4 and hence the two reactions cannot both involve T1(11). We therefore conclude that either the Tl(1)-Tl(II1) exchange reaction or the Ce(1V)Tl(1) reaction, or both, must involve a two electron transfer of some sort, probably an oxygen atom transfer.
2.0 2.4
t&,
-s 2.8 Ai
I
3.2
3.6 4 .O L
6
7
I
I
I
I
8
9
10
11
I 12
13
PH (PD). Fig. 1.-The hydrolysis of some cinnamob-1 derivatives : ( A ) cinnamoyl-chymotrypsin in H 2 0 ; ( B ) cinnamol-Ichymotrypsin in DzO; ( C ) cinnainoylimidazole; ( D ) 0cinnamoyl-iV-acetylserinamidein 8 M urea ; ( E ) cinnanioylchymotrypsin in 8 J.1 urea.
a base with an apparent pKa of approximately 7 . Since bell-shaped pH-rate profiles are found in most enzymatic processes, we wished to investigate carefully the possibility of a bell-shaped curve in a-chymotrypsin catalysis. We further wished to investigate the function of the base of pKa 7 by observations of the kinetic effect of D20. The kinetics of the deacylation of cinnamoyl-cuchymotrypsin3 was determined because of the simplicity of the system5 The hydrolysis of cin(4) (a) R. J. Prestwood and A. C . Wahl, J . A m . Chem. Soc., 7 1 , namoyl-a-chymotrypsin (Curve A of Fig. 1) shows 3137 (1949); (b) G. Harbottle and R. W. Dodson, ibid., 73, 2442 the usual log k-pH profile with dependence on a (1951). Since the conditions used by these workers were diderent basic group of apparent pK 7.1. Significantly the from those employed here, the exchange was measured under our excurve remains flat up to pH 12, in spite of considerperimental conditions. When T l ( I ) = 0.0086 f and Tl(II1) = 0.0077 f the half life of the exchange is 0.25 hour, while the Ce(IV)-TI(I) reable denaturation of the enzyme. This result action under the same conditions with Ce(IV) = 0.060 has a half time rules out the participation of the tyrosine hydroxyl of 10 hours. groups (pK 10.0) and the c-ainmonium ions of DEPARTMEST OF CHEMISTRY J. W.GRYDER lysine ($JK 10.2) but leaves the possibility of the THEJOHNS HOPKIKS UNIVERSITY MARYC. DORFMAN participation of a group of pKa above 12.3 as a n 18, MARYLAND BALTIMORE (unobserved) general acid catalyst. RECEIVED J A N U A R Y 14, 1961 The hydrolysis of h--cinnamoylimidazole (Curve C) exhibits the usual base catalysis. The log THE INFLUENCE OF PH AND DEUTERIUM OXIDE k-pH profiles for the alkaline hydrolyses of 0ON THE KINETICS OF a-CHYMOTRYPSIN-CATAcinnamoyl-N-acetylserinamideand cinnamoyl-aLYZED REACTIONSi~2 chymotrypsin in S M urea are Curves D and E. Sir: respectively. The acylenzyme loses its special The action of a-chymotrypsin on phenyl esters hydrolytic properties in S Jf urea because of dehas been shown to proceed in three steps: (1) naturation, as expected, and exhibits hydrolytic adsorption of the substrate on the enyzme; ( 2 ) behavior (4.0 X l./mole sec.) similar to that acylation of the enzyme releasing the phenol; of 0-cinnamoyl-E-acetylserinamide(5.4 X l o p 2 and (3) deacylation of the acyl-enzyme producing I.,/’mole sec.). These rate data indicate that the carboxylic acid and regenerating the e n ~ y m e . ~cinnamoyl-a-chymotrypsin ,~ structurally resembles Most studies of the effect of pH on a-chymotrypsin an ester (of serine)6and not a cinnamoylimidazole action may be analyzed in terms of dependency on derivative, confirming earlier degradative evidence. (1) This research was supported by Grant H-572G of the National Institutes of Health, and by grants from the Upjohn Company, and the Lilly Research Laboratories. (2) Paper VI in the series “The Mechanism of Action of Proteolytic Enzymes”; previous paper, W. A. Glasson and M. L. Bender, J . A m . Chem. Soc., 83, 3 3 3 6 (1960). ( 3 ) G. R. Schonbaum, K . Xakamura and M. L . Bender, tbid., 8 1 , 4746 (1954). (4) M . L . Bender, Chem. Reus., 60, 53 (1960).
(5) It can be shown t h a t the catalytic processes of both the acylation and deacylation steps must utilize the same enzymatic components. from a consideration of or-chymotrypsin-catalyzed isotopic exchange reactions and the principle of microscopic reversibility. See R I . 1,. Bender, G . R. Schonbaum and G. A. Hamilton, J . Polylnzr Sci , in press (1961). (6) Similar behavior of acetyl-chymotrypsin in 8 hf urea has been noted: B. M . Anderson, E. H . Cordes and W. P. Jencks, J . B d . C h e m . , in press (1961).
COMMUNICATIONS TO THE EDITOR
1256
Deuterium oxide causes two effects in the deacylation of cinnamoyl-a-chymotrypsin (Curve B): the apparent pKa is 7.7 in DzO; and the rate constant a t any pH(pD) is 2.5 fold more in HzO than in D20. The shift in the pKa of the catalytically important group in D20 is expected.’ X substantial decrease in rate in D 2 0 also is found in the acylation of chymotrypsin using p-nitrophenyl trimethylacetate and in the hydrolysis of the specific substrate, N-acetyl-L-tryptophan methyl ester (kH/kD = 2.83).8 These isotope effects cannot be attributable to a pathway involving nucleophilic catalysis alone, which would be expected to exhibit essentially no D20 isotope eff e ~ t . ~ . ~ ~The ’ O deuterium oxide and pH results are consistent with a mechanism involving general basic catalysis by a group of pK, 7.1, or a mechanism involving nucleophilic catalysis together with general acid catalysis by a species of pSa above 12.5. In either case k H / k D would reflect a slow proton transfer and be approximately 2-3.6$11 Acylation and deacylation are mechanistically similar : they exhibit similar fiH dependencies, deuterium isotope effects and effects of structure on reactivity.’* Let LIS assume that acylation and deacylation correctly describe the catalytic process, and that the a-chymotrypsin-catalyzed isotopic exchange reaction2 proceeds through the same steps as the hydrolysis reaction (Eq. 1). Then i t follows that the “acylation” and “deacylation” steps (I and 2) of the isotopic exchange must be identical. (Step 1 = step -2, but since RCOEn 0
II
1
0
RCOMe-En 7 -RdEn Me013
2
0
\I * 7- RCOMe9En
(1)
MeOH*
is also an alkyl ester, step 1 = step 2). A mechanism meeting these rcquirements must employ a t least two siinultaneous catalytic functions such as general acidic and general basic catalysis (Eq. 2).13 In acylation B and HA will be operative while in deacylation the kinetically equivalent coinbination of BHf and A- will be operative. It is suggested that this forin of catalysis be called “conjugate” catalysis for a base and its conjugate acid are operative in the two catalytic steps, as are an acid antl its conjugate base.IJ Catalysis by a lone general base6 (c 2 , in Eq. 2) would fit the kinetic results but would violate the chcmical syninietry required 1-)y the exchange ( 7 ) T h e s h i f t of 0.13 t 0.1 pk’ unit is n-ithin the exjierimental el-ror of the predicted shift of 0.~56 Ph’ unit for a group of pK, 7: K. P. Bell, “ T h e Proton in Chemistry,” Cornell University Press, Ithaca, N. Y.,1D53, p. 1S8. ( 8 ) J. F. Thompson, Arch. Biochem. Biophys.. 90, 1 ( l Q G O ) , reports k ” / k D = 2 . 5 f o r the fumarase-catalyzed conversion of fumarute to malate. (9) Unpublished observations of h l . L. Bender and X. C . A-eveu. (10) DxG stabilizes the helical form of ribonuclease, relative to its uncoiled form: J . Hermans and H. A. Scheraga. Biochim. et B i o p h y s . Acta. 36, 534 (1959). (11) I-. Zerner and M. L. Bender, J. A m . Chem. Soc., 83, in press (1961): Y. Pocker, Proc. Chem. Soc., 17 (1960); A . R. Butler and V. Gold, ihid., 15 (1960). (12) Unpublished observations of C. A. Hamilton and K. Nakamura. (13) This treatment assumes no tetracovalcnt intermediates such as RC(OH)(OR)z. (14) A referee has suggested t h a t in addition t o an enzymntic grriup, IIA could be a water niolecule.
o
-BH: .-((”-
rBH
2-c-R oar:
-CI12’
I
i
A1
I
H L--
Vol. 83
?
‘0
7-R
/
onG
(2;
H
.4 ___----J
reaction A lone general base would remove a proton from the attacking alcohol in step 1 (but not in step 2) and its conjugate acid (EH+) would donate a proton to the alkoxy1 group of the ester in step 2 (but not in step l ) , whereas in this exchange chemical symmetry requires that exactly the same processes take place in both halves of the over-all exchange reaction. Therefore i t must be concluded that a single kind of catalysis (either acid, base or nucleophile) is not sufficient and that one must postulate a scheme such as eq. 2. If extrapolation to the hydrolytic system is allowed (substitution of H20for MeOH in the deacylation step) the cnzynzut ic participants in acylation and deacylation must still bear the same relationship to one another, requiring that a general base (or nucleophile) and a general acid again be involved. (15) Alfred P Sloan Foundation Research Fellow. ( l G ) Department of Chemistry, Northwestern Universlty hIYRON L. BENDERiJ”’ DEPARTMENTS O F CHEMISTRY ILLINOIS INSTITUTE OF TECHNOLOGY CHICAGO 16, ILLINOIS, AND GREGORY R SCHO~BAUM GORDON H A M I L ~ O Y NORTHWESTERN UNIVERSITY EVASSTON, ILLINOIS BURT ZERNER RECEIVED JANUARY 3, 1961
ORGANIC DISULFIDES AND RELATED SUBSTANCES. 111. ONE-STEP PREPARATION OF SULFINIC ESTERS FROM LEAD TETRAACETATE AND DISULFIDES OR THIOLS’
Sir: We wish to report a novel means of prqmiiig suliinic esters by oxidizing disulfides with lexd tetraacetate (I) in chloroform-methanol, probably according to the equation RSSR t- 3Pb(OAc)r + 4CH30I-I --+ 1
BRS(0)OCHa
+ 3Pb(0.4~)?+ 4AcOH + ~ A C ~ C I I X
Thiols also can be used, since the agent I oxidizes them to disulfides.2 In view of the stability antl ready availability of disulfides and thiols, the present process should make sulfinic esters much (1) Research sugpotted by the Office of Ordnance Icescnrch, U . S. Artily. Presented a t the Southeastern Regional Meeting of the Arnericnn Chemical Society, Uirniingham, Ala,, Xov. :3-5, 1960. Part 11, D. E. Pearson, D. Cairle and L. Field, J. O ~ EC. h e m . , 2 5 , 867 il!Iljl))
( 2 ) I.. Fieid and J. E. Laason, J . A i n . Client. Soc., 80, 636 (1636).
