2856
HAROLD R. ALMOND, JR., RICHARD J. KERRAND CARLNIEMANN
suspended in 100 ml. of chloroform, the slurry added t o 500 ml. of chloroform previously saturated with dry gaseous ammonia, the precipitated ammonium chloride removed by filtration, the filtrate evaporated to dryness in z'ucuo and the residue dried in z'ucuo over phosphorus pentoxide t u give 29.6 g . of crude L-tyrosine ethyl ester; yield 59.47,. dcetylation of the crude ester with acetyl chloride, under Schotten-Bauman conditions, gave 29.0 g . (83y0)of acetylL-tyrosine ethyl ester. This product was dissolved in absolute ethanol and the solution added to 3.8 g. of hydrazine in the same solvent. The reaction mixture was heated under refluxing conditions for 2 h r . , cooled, the precipitated product collected, recrystallized twice from absolute methanol and dried in vucuo over phosphorus pentoxide to give 27.4 g. (83yo)of or-S-acetyl-L-t?-rosinliydrazide, m.p. 227223", [ ~ x ] * ~ . ~ D 4f0 0. 8. B o ( c 2 . 0 2 5 ~in , Methyl Cellosolve). Anal. Calcd. for CllH1503N~( 2 3 7 ) : C, 55.7; H , 6.4; S , 17.7. Found: C, 5 5 . 6 ; H, 6 . 5 ; X, 17.8. Enzyme Solutions.-An Xrrnour preparation, lot no. 00592, was used throughout. T h e stock solutions were prepared as before5 except t h a t 50 mg. of the enzyme preparation was dissolved in 5.0 ml. of distilled water. One ml. of the enzyme stock solution diluted 1: 10 led to a final concentration of 1 mg. per ml. or, when based upon a nitrogen content of 14.5%, t o a concentration of 0.145 mg. of proteinM.2iJ nitrogen per ml. or 4.55 X Buffer Solution.--A THA4M-HCl buffer stock solution, 0.2 ;M in the amine component, was prepared as b e f ~ r e . ~ Enzymatic Reaction Systems and their Analysis.-The reaction systems were established essentially as described previously.5 The analyses were conducted under conditirnis
VOl. 81
where the final concentration of aldehyde was 2.684 X lo-*
M and the hydrochloric acid concentration 0.167 N . In
practice a n acidic aldehyde reagent was prepared immediately before use by mixing equal volumes of the acid and aldehyde reagent solutions, vide ante. -12.0-ml. aliquot of the acidic aldehyde reagcnt was introduced into a series of 10.0-ml. G.S. volumetric flasks and sufficient distilled water added t o each flask to bring the volume to 9.0 ml. At selected time intervals, usually two minutes, a 1.0-ml. aliquot of the reaction mixture was transferred to a flask, the solution equilibrated at 25.0 i 0.1' for exactly 20 minutes whereupon the optical density was determined at 455 nip as indicated above except that in this iiistance the blank contained all of the components of the reaction and analyses systems other than the reaction products. T h e blank was prepared by dilutiiig the acidic aldehyde reagent with the buffered specific substrate solution5 and then adding the enzl-me solution. \Vlien it became evident that the optical density would soon exceed a value of 1.1, larger volumetric flasks weie substituted for the 10.0-ml. flasks used initially and the optical density currected aiid recorded as its equivalent in a 10.0-1n1. flask. n'henever this latter practice was followed, care ivas taken to maintain the final acid and aldehyde concentrations a t a constant value by proportionally increasing the amount of acidic aldehyde reagent added to each flask, i.t,.,5.0 ml. t o a 25.0 ml. and 10.0 ml. t o a 50.0 ml. flask, and proportionally diluting the acidic aldehyde reagent before introducing the 1.0-ml. aliquot obtained from the reaction sJ-stem. Further experimental details are summarized in Table I . PAS4DES.+, C.\I.IFORNI.I
.-
[ C ' ~ ~ \ T K I H l ~ ~ I .K l OON.
2414
FROM T H E (;ATES
ASD
CKELLIS h B O K A T l O K I E S TECHSOLOGY]
OF
CtIl*;MlSl'R\~, CALlFORSIA
I S S T 1 T l ; T E Of'
The Apparent Ionization Constants of a Series of Phenylalanine Derivatives' BY HAROLD R. XL~IOND,JR., RICHARD J. KERRAXD CAXL SIEMXNK: RECEIVEDSOVEMBER 20, 1958 The apparent ionization constants of the a-ammonium groups present in DL-phenylalanine and its monoprotonated amide, thioamide, amidoxime, hydrazide, methyl ester and hydroxamide have been determined in aqueous solutions at 25.0 f 0.1" m d 0.05.0.20 and 0.20 M in sodium chloride. The values of p K O ~ ( r n ~ were - ) observed to increase from 6.78 i 0.03 t o 9.15 f 0.01 in the order -COSHOH < -COzCHa 5 -CONHSH, A - C ( S O H ) S H 2 < --CSNHz < -CONHs dium ( m ) and weak ( w ) . PASADENA, CALIF.
DEPARTMENTS O F BIOCHEMISTRY .4NU
y.4LE
UUIVERSITY]
Imidazole Catalysis. V. The Intramolecular Participation of the Imidazolyl Group in the Hydrolysis of Some Esters and the Amide of ~-(4-Imidazolyl)-butyricAcid and 4-(2 '-Acetoxyethyl) -imidazole2 BY 'I"0JfAs
c. RRUICE' AND JULIAN
AI. STURTEV.\NT
RECEIVED DECEMBER 13, 1958 The synthesis of a number of imidazoles including r-(4-imidazolyl)-butyric acid (V) and four of its phenyl esters as well as the methyl ester and amide are recorded. Also, a new method fcr the easy preparation of N-acyl imidazoles is noted. The phenyl esters of V solvolyze rapidly in water due to the very effective anchimeric assistance of the neutral imidazolyl group. The fact that the P-nitrophenyl ester of V exhibits a rate of solvolysis almost identical to that of the a-chymotrypsinp-nitrophenyl acetate complex is discussed in the light of the involvement of an imidazolyl group in both processes. The change in mechanism in going from inter- to intramolecular catalysis of substituted phenyl acetates is discussed in terms of the nucleophilic attack becoming concerted with the dissociation of the imidazolium species so that the rate-determining step becomes the collapse of the tetrahedral intermediate in the intramolecular reactions whereas in the bimolecular reaction? the tetrahedral intermediate is a t a very low and steady state concentration. Unlike the methyl ester of V, 4-(2'-acetoxyethyl)-imidazole undergoes hydrolysis with imidazole participation. This is the first reported instance cf the nucleophilic catalysis of the hydrolysis of a n aliphatic ester by a n imidazole. In the hydrolysis of the amide of V the protonated imidazolyl group participates. The similarity between the effectiveness of the imidazole and carboxyl anion and imidazoliurn and carboxyl groups as anchimeric participants in ester and amide hydrolysis is pointed out.
Considerable evidence has been accumulated in realized through incorporation of the nucleophilic recent years to indicate that esters and amides are or electrophilic participants into polymers to which catalytically hydrolyzed by esteratic enzymes the substrate becomes b o ~ n d . ~Due . ~ to the ready through a double displacement reaction involving availability of suitably substituted esters and an acylated enzyme intermediate. The formation amides, the carboxyl and carboxylate groups have received particular attention as intramolecular participants in ester and amide hydrolysis.'-" ( a ) EnzH + RCOX In the case of numerous esteratic enzymes, there k.k1 ., E n z H " ' R C 0 X --j. is much evidence to indicate that a non-protonated 0 imidazolyl group of a histidine residuels and an 11 k2 aliphatic hydroxyl group of a serine residue'Y Enz-CR' ' S H -+- EnzCOR + XH (1) '
[ ]
0
I1
( h ) Enz-CR
ka + H20-+EnzH + RCOOH
of acyl-enzyme in l a takes place after formation of
an enzyme-substrate complex, and undoubtedly involves the participation of specific amino-acid side chains (intracomplex participation) in the displacement of X ; in a t least one case4 the formation of acyl-enzyme is kinetically first order in the enzyme-substrate complex. It follows, therefore, that appropriate models for esteratic enzymes should be sought among hydrolytic reactions which proceed via first-order processes with assistance of an intracomplex or intramolecular nature. Intracomplex participation, in the catalysis of the hydrolysis of amides and esters, has been (1) Contribution No. 1523. (2) For previous papers in this series see: (a) T.C. Bruice and G. L. Schmir, THIS J O U R N A L , 79, 1663 (1957); (b) 80, 148 (1958); (c) G.L. Schmir and T.C. Bruice, i b i d . , 80, 1173 (1958); (d) T.C. Bruice and R . Lapinski, ibid., 80, 2265 (1958). (3) Inquiries t o this author should h e addressed t o t h e Department of Physiological Chemistry, T h e Johns Hopkins School of Medicine, Baltimore 5 , Md. (4) H.Gutfreund and J. M. Sturtevant, Biochem. J . , 63,756 (1956).
(5) J . R. Whitaker and F. E. Deatherage, 'I'HIS J O I I R N A L , 77, 5298 (1955); 5.C Paulson, F. E. Deatherai:e a n d l? F Almy i b b * l . ,7 5 , 3039 (1953) (6) H . Samelsun and I,. 1'. Hammett, ibi,!., 78, 324 ( I Y j l ; ) . (7) L. J. Edwards, T r a i ~ i F . n i o d n y S o < - . , 46, 7 2 3 ( l W 3 , i b " J 48, 696 (1952). (8) E . R . Garrett, 'hiis J O U R N A L , 79, :3101 ( l ! l . 7 7 ) (9) E. R. Garrett, ibid., 79, 5206 (1957). (10) P. E. Zimmering, E . W.\Yeithead and H . \ I < r r a n e t z . f5iorheiPl Biophrs. A d c , 26, 37ti (1957). (11) E. W. n'esthead and H MoranctL, TIIISJ u t l R N A 1 2 . 8 0 , ?37 (1958). (12) H . hlorawetz and I . Oreskes, i b i d . , 80, 2591 (1958). (13) RI. L. Bender. Y . Chow and F. Chloupek. i b i d . , 8 0 , 3880 (1958). (14) 31. J,, Render. I' Chlouprk and SI C. S e v e u , { b i d , 8 0 , ,7384 (1958). (15) hl. L . Bender and M .C . S e v e u , ibirf . 8 0 , 5.188 f I 9 5 8 ) . (IO) E R. Garrett, i b i d . , 8 0 , 4049 (19,523). (17) S . J . Leach and H. Lindley, T v ,re recent studie4 ( 1 8 ) For pertinent references see ref , 529 (195ti); V see: D. R. Davies and A . I.. Green. Ri Massey and B. S . Hartley, Bioc.hiir;. B , 2 1 , 301 (1950); €3. S. Hartley, Biocheni. J . , 64, 2 i I' Bid. C h r m . , 226, 873 (19.57); I,. \V ihid , 2 2 1 , L"?; : l ( l > l > ) , C, H P i y o n .ind $I X?iimth. i h i d , , 226, 10.10 (19573. ( I n ) H. Gutfreund and J hl turtevant. rei. 4 and Pior. "Val. Acod. K . Schaffer, C. S. M a y and W. H . SC;. SJ, 411, 719 (1956); Summerson, J . B i d . Chem., 202, 67 (19.53); R. A. Oosterbaan, P.
(u.
