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The inertness of the ammonium salt of N-acetyl-L -cysteic acid carboxamide in systems containing alpha-chymotrypsin
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D-Glucose o b t a i n e d from t h e p o l y s a c c h a r i d e in each case still h a d m u c h of t h e a s y m m e t r i c labeling of t h e c o r r e s p o n d i n g s u b s t r a t e . T h e s m a l l e r degree of isotope r e d i s t r i b u t i o n in t h e second e x p e r i m e n t is p r e s u m a b l y due to t h e s h o r t e r i n c u b a t i o n time. T h e labeling p a t t e r n in t h e L-Iucose closely parallels t h a t of t h e o-glucose. W i t h glucose-I-14C as t h e sole carbon source t h e fucose is p r i m a r i l y labeled in C-I, w h e r e a s C-6 is p r e d o m i n a n t l y labeled w h e n glucose-6-14C is t h e c a r b o n source. I n t h e second e x p e r i m e n t , however, t h e r e w a s a s o m e w h a t g r e a t e r r e d i s t r i b u t i o n of isotope in fucose t h a n was o b s e r v e d in glucose. T h e latter o b s e r v a t i o n s u g g e s t s t h e presence of a minor p a t h w a y for fucose b i o s y n t h e s i s from small f r a g m e n t s which c a n give rise to b o t h t h e top a n d b o t t o m h a l v e s of t h e molecule. However, t h e a s y m m e t r i c labeling observed in L-fucose indicates t h a t in t h e conversion of Dglucose to t h e d e o x y p e n t o s e either n o n - i n t e r c o n v e r t i b l e small molecules derived from glucose a c t as i n t e r m e d i a t e s or utilization of t h e i n t a c t hexose c a r b o n - s k e l e t o n is involved. STANTON SEGAL National Institute o/ Arthritis and Metabolic Diseases, YALE J. TOPPER
National Institutes o/Health, Public Health Service, United States Department o/Health, Education and Wel[are, Bethesda, Md. (U.S.A.) a j . F. WILKINSON, W. F. DUDMAN AND G. O. ASPINALL, Biochem. J., 59 (1955) 446. E. G. V. PERCIVAL AND A. G. R o s s , J. Chem. Soc., (~95 o) 717 . s h . B E s m c H , E. A. KABAT AND A. E. BEZER, J. Am. Chem. Soc., 69 (I947) 2163. Z. DlscriE AND L. B. SHETTLES, J. Biol. Chem., I75 (1948) 595. s D. M. WALDEON, Nature, 17o (1952) 461. F. EZSENeZE~, Jr., j . Am. Chem. Sot., 76 (I954) 5152. 7 j . M. PASSMANr¢, N. S. RADIN ArCD J. A. D. COOPER, Anal. Chem., 28 (1956) 484 . Received April 26th, z957
The inertness of the ammonium salt of N-acetyI-L-cysteic acid carboxamide in systems containing alpha-chymotrypsin According to SANGER AND THOMPSON I a - c h y m o t r y p s i n , or possibly a n o t h e r closely associated e n z y m e w h i c h is also inhibited b y diisopropylfluorophosphate, causes t h e hydrolysis of three of t h e t w e n t y peptide b o n d s p r e s e n t in t h e oxidized A-chain of bovine insulin, i.e., GlyIleu-VaI-Glu-Glu (NH~)-CySO~H-CySOsH-AIa-Ser-VaI-CySOsH-~-Ser-Leu-TyryGIu (N Hz)-Leu-GIuA s p ( N H ~ ) - T y r ¥ C y S O s H - A s p ( N H ~ ) , w h e n a n a q u e o u s solution of this s u b s t a n c e a n d crystalline a - c h y m o t r y p s i n , a d j u s t e d to p H 7.5 with a m m o n i u m hydroxide, is allowed to s t a n d a t 37 ° for 24 h. While a p r e c e d e n t for t h e h y d r o l y t i c cleavage of t h e two Peptide bonds involving t h e c a r b o x y l g r o u p s of t h e two tyrosine residues can be found a m o n g t h e m a n y s y n t h e t i c specific s u b s t r a t e s of a - c h y m o t r y p s i n which are simple peptides or a m i d e s derived from a v a r i e t y of a - N - a c y l a t e d L-tyrosines ~, a, no similar derivatives involving t h e c a r b o x y l g r o u p of an N - a c y l a t e d L-cysteic acid h a v e been e x a m i n e d for specific s u b s t r a t e activity. Therefore, it a p p e a r e d desirable to p r e p a r e s u c h a d e r i v a t i v e a n d to e x a m i n e its behavior with a - c h y m o t r y p s i n . T h e a m m o n i u m salt of N-acetyl-L-cysteic acid c a r b o x a m i d e was prepared. W h e n a q u e o u s solutions of this c o m p o u n d a n d crystalline a - c h y m o t r y p s i n , a d j u s t e d to p H 6.2, 6.8, 7.3, 7.9 or 8.3 with a q u e o u s N a O H , were allowed to s t a n d a t 25 -~ for as long as 27. 5 h, t h e e x t e n t of a p p a r e n t h y d r o l y s i s was no more t h a n would h a v e been observed h a d t h e i n t e n d e d specific s u b s t r a t e been a b s e n t (Table I). T h e absence of a n y significant h y d r o l y s i s raised t h e q u e s t i o n w h e t h e r t h e lack of reactivity of N-acetyl-L-cysteate c a r b o x a m i d e was due to an inability of this a n i o n to c o m b i n e with t h e c a t a l y t i c a l l y active site of t h e e n z y m e or a l t e r n a t i v e l y was due to c o m b i n a t i o n with t h e active site in a m o d e or m o d e s which did n o t lead to t h e s u b s e q u e n t f o r m a t i o n of reaction products. To a n s w e r this q u e s t i o n t h e a m m o n i u m salt of N-acetyl-L-cysteic acid c a r b o x a m i d e was e x a m i n e d with respect to its ability to f u n c t i o n as a c o m p e t i t i v e inhibitor in t h e a - c h y m o t r y p s i n - c a t a l y z e d h y d r o l y s i s of a - N - a c e t y l - L - t y r o s i n e h y d r a z i d e 5 in a q u e o u s solutions a t 25 ° a n d p H 7.9 a n d 0. 5 M in t h e T H A M ( t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ) c o m p o n e n t of a T H A M - H C I buffer. It will be seen from t h e d a t a given in Table II t h a t no evidence was obtained for c o m p e t i t i v e inhibition by t h e N - a c e t y l - L - c y s t e a t e c a r b o x a m i d e a n i o n a n d it m a y be concluded t h a t , if this c o m p o u n d can so function, t h e v a l u e of /t"I (the e n z y m e - i n h i b i t o r dissociation c o n s t a n t ) , is s u b s t a n t i a l l y g r e a t e r t h a n o.I J14. T h e i n e r t n e s s of t h e a m m o n i u m salt of N-acetyl-L-cysteic acid c a r b o x a m i d e in t h e a b o v e
v~I.. 2 5
(I()57)
421
sHoRT CO.XIMVNIC.\IIONS "I'A Bl.l'; I .\CTION OF a-CH\'MOFRYP.qIN UPON I'llV AMMONIUM S.\I.T O1. ~-.'\CET'~'I.-I.-C\'NTI';IC A('II) CARBOXAMII)F"
pH
.
.
2. 5 h
.
.
.
.
.
.
h'..5 h
.
.
.
.
22 k
27.5 k
b.2 6.8
o.o5
~.55
°.55
o.8
1.1
1. 4
0.55 1.1
7.3 7.95 8. 3
o.5 o.i~; t.o
0.5 1.3 I. 7
J .5 2-5 3.o
x .5 3.o 3.3
* In a q u e o u s s o l u t i o n s a t 25:' w i t h o.1444 mg c h y m o t r y p s i n N / m l 0.2 34 s u b s t r a t e . ** Based upon the formal t i t r a t i o n described by }iUANG AXD NIE.MANN4 a nd c orre c t e d for a b l a n k t i t e r for[t = o. TABLE
II
INFLUENCE OF THE AMMONIU.M SALT OF N-ACETYL-L-CYSTEIC ACID CARHOXAMII)E (I) UPON THE a-CHYMOTRYPSIN-CATALYZEI) HYDROLYSI.'-; O F a-~'-ACETYL-L-TYROSINEHYDRAZIDE" (~)
[s]o
[I.: i': Io*.lI
"< Io'%'~1 mi~t
X. ' 7
O.O lo.26
0.53 0.50
1.0~)
O.O
o.09
0.88
1.69 2.53 -'.53 4.23 4.23 6.67
[o.20 o.o lo.20 0.o xo.20 o.o
O.bl 1.05 l.XO 1.79 1.70 2.4 I
0.07
10.26
-.63
;< lOS3I
I .27 I .(it)
t.o"*
. V°l]V°~
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0.98 1.0¢1
* In a q u e o u s solutions a t 25 ° and pH 7.9 a n d o . 5 M in t h e T H A M c o m p o n e n t of a T H A M - H C I buffer w i t h o. I444 m g c h y m o t r y p s i n N/ml. "* I n i t i a l v e l o c i t y c a l c u l a t e d from a least s q u a r e s lit of 9 p o i n t s o b s e r v e d from t = 2 to t = x8 rain u n d e r c o n d i t i o n s where In [Sjt v s t. a p p e a r e d to be linear, i.e., for a t o t a l e x t e n t of r e a c t i o n of from 6 to 9 %. W h i l e these v a l u e s are a d e q u a t e for the c o m p a r i s o n m a d e in t h i s s t u d y , t h e i r use for t h e e v a l u a t i o n of K s and k 3 is q u e s t i o n a b l e because of t he low i ni t i a l s u b s t r a t e concentration. s y s t e m s a n d t h e r e p o r t e d c l e a v a g e of a p e p t i d e bond i n v o l v i n g t h e c a r b o x y l g r o u p of a c y s t e i c acid residue p r e s e n t in t h e o x i d i z e d A-chain of b o v i n e insulin I m a y be e x p l a i n e d in s e v e r a l w a ys . The fact t h a t only one of the four p e p t i d e bonds i n v o l v i n g t h e c a r b o x y l groups of t h e four c y s t e i c acid residues p r e s e n t in the p o l y p e p t i d e was r e p o r t e d to be h y d r o l y z e d by c r y s t a l l i n e a - c h y m o t r y p s i n 1 a n d t h a t this p a r t i c u l a r p e p t i d e bond also i n v o l v e d t h e a - a m i n o g r o u p of a n a d j a c e n t seryl resid ue I s u g g e s t s t h e p o s s i b i l i t y t h a t the r e p o r t e d c l e a v a g e a c t u a l l y i n v o l v e d a n e s t e r t y p e of bond w h i c h could h a v e arisen, a t least in part, by a N- to O-acyl m i g r a t i o n e d u r i n g t h e p r e p a r a t i o n of th e peptide. Since t h e o b s e r v a t i o n s of SANGER AnD TrtOMPSON l were q u a l i t a t i v e in n a t u r e a n d since it is k n o w n ~ t h a t a c y l a t e d a - a m i n o acid esters are m u c h more s u s c e p t i b l e to an a - c h y m o t r y p s i n - c a t a l y z e d h y d r o l y s i s t h a n are t h e c o r r e s p o n d i n g a mi de s , it is possible t h a t t h e a n o m a l y n o t e d a b o v e is more a p p a r e n t t h a n real. A l t e r n a t i v e l y , it is possible t h a t t he re are, in a - c h y m o t r y p s i n , accessory c o m b i n i n g sites a t loci r e m o v e d from t h e c a t a l y t i c a l l y a c t i v e site of the e n z y m e . Thus, c o n c e p t s of specificity, which are based upon t h e i n t e r a c t i o n or l a c k of i n t e r a c t i o n of r e l a t i v e l y s m a l l specific s u b s t r a t e molecules w i t h t h e c a t a l y t i c a l l y a c t i v e site ot t h e e n z y m e , b u t wh ich c a n not s u b t e n d such accessory c o m b i n i n g sites, can not be e x t e n d e d to t h e l a r g e r specific s u b s t r a t e m o l e c u l e s which in a d d i t i o n c a n i n t e r a c t w i t h one or more of t h e p o s t u l a t e d accessory c o m b i n i n g sites. Experimental. L-Cystine d i m e t h y l ester d i h y d r o c h l o r i d e was p r e p a r e d in 89 ~o yield from i.-cystine. The d i e s t e r d i h y d r o c h l o r i d e , o.l mole, was s u s p e n d e d in 2 1 d r y t e t r a h y d r o f u r a n , t h e s u s p e n s i o n cooled to - - 5 °, a n d l.o mole d r y r e d i s t i l l e d t r i e t h y l a m i n e a d d e d to t h e s us pe ns i on 28
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followed b y t h e slow addition of o.22 mole freshly distilled acetyl chloride. A f t e r 4 h, t h e precipit a t e d t r i e t h y l a m m o n i u m chloride was r e m o v e d b y filtration a n d t h e filtrate e v a p o r a t e d to give 71 To of crude N,N'-diacetyl-L-cystine d i m e t h y l ester. 0.05 mole of this p r o d u c t was dissolved in a m i x t u r e of 250 ml distilled w a t e r a n d 60 ml conc. HCI a n d to this solution Br~ was added, slowly a n d w i t h stirring, until a slight excess was present. A p p r o x i m a t e l y 0.25 mole was required. T h e resulting solution was c o n c e n t r a t e d in vacuo to give 75 % of crude N-acetyl-L-cysteic acid c a r b o x y m e t h y l ester, m.p. z86 ° with decomp. A solution of 0.o 4 mole of t h e crude ester in t 1 d r y m e t h a n o l was s a t u r a t e d with a n h y d r o u s a m m o n i a a n d t h e reaction m i x t u r e m a i n t a i n e d a t 25 ° in a sealed vessel for 5 days. T h e reaction m i x t u r e was t h e n e v a p o r a t e d to d r y n e s s a n d t h e residue recrystallized from a n h y d r o u s e t h a n o l to give t h e a m m o n i u m salt of N-acetyl-L-cysteic acid c a r b o x a m i d e , m.p. 2 o l - 2 o 2 °, [a]~ = - - 9 . 3 ° (in water). Yield from L-cystine, 18 %. Anal. Calcd. for CsHz3OsNsS (227): C, 26.4; I-I, 5.7; N, I8.5; S, z4.z. F o u n d : C, 26.4; H, 5.8: N, z8.5; S, 14.o. T h e conditions e m p l o y e d in e x a m i n i n g t h e action of a - c h y m o t r y p s i n on t h e a m m o n i u m salt of N-acetyl-L-cysteic acid c a r b o x a m i d e are s u m m a r i z e d in Table I. T h e a - c h y m o t r y p s i n was an A r m o u r preparation, lot No. 00592. T h e procedure e m p l o y e d for o b s e r v i n g t h e effect of a d d e d a m m o n i u m N - a c e t y l - L - c y s t e a t e c a r b o x a m i d c u p o n t h e a - c h y m o t r y p s i n - c a t a l y z e d h y d r o l y s i s of a - N - a c e t y l - L - t y r o s i n e h y d r a z i d c (Table II) was identical to t h a t described p r e v i o u s l y 4 a n d again t h e e n z y m e p r e p a r a t i o n was crystalline a - c h y m o t r y p s i n , A r m o u r lot No. 00592. This investigation was s u p p o r t e d in p a r t by a g r a n t from the National I n s t i t u t e s of H e a l t h , Public H e a l t h Service. Microanalyses b y Dr. A. EL~K. HOWARD F. MOWER Gates and Crellin Laboratories o/Chemistry*, CARL NIEMANN
Cali/ornia Institute o[ Technology, Pasadena, Call]. (U.S.A.) 1 F. 2 H. s R. 4 H. 5 R. * D.
SANGER AND E. 0 . P. THOMPSON, Biochem. J., 53 (I953) 366. NEURATH AND G. W. SCHWERT, Chem. Revs., 46 (z95o) 69. J. FOSTER AND C. NIEMANN, .]. Am. Chem. Soc., 77 (I955) t886. T. HUANG AND C. NIEMANN, ibid., 73 (1951) I541. LUTWACK, H. F. MOWER AND C. NIEMANN, ibid., 79 (I957) 2179. F. ELLIOTT, 13iochem. J., 5 o (I952) 542. Received April 18th, z957 * Contribution No. 2182.
3-Methoxy-4-hydroxy-D-mandelicacid, a urinary metabolite of norepinephrine* O n e of t h e phenolic acids in h u m a n urine, c o m p o u n d zo 1, a p p e a r s to be of e n d o g e n o u s origin, since its e x c r e t i o n is n o t affected b y d i e t a r y changes. M a n y of t h e q u a l i t a t i v e reactions of t h i s s u b s t a n c e were observed to be similar to t h o s e of s o m e c o m p o u n d s c o n t a i n i n g t h e 3 - m e t h o x y 4 - h y d r o x y p h e n y l group. T h i s fact, a l o n g with t h e recent o b s e r v a t i o n t h a t h o m o p r o t o c a t e c h u i c acid u n d e r g o e s biological m e t h y l a t i o n to homovanillic acid 2, a n d t h e solubility c h a r a c t e r i s t i c s of t h e u n k n o w n s u b s t a n c e as indicated b y its c h r o m a t o g r a p h i c b e h a v i o r were s u g g e s t i v e t h a t it m i g h t be 3 - m e t h o x y - 4 - h y d r o x y m a n d e l i c acid (I). (I) m i g h t be expected to be f o r m e d b y t h e action of a m i n e oxidase u p o n n o r e p i n e p h r i n e or e p i n e p h r i n e s, followed by m e t h y l a t i o n of t h e r e s u l t i n g 3 , 4 - d i h y d r o x y m a n d e l i c acid. T h e following results indicate t h a t (I) is, indeed, a n i m p o r t a n t u r i n a r y m e t a b o l i t e of n o r e p i n e p h r i n e : (I) T h e p a r e n t e r a l a d m i n i s t r a t i o n of n o r e p i n e p h r i n e leads to a n increased a m o u n t of ( I ) i n t h e urine**; (2) orally ingested 3,4-dihydroxy-DL-mandelic acid gives rise to a n increased excretion of (I); a n d (3) t h r e e p a t i e n t s w i t h p h e o c h r o m o c y t o m a s excreted g r e a t l y increased a m o u n t s of (I) p r e o p e r a t i v e l y a n d n o r m a l a m o u n t s p o s t o p e r a t i v e l y * * * . A u t h e n t i c DL-(I) was p r e p a r e d b y t h e m e t h o d of GARDNER AND HIBBERT4; m . p . z29--z3 O° dec. §. L- a n d D-(I) were p r e p a r e d b y fractional crystallization of t h e cinchonine salt of DE-(I). T h e * T h i s w o r k w a s s u p p o r t e d b y research g r a n t s from t h e N a t i o n a l I n s t i t u t e of H e a l t h , U.S. Public H e a l t h Service. ** W e are i n d e b t e d to Dr. HAROLD BROWN a n d t h e resident staff of t h e V e t e r a n s A d m i n i s t r a t i o n Hospital, Salt L a k e City, U t a h , for p r o v i d i n g m a t e r i a l f r o m p a t i e n t s . *** W e are g r a t e f u l to Dr. JoHN WALDO, Salt L a k e City, Drs. J. G. Or.SEN AND PAUL SOUTHWICK, Ogden, U t a h , a n d Dr. HAROLD BROWN, for p r o v i d i n g u r i n e f r o m t h e s e p a t i e n t s . J All m e l t i n g p o i n t s were m a d e in open capillary t u b e s a n d are uncorrected.
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D-Glucose o b t a i n e d from t h e p o l y s a c c h a r i d e in each case still h a d m u c h of t h e a s y m m e t r i c labeling of t h e c o r r e s p o n d i n g s u b s t r a t e . T h e s m a l l e r degree of isotope r e d i s t r i b u t i o n in t h e second e x p e r i m e n t is p r e s u m a b l y due to t h e s h o r t e r i n c u b a t i o n time. T h e labeling p a t t e r n in t h e L-Iucose closely parallels t h a t of t h e o-glucose. W i t h glucose-I-14C as t h e sole carbon source t h e fucose is p r i m a r i l y labeled in C-I, w h e r e a s C-6 is p r e d o m i n a n t l y labeled w h e n glucose-6-14C is t h e c a r b o n source. I n t h e second e x p e r i m e n t , however, t h e r e w a s a s o m e w h a t g r e a t e r r e d i s t r i b u t i o n of isotope in fucose t h a n was o b s e r v e d in glucose. T h e latter o b s e r v a t i o n s u g g e s t s t h e presence of a minor p a t h w a y for fucose b i o s y n t h e s i s from small f r a g m e n t s which c a n give rise to b o t h t h e top a n d b o t t o m h a l v e s of t h e molecule. However, t h e a s y m m e t r i c labeling observed in L-fucose indicates t h a t in t h e conversion of Dglucose to t h e d e o x y p e n t o s e either n o n - i n t e r c o n v e r t i b l e small molecules derived from glucose a c t as i n t e r m e d i a t e s or utilization of t h e i n t a c t hexose c a r b o n - s k e l e t o n is involved. STANTON SEGAL National Institute o/ Arthritis and Metabolic Diseases, YALE J. TOPPER
National Institutes o/Health, Public Health Service, United States Department o/Health, Education and Wel[are, Bethesda, Md. (U.S.A.) a j . F. WILKINSON, W. F. DUDMAN AND G. O. ASPINALL, Biochem. J., 59 (1955) 446. E. G. V. PERCIVAL AND A. G. R o s s , J. Chem. Soc., (~95 o) 717 . s h . B E s m c H , E. A. KABAT AND A. E. BEZER, J. Am. Chem. Soc., 69 (I947) 2163. Z. DlscriE AND L. B. SHETTLES, J. Biol. Chem., I75 (1948) 595. s D. M. WALDEON, Nature, 17o (1952) 461. F. EZSENeZE~, Jr., j . Am. Chem. Sot., 76 (I954) 5152. 7 j . M. PASSMANr¢, N. S. RADIN ArCD J. A. D. COOPER, Anal. Chem., 28 (1956) 484 . Received April 26th, z957
The inertness of the ammonium salt of N-acetyI-L-cysteic acid carboxamide in systems containing alpha-chymotrypsin According to SANGER AND THOMPSON I a - c h y m o t r y p s i n , or possibly a n o t h e r closely associated e n z y m e w h i c h is also inhibited b y diisopropylfluorophosphate, causes t h e hydrolysis of three of t h e t w e n t y peptide b o n d s p r e s e n t in t h e oxidized A-chain of bovine insulin, i.e., GlyIleu-VaI-Glu-Glu (NH~)-CySO~H-CySOsH-AIa-Ser-VaI-CySOsH-~-Ser-Leu-TyryGIu (N Hz)-Leu-GIuA s p ( N H ~ ) - T y r ¥ C y S O s H - A s p ( N H ~ ) , w h e n a n a q u e o u s solution of this s u b s t a n c e a n d crystalline a - c h y m o t r y p s i n , a d j u s t e d to p H 7.5 with a m m o n i u m hydroxide, is allowed to s t a n d a t 37 ° for 24 h. While a p r e c e d e n t for t h e h y d r o l y t i c cleavage of t h e two Peptide bonds involving t h e c a r b o x y l g r o u p s of t h e two tyrosine residues can be found a m o n g t h e m a n y s y n t h e t i c specific s u b s t r a t e s of a - c h y m o t r y p s i n which are simple peptides or a m i d e s derived from a v a r i e t y of a - N - a c y l a t e d L-tyrosines ~, a, no similar derivatives involving t h e c a r b o x y l g r o u p of an N - a c y l a t e d L-cysteic acid h a v e been e x a m i n e d for specific s u b s t r a t e activity. Therefore, it a p p e a r e d desirable to p r e p a r e s u c h a d e r i v a t i v e a n d to e x a m i n e its behavior with a - c h y m o t r y p s i n . T h e a m m o n i u m salt of N-acetyl-L-cysteic acid c a r b o x a m i d e was prepared. W h e n a q u e o u s solutions of this c o m p o u n d a n d crystalline a - c h y m o t r y p s i n , a d j u s t e d to p H 6.2, 6.8, 7.3, 7.9 or 8.3 with a q u e o u s N a O H , were allowed to s t a n d a t 25 -~ for as long as 27. 5 h, t h e e x t e n t of a p p a r e n t h y d r o l y s i s was no more t h a n would h a v e been observed h a d t h e i n t e n d e d specific s u b s t r a t e been a b s e n t (Table I). T h e absence of a n y significant h y d r o l y s i s raised t h e q u e s t i o n w h e t h e r t h e lack of reactivity of N-acetyl-L-cysteate c a r b o x a m i d e was due to an inability of this a n i o n to c o m b i n e with t h e c a t a l y t i c a l l y active site of t h e e n z y m e or a l t e r n a t i v e l y was due to c o m b i n a t i o n with t h e active site in a m o d e or m o d e s which did n o t lead to t h e s u b s e q u e n t f o r m a t i o n of reaction products. To a n s w e r this q u e s t i o n t h e a m m o n i u m salt of N-acetyl-L-cysteic acid c a r b o x a m i d e was e x a m i n e d with respect to its ability to f u n c t i o n as a c o m p e t i t i v e inhibitor in t h e a - c h y m o t r y p s i n - c a t a l y z e d h y d r o l y s i s of a - N - a c e t y l - L - t y r o s i n e h y d r a z i d e 5 in a q u e o u s solutions a t 25 ° a n d p H 7.9 a n d 0. 5 M in t h e T H A M ( t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ) c o m p o n e n t of a T H A M - H C I buffer. It will be seen from t h e d a t a given in Table II t h a t no evidence was obtained for c o m p e t i t i v e inhibition by t h e N - a c e t y l - L - c y s t e a t e c a r b o x a m i d e a n i o n a n d it m a y be concluded t h a t , if this c o m p o u n d can so function, t h e v a l u e of /t"I (the e n z y m e - i n h i b i t o r dissociation c o n s t a n t ) , is s u b s t a n t i a l l y g r e a t e r t h a n o.I J14. T h e i n e r t n e s s of t h e a m m o n i u m salt of N-acetyl-L-cysteic acid c a r b o x a m i d e in t h e a b o v e
v~I.. 2 5
(I()57)
421
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pH
.
.
2. 5 h
.
.
.
.
.
.
h'..5 h
.
.
.
.
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27.5 k
b.2 6.8
o.o5
~.55
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o.8
1.1
1. 4
0.55 1.1
7.3 7.95 8. 3
o.5 o.i~; t.o
0.5 1.3 I. 7
J .5 2-5 3.o
x .5 3.o 3.3
* In a q u e o u s s o l u t i o n s a t 25:' w i t h o.1444 mg c h y m o t r y p s i n N / m l 0.2 34 s u b s t r a t e . ** Based upon the formal t i t r a t i o n described by }iUANG AXD NIE.MANN4 a nd c orre c t e d for a b l a n k t i t e r for[t = o. TABLE
II
INFLUENCE OF THE AMMONIU.M SALT OF N-ACETYL-L-CYSTEIC ACID CARHOXAMII)E (I) UPON THE a-CHYMOTRYPSIN-CATALYZEI) HYDROLYSI.'-; O F a-~'-ACETYL-L-TYROSINEHYDRAZIDE" (~)
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0.53 0.50
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O.O
o.09
0.88
1.69 2.53 -'.53 4.23 4.23 6.67
[o.20 o.o lo.20 0.o xo.20 o.o
O.bl 1.05 l.XO 1.79 1.70 2.4 I
0.07
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-.63
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* In a q u e o u s solutions a t 25 ° and pH 7.9 a n d o . 5 M in t h e T H A M c o m p o n e n t of a T H A M - H C I buffer w i t h o. I444 m g c h y m o t r y p s i n N/ml. "* I n i t i a l v e l o c i t y c a l c u l a t e d from a least s q u a r e s lit of 9 p o i n t s o b s e r v e d from t = 2 to t = x8 rain u n d e r c o n d i t i o n s where In [Sjt v s t. a p p e a r e d to be linear, i.e., for a t o t a l e x t e n t of r e a c t i o n of from 6 to 9 %. W h i l e these v a l u e s are a d e q u a t e for the c o m p a r i s o n m a d e in t h i s s t u d y , t h e i r use for t h e e v a l u a t i o n of K s and k 3 is q u e s t i o n a b l e because of t he low i ni t i a l s u b s t r a t e concentration. s y s t e m s a n d t h e r e p o r t e d c l e a v a g e of a p e p t i d e bond i n v o l v i n g t h e c a r b o x y l g r o u p of a c y s t e i c acid residue p r e s e n t in t h e o x i d i z e d A-chain of b o v i n e insulin I m a y be e x p l a i n e d in s e v e r a l w a ys . The fact t h a t only one of the four p e p t i d e bonds i n v o l v i n g t h e c a r b o x y l groups of t h e four c y s t e i c acid residues p r e s e n t in the p o l y p e p t i d e was r e p o r t e d to be h y d r o l y z e d by c r y s t a l l i n e a - c h y m o t r y p s i n 1 a n d t h a t this p a r t i c u l a r p e p t i d e bond also i n v o l v e d t h e a - a m i n o g r o u p of a n a d j a c e n t seryl resid ue I s u g g e s t s t h e p o s s i b i l i t y t h a t the r e p o r t e d c l e a v a g e a c t u a l l y i n v o l v e d a n e s t e r t y p e of bond w h i c h could h a v e arisen, a t least in part, by a N- to O-acyl m i g r a t i o n e d u r i n g t h e p r e p a r a t i o n of th e peptide. Since t h e o b s e r v a t i o n s of SANGER AnD TrtOMPSON l were q u a l i t a t i v e in n a t u r e a n d since it is k n o w n ~ t h a t a c y l a t e d a - a m i n o acid esters are m u c h more s u s c e p t i b l e to an a - c h y m o t r y p s i n - c a t a l y z e d h y d r o l y s i s t h a n are t h e c o r r e s p o n d i n g a mi de s , it is possible t h a t t h e a n o m a l y n o t e d a b o v e is more a p p a r e n t t h a n real. A l t e r n a t i v e l y , it is possible t h a t t he re are, in a - c h y m o t r y p s i n , accessory c o m b i n i n g sites a t loci r e m o v e d from t h e c a t a l y t i c a l l y a c t i v e site of the e n z y m e . Thus, c o n c e p t s of specificity, which are based upon t h e i n t e r a c t i o n or l a c k of i n t e r a c t i o n of r e l a t i v e l y s m a l l specific s u b s t r a t e molecules w i t h t h e c a t a l y t i c a l l y a c t i v e site ot t h e e n z y m e , b u t wh ich c a n not s u b t e n d such accessory c o m b i n i n g sites, can not be e x t e n d e d to t h e l a r g e r specific s u b s t r a t e m o l e c u l e s which in a d d i t i o n c a n i n t e r a c t w i t h one or more of t h e p o s t u l a t e d accessory c o m b i n i n g sites. Experimental. L-Cystine d i m e t h y l ester d i h y d r o c h l o r i d e was p r e p a r e d in 89 ~o yield from i.-cystine. The d i e s t e r d i h y d r o c h l o r i d e , o.l mole, was s u s p e n d e d in 2 1 d r y t e t r a h y d r o f u r a n , t h e s u s p e n s i o n cooled to - - 5 °, a n d l.o mole d r y r e d i s t i l l e d t r i e t h y l a m i n e a d d e d to t h e s us pe ns i on 28
422
SHORT COMMUNICATIONS
VOL. 25 (Z957)
followed b y t h e slow addition of o.22 mole freshly distilled acetyl chloride. A f t e r 4 h, t h e precipit a t e d t r i e t h y l a m m o n i u m chloride was r e m o v e d b y filtration a n d t h e filtrate e v a p o r a t e d to give 71 To of crude N,N'-diacetyl-L-cystine d i m e t h y l ester. 0.05 mole of this p r o d u c t was dissolved in a m i x t u r e of 250 ml distilled w a t e r a n d 60 ml conc. HCI a n d to this solution Br~ was added, slowly a n d w i t h stirring, until a slight excess was present. A p p r o x i m a t e l y 0.25 mole was required. T h e resulting solution was c o n c e n t r a t e d in vacuo to give 75 % of crude N-acetyl-L-cysteic acid c a r b o x y m e t h y l ester, m.p. z86 ° with decomp. A solution of 0.o 4 mole of t h e crude ester in t 1 d r y m e t h a n o l was s a t u r a t e d with a n h y d r o u s a m m o n i a a n d t h e reaction m i x t u r e m a i n t a i n e d a t 25 ° in a sealed vessel for 5 days. T h e reaction m i x t u r e was t h e n e v a p o r a t e d to d r y n e s s a n d t h e residue recrystallized from a n h y d r o u s e t h a n o l to give t h e a m m o n i u m salt of N-acetyl-L-cysteic acid c a r b o x a m i d e , m.p. 2 o l - 2 o 2 °, [a]~ = - - 9 . 3 ° (in water). Yield from L-cystine, 18 %. Anal. Calcd. for CsHz3OsNsS (227): C, 26.4; I-I, 5.7; N, I8.5; S, z4.z. F o u n d : C, 26.4; H, 5.8: N, z8.5; S, 14.o. T h e conditions e m p l o y e d in e x a m i n i n g t h e action of a - c h y m o t r y p s i n on t h e a m m o n i u m salt of N-acetyl-L-cysteic acid c a r b o x a m i d e are s u m m a r i z e d in Table I. T h e a - c h y m o t r y p s i n was an A r m o u r preparation, lot No. 00592. T h e procedure e m p l o y e d for o b s e r v i n g t h e effect of a d d e d a m m o n i u m N - a c e t y l - L - c y s t e a t e c a r b o x a m i d c u p o n t h e a - c h y m o t r y p s i n - c a t a l y z e d h y d r o l y s i s of a - N - a c e t y l - L - t y r o s i n e h y d r a z i d c (Table II) was identical to t h a t described p r e v i o u s l y 4 a n d again t h e e n z y m e p r e p a r a t i o n was crystalline a - c h y m o t r y p s i n , A r m o u r lot No. 00592. This investigation was s u p p o r t e d in p a r t by a g r a n t from the National I n s t i t u t e s of H e a l t h , Public H e a l t h Service. Microanalyses b y Dr. A. EL~K. HOWARD F. MOWER Gates and Crellin Laboratories o/Chemistry*, CARL NIEMANN
Cali/ornia Institute o[ Technology, Pasadena, Call]. (U.S.A.) 1 F. 2 H. s R. 4 H. 5 R. * D.
SANGER AND E. 0 . P. THOMPSON, Biochem. J., 53 (I953) 366. NEURATH AND G. W. SCHWERT, Chem. Revs., 46 (z95o) 69. J. FOSTER AND C. NIEMANN, .]. Am. Chem. Soc., 77 (I955) t886. T. HUANG AND C. NIEMANN, ibid., 73 (1951) I541. LUTWACK, H. F. MOWER AND C. NIEMANN, ibid., 79 (I957) 2179. F. ELLIOTT, 13iochem. J., 5 o (I952) 542. Received April 18th, z957 * Contribution No. 2182.
3-Methoxy-4-hydroxy-D-mandelicacid, a urinary metabolite of norepinephrine* O n e of t h e phenolic acids in h u m a n urine, c o m p o u n d zo 1, a p p e a r s to be of e n d o g e n o u s origin, since its e x c r e t i o n is n o t affected b y d i e t a r y changes. M a n y of t h e q u a l i t a t i v e reactions of t h i s s u b s t a n c e were observed to be similar to t h o s e of s o m e c o m p o u n d s c o n t a i n i n g t h e 3 - m e t h o x y 4 - h y d r o x y p h e n y l group. T h i s fact, a l o n g with t h e recent o b s e r v a t i o n t h a t h o m o p r o t o c a t e c h u i c acid u n d e r g o e s biological m e t h y l a t i o n to homovanillic acid 2, a n d t h e solubility c h a r a c t e r i s t i c s of t h e u n k n o w n s u b s t a n c e as indicated b y its c h r o m a t o g r a p h i c b e h a v i o r were s u g g e s t i v e t h a t it m i g h t be 3 - m e t h o x y - 4 - h y d r o x y m a n d e l i c acid (I). (I) m i g h t be expected to be f o r m e d b y t h e action of a m i n e oxidase u p o n n o r e p i n e p h r i n e or e p i n e p h r i n e s, followed by m e t h y l a t i o n of t h e r e s u l t i n g 3 , 4 - d i h y d r o x y m a n d e l i c acid. T h e following results indicate t h a t (I) is, indeed, a n i m p o r t a n t u r i n a r y m e t a b o l i t e of n o r e p i n e p h r i n e : (I) T h e p a r e n t e r a l a d m i n i s t r a t i o n of n o r e p i n e p h r i n e leads to a n increased a m o u n t of ( I ) i n t h e urine**; (2) orally ingested 3,4-dihydroxy-DL-mandelic acid gives rise to a n increased excretion of (I); a n d (3) t h r e e p a t i e n t s w i t h p h e o c h r o m o c y t o m a s excreted g r e a t l y increased a m o u n t s of (I) p r e o p e r a t i v e l y a n d n o r m a l a m o u n t s p o s t o p e r a t i v e l y * * * . A u t h e n t i c DL-(I) was p r e p a r e d b y t h e m e t h o d of GARDNER AND HIBBERT4; m . p . z29--z3 O° dec. §. L- a n d D-(I) were p r e p a r e d b y fractional crystallization of t h e cinchonine salt of DE-(I). T h e * T h i s w o r k w a s s u p p o r t e d b y research g r a n t s from t h e N a t i o n a l I n s t i t u t e of H e a l t h , U.S. Public H e a l t h Service. ** W e are i n d e b t e d to Dr. HAROLD BROWN a n d t h e resident staff of t h e V e t e r a n s A d m i n i s t r a t i o n Hospital, Salt L a k e City, U t a h , for p r o v i d i n g m a t e r i a l f r o m p a t i e n t s . *** W e are g r a t e f u l to Dr. JoHN WALDO, Salt L a k e City, Drs. J. G. Or.SEN AND PAUL SOUTHWICK, Ogden, U t a h , a n d Dr. HAROLD BROWN, for p r o v i d i n g u r i n e f r o m t h e s e p a t i e n t s . J All m e l t i n g p o i n t s were m a d e in open capillary t u b e s a n d are uncorrected.