Oct 18, 1971 - effective inhibitors of carboxypeptidase. A. The âL ... The activity of carboxypeptidase. A from beef pancreas ( .... B 160 times more tightly than 3-.
Synthesis
of Fatty
atoms of acids during oxidation or the formation of acids by the reversal of /3 oxidation (or both), reported to be catalyzed by the fatty acid synthetase itself (8). The possibility that the enzymes causing @ reduction found in the cytoplasm are of mitochondrial origin can be ruled out as neither leakage nor release of the P-oxidative enzymes occurred when rabbit, mammary mitochondria were either subjected to repeated homogenization or were disrupted by freezing and thawing (1). The mitochondrial enzymes stayed bound to the mitochondrial membranes and even when disrupted were removed on ultracentrifugation. Similarly, Williamson et al. (9) have established that acetoacetyl-CoA thiolase, found in rat liver cytoplasm, did not arise as a result of mitochondrial disruption as there was no leakage of glutamate dehydrogenase during the homogenization procedure. It is concluded from the results that in the mammalian liver and mammary tissue the important primer in the fatty acid synthetase-catalyzed reactions is butyryl-CoR and not acetyl-CoA and that the major pathway for the synthesis of fatty acids in these tissues involves the reactions shown in Scheme 1. To what extent these reactions actually occur under physiological conditions remains to be established. REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9.
A. K. N., AND KUMAR, S. (1969) Arch. Biochem. Biophys., 134, 563. LIN, C. T., AND KUMAR, S. (1971) J. Biol. Chem., 246, 3284. NANDEDKAR, A. K. N., SCHIRMER, E. W., PYNADATH, T. I., AND KUMAR, S. (1969) Arch. B&hem. Biophys., 134, 554. SMITH, S., AND ABRAHAM, S. (1971) J. Biol. Chem., 246, 2537. Biophys., HUANG, K. I’., AND STUMPF, P. K. (1971) Arch. B&hem. 143, 412. WAKIL, S. J. (1961) J. Lipid. Res., 2, 1. GINGER, L. G. (1944) J. Biol. Chem., 156, 453. CAREY, E. M., AND DILS, R. (1970) Biochim. Biophus. Acta, 210, 388. !J~ILI.IAMSON, D. H., BATES, M. W., AND KREBS, H. A. (1968) Biothem. J., 108, 353. XANDEDKAR,
A Potent Reversible Inhibitor Carboxypeptidase A* (Received
October
18, 1971)
From the Department of Biochemistry, University 27514 Carolina, Chapel Hill, North Carolina
of North
L.
1).
BYERS
AND
for publication,
of
R.
WOLFERTDEN
SUMMARY Benzylsuccinic acid and closely related compounds are effective inhibitors of carboxypeptidase A. The “L” isomer of benzylsuccinic acid, Z(R)-benzyl-3carboxypropionic acid, appears to be bound by the enzyme several orders of magnitude more tightly than known substrates and inhibitors. This inhibitor may resemble the collected substrates for the reverse reaction, combining their individual binding characteristics in a single molecule. Reversible inhibitors are useful for probing the binding properties of enzymes and may also help in elucidating mechanisms of catalysis (1). Unusual potency is expected of inhibitors * This work was supported in part by Research Grants GM-12725 and GY-18325, a predoctoral fellowship (G&1-49094), and a research career development award (Ahf-08560) from the National Institutes of Health, United States Public Health Service.
Acids
Vol.
247, No.
2
bearing a structural resemblance to chemically activated intermediates in catalysis or incorporating the binding characteristics of several reactants (in a multisubstrate reaction) in a single inhibitor molecule. We wish to report an inhibitor of carboxypeptidase A, apparently of the latter type, which is considerably more tightly bound than any reversible inhibitor of this enzyme previously described. The present inhibitor, 2(R)-benzyl-3-carboxypropionic acid (I), was prepared by a procedure similar to that described by Cohen and Milovanovic (2). Racemic 2-benzyl-3-carboxypropionic acid was first converted to the dimethyl ester (b.p. 145-153” per 3.5 mm; literature 148-151” per 3 mm (3)). Stereoselective hydrolysis with chymotrypsin gave 2(n)-benzyl-3carbomethoxypropionic acid, which yielded I (m.p. 160-161”, [o(] $ = +26”) upon alkaline hydrolysis. 2(S)-Benzyl-3-carboxypropionic acid (m.p. 161-162”, [al]? = -26”) was obtained by alkaline hydrolysis of the chymotrypsin-resistant portion of the racemic dimethyl ester. The properties of both enantiomers were identical with those reported by Cohen and Nilovanovic (2). Other inhibitors were obtained as shown in the footnotes to Table I. The activity of carboxypeptidase A from beef pancreas (the crystalline Anson preparation, obtained from Sigma) was determined spectrophotometrically with the substrates hippurylL-phenylalanine and hippuryl-L-phenyllactate at 254 nm (7) and with the substrate carbobenzyloxyglycylglycyl-L-phenylalanine at 228 nm where this substrate gave Ae = -330 for hyTABLE Inhibitors
I
of Carborypeptidase
Aa
Inhibitor
L-Benzylsuccinic acid (I) n-Benzylsuccinic acid on-Benzylsuccinic acid 2(R)-Benzyl-3.carbomethoxypropionic ester of I) n-2-Methyl-3.phenylpropionic acidb 3.l’henylpropionic acid (II)” on-2-Benzylglutaric acidd Benzylmalonic acid” un-Phenylsuccinic acide Succinic acid” L-Phenylalanine” Hippuric scidf Carbobenxyloxyglycine Carbobenzyloxyglycylglycine~ Hippuryl+phenylalaninee (K,) Carbobenzyloxyglycyl-n-phenylalanineg Carbobenzyloxyglycylglycyl-n-phenylalanineg M?n) Hippuryl-L-phenyllactic acid (lcm)
Ki
acid
(half-
6 5 1.1 G
X x x x
;“,- h 10-e 10-S 10-G h
1.4 1.0 5 6 2 4 5 8 3
x x x X x x x X x
lo-3h 10-&h 10-G 1O-5 i 10-4 j 10-4 i 10” lO+h 10-Z h. f
>10-2
(Ii,)
h.
2 x 2 x 8 x
10-S 10-z 10-d
2 x
10-d
i
a Determined from double reciprocal plots using hippuryl-L-phenylalnnine as substrate in Tris-HCl buffer (0.025 M, pH 7.5, containing sufficient NaCl to maintain ionic strength 0.5) at 25’, unless otherwise noted. b Prepared by the action of methyl iodide on diethylbenzylmalonnte in sodium ethoxide, followed by hydrolysis, decarboxylation, and resolution by the method of Schrecker (4). c Obtained from Eastman Co. and recrystallized. d Prepared by the method of Ansell and Hey (5). e Obtained from Aldrich Co. and recrystallized. f Obtained from Sigma. 0 Obtained from Cycle Co. h Also tested using carbobenzyloxyglycylglycyl-L-phenylalanine as substrate, with similar results. i Ki estimated from the concentration of inhibitor required to produce 50% inhibition. j Value also obtained by Auld and Vallee (6).
Issue
of January
L. D. Byers and R. Wolfenclen
25, 1972
drolysis, in Tris-HCl buffer (0.025 M, pH 7.5, containing 0.5 M NaCl) at 25”. Racemic 2-benzylGcarboxypropionic acid was found to produce linear inhibition, apparently competitive, against all three substrates, with a constant K i value of 1.1 + 0.1 KM. The R enantiomer, I, was mainly responsible for the inhibition, with K; = 0.6 pM. The X enantiomer and the half-ester of the R enantiomer were an order of magnitude less effective, and other modifications resulted in extensive losses of inhibitory power (Table I). Typical results are shown in Figs. 1 and 2. Inhibition by I was reversed by dilution or dialysis, and no irreversible loss of activity was observed after 60-hours incubation of the enzyme with saturating concentrations of inhibitor. The enzyme was effectively protected by I against inactivation by the irreversible inhibitor 1-cyclohexyl-3-(Zmorpholinoethyl)carbodiimide metho-p-toluenesulfonate (8). Concentrated enzyme was treated with the inhibitor (0.016 M) in 2-(N-morpholino)ethanesulfonate buffer (0.05 M, pH 6.0, containing 1 M NaCl) at 4” (8), and aliquots removed at various time intervals were tested for remaining activity after dilution. After treatment of the enzyme (1.0 X 10m4 M) under these conditions, no detectable esterase or peptidase activity remained after 1 hour; however a slight excess of I (1.06 X 10-d M) afforded almost complete protection, reducing the rate of inactivation at 4” by at least two orders of magnitude. Ph Ph Ph Ph
-()(y&-H
-(y&b-II
-ooc-L-H
Am LO k Srtbstrate
HO-C=0
(1)
bH? I
AH2
Lx2
lm
AH2
-WC-h-11 iI
AIT2
L
607
I
I
I
I
1 2
I 4
1 6
I i-3
[s]-’
I IO
1 12
x
FIG. 1. Double reciprocal plot of the rate of the catalyzed hydrolysis of hippuryl-L-phenyllactic acid 0.025 M, pH 7.5, containing 0.5 M NaCl at 25’) as strate concentration in the absence (0) of inhibitor acid and in the presence of inhibitor at concentrations 2.7 PM (U). Total enzyme concentration is 1.6 X observed velocity (in molar per min) divided by the tion (in molar).
I
I
I I
I 2
carboxypeptidase(in Tris-HCI buffer, a function of subDL-benzylsuccinic of 1.3 PM (A) and 10-s M. k&s is the enzyme concentra-
HO-C=0 k Products
uu
The inhibitor I may be termed “n-benzylsuccinate” in view of It is its absolute stereochemical relationship to L-phenylalanine. bound by carboxypeptidase B 160 times more tightly than 3phenylpropionate (II), the most effective reversible inhibitor of carboxypeptidase A previously known (9), and 3000 times more tightly than the substrate carbobenzyloxyglycylglycyl-L-phenylalanine, for which K, appears very likely to be a true dissociation constant (6, 10). The unusual potency of I as an inhibitor is presumably associated with the presence of a second carboxylic acid group, which distinguishes this compound from substrates. The distance separating the two carboxylic acid groups is also important, as shown by the weaker binding of benzylglutaric and benzylmalonic acids (Table I). It does not appear likely that tight binding of I results from simple chelation of the zinc atom at the active site: the half-ester of I is only 5 times less effective than I (Table I), and succinic acid derivatives are not unusually effective as chelating agents for divalent cations (11). A more attractive explanation for the effectiveness of I is its apparent resemblance to the pair of substrates for the reverse reaction, i.e. the products of peptide hydrolysis. Exempted from the entropic requirement of gathering two ligands from solution, the enzyme exhibits an affinity for I which is comparable with its combined affinity for pairs of products such as hippuric acid and L-phenylalanine (Table I). This situation, analogous to the affinity of polyvalent cations for chelating agents, has been
[s]-’
I 3
x lo-3
FIG. 2. Double reciprocal plot of the rate of the catalyzed hydrolysis of carbobenzyloxyglycylglycyl-L-phenylalanine function of substrate concentration in the absence L-benzylsuccinic acid and in the presence of inhibitor Assay conditions of 0.52 PM (A) and 1.32 ELM (0). noted for Fig. 1 with an enzyme concentration of 5.6
carboxypeptidaseas a (0) of inhibitor at concentrations are the same X 1Om8 M.
as
discussed elsewhere (l), and a similar interpretation has been made of the effectiveness of a phosphonate inhibitor of aspartyl Inhibitors of this kind differ from transitranscarbamylase (12). tion state analogs which are believed to resemble chemically
Inhibition
60s
of Carboxypeptidase
A
Vol.
247,
No.
2
activated intermediates in catalysis. They may nevertheless provide useful mechanistic information by suggesting the spatial relationship between productively-bound substrates on the enzyme.’ REFERENCES 1. WOLFENDEN, R., (1972) AC&. Chem. Res. 6, 10. 2. COHEN, S., AND MILOVANOWC, A. (1968) J. Amer. Chem. SW., 90, 3495. 3. MATGUDA, S., YAMAUCHI, T., AND MORI, K. (1965) J. Chem. SW. (Japan), 58, 60. 4. SCHRECKER, A. W. (1951) J. Org. Chem. 22, 33. 5. ANSELL, M. F., AND HEY, D. H. (1950) J. Chem. Sot. (London), 1683. AULD, D. S., AND VALLEE, B. L. (1970) Biochemistry, 9, 602. ;: MCCLURE, W. O., NEURATR, H., AND WALSH, K. A. (1964) Biochemistrz/, 3, 1897. 8. RIORDAN, J. F., AND HAYASHIDA, H. (1970) B&hem. Biophys. Res. Commun. 41, 122. 9. ELKINS-KAUFMAN, E., AND NEURATH, H. (1949) J. Biol. Chem., 178, 645. B. L. (1970) Biochemistry, 9, 4352. 10. AULD, D. S., AND VALLEE, Il. MARTELL, A. E. (Editor) (1964) Stabilitzl constants, Chemical Societv,_ London, Special Publication.No. 17. 12. COLLINS, K. D., AND STARK, G. R. (1971) J. Biol. Chem., 246, 6599. 13. VALIZE, A. L. (1964) Fed, Proc., 32, 10. 14. HARTSUCK, J. A., AND LIPSCOMB, W. N. (1971) in P. D. BOYER (Editor), The Enzymes, Vol. 5, p. 1. Academic Press, New York. 15. KAISER, B. L., AND KAISER, E. T. (1969) Proc. Nat. Aead. Sci. U. S. A., 64, 36. i It has not been clearly established whether carboxypeptidase A acts through a single or a double displacement mechanism (13-15). The present inhibitor (as drawn) bears an apparent resemblance to products in the relative positions in which they might be found on the ensyme immediHowever, it is also ately following direct water attack on the substrate. possible that during a double displacement reaction (involving, for example, an acyl-enzyme intermediate) the bound products might fortuitously assume a configuration similar to that of bound n-benzylsuccinate.
X-ray Crystallographic Cobrotoxin”
Study
(Received CHI-HSIANG LEE
CHEN
for publication,
WONG,
From National China CHUNG
of
TSE WEN
Tsing
Hua
November
CHANG,
University,
1, 1971)
AND TSONG Hsinchu,
JEN
Taiwan,
YANG
From Kaohsiung China
Medical
College,
Kaohsiung,
Taiwan,
SUMMARY
Single crystals of the protein cobrotoxin have been grown to usable size for X-ray diffraction study. The space group determined is either P4121 or P4&. The cell dimensions determined are a = b = 40.40 A and c = 71.16 A. There are 8 molecules per unit cell. Cobrotoxin is the main neurotoxin extracted and purified from the venom of Formosan cobra, Nuja nuja &a (1, 2). It has a molecular weight of 6950 with 62 amino acid residues, and * This work lic of China.
was supported
by the National
Science Council, Repub-
FIG. 1.
its amino acid sequence has also been determined (3). Cobrotoxin is known to block the neuromuscular transmission at the end-plate (4). To work out its complete three-dimensional structure is of great importance in studying such biological functions. Single crystals of cobrotoxin were grown to the average size of 0.1 X 0.1 X 2 mm3 in the shape of a tetragonal prism (Fig. 1). The optimum conditions for growing such single crystals were The purified and lypohilized protein found to be the following. is dissolved in an aqueous solution of ammonium sulfate and ammonium chloride at pH 3.24. The solution was prepared by mixing 1 part (by volume) of saturated ammonium chloride solution with 2 parts of saturated ammonium sulfate solution and adjusting to the desired pH value by introducing 6 N HCl dropwise. The protein concentration was about 5 mg per ml. After the protein was dissolved, 2 ~1 of pyridine were added to each milliAfter the pyridine-treated protein liter of the protein solution. They solution had stood for a week, selected seeds were added. grow to the usable size for X-ray diffraction study in 6 to 8 months. Routine precession pictures have been taken with CuKar radiation on a Rigaku Denki rotating anode X-ray generator. Photographs of OkG show for Ok0 only k = 2n and for OOl, C = 4n are present, thus indicating the space group to be either P4121 or P4~2~. The cell dimensions determined from the photographs were: a = b = 40.40 Z!Z 0.06 A and c = 71.16 rt 0.14 A. The pictures show a resolution of better than 2 A. By comparing the volume of the unit cell with that of erabutoxin (5), we have deduced that there are 8 cobrotoxin molecules per unit cell. We are now undertaking the collection of its complete threedimensional data as well as those of its heavy atom derivatives on a Syntex Pi autodiffractometer. REFERENCES 1. YANG, C. C. (1964) J. Formosan Med. Ass. 63, 325. 2. YANG, C. C. (1965) J. B&Z. Chem., 240, 1616. 3. YANG, C. C., YANG, H. J., AND HUANG, J. S. (1969) Biochim. Biophys. Acta, 188, 65. 4. CHANG. C. C., AND LEE, C. Y. (1965) Brit. J. Pharmacol. Chemother., 28, 172. 5. Low, B. W., POTTER, R., JACKSON, R. B., TAMIYA, N., AND SATO, S. (1971) J. Bill. Chem., 246,4366.
