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The specificity of transglutaminase
ARCHIVES
OF
BIOCHEMISTRY
AND
The
BIOPHYSICS
Specificity
II. Structural JACK Department
of Biochemistry, Pharmacology,
Columbia New York
(1968)
of Transglutaminase
Requirements
H. PINCUS2
Received
126, 44-52
of the Amine HElNRICH
AND
WAELSCH3
University College of Physicians State Psychiatric Institute, New
December
11, 1967; accepted
Substrate’
January
and Surgeons, and Department York, New York 10032
of
22, 1968
The blocked tripeptide carbobenzoxy-L-alanyl-L-glutaminyl-L-valine-ethyl ester w&s used as an amine acceptor to determine the apparent K, values of some amines and related compounds in the transglutaminase reaction. A marked degree of specificity for the amine was observed; the two substrates with the lowest apparent K, of those test,ed were histamine and glycine ethyl ester. Larger homoglycine peptides did not inhibit histamine incorporation into the synthetic tripeptide as well as glycine ethyl es&-. The data presented here suggest that there are two sites on the enzyme, one that binds a glutamine residue within the protein substrate and a second that binds the amine.
In the previous paper (1) the factors affecting the reactivity of a protein substrate in the transglutaminase reaction were discussed.The second substrate involved in the enzyme reaction is an amine4 (2). The initial work on the amine specificity of the enzyme by Clarke e2 al. (3) showed that most compounds that possess a primary amino group could be incorporated into proteins, although to somewhat different degrees. However, each of these was tested at only one concentration and initial rates of amine incorporation were not determined. It, is therefore difficult to draw any definite conclusions concerning the relative reactivity ef the different compounds, nor can it be determined whether the enzyme possesses a binding site for the amine or whether
the amine competes with water in a nucleophilic displacement reaction. The present work describes the use of a single glutamine containing peptide, carbobenzoxy - L - alanyl - L - glutaminyl - L - valine ethyl ester, as the amine acceptor in order to determine the apparent K,,, values of several amines and thereby compare their relative reactivities in the transglutaminase reaction. The results indicate that histamine is the best biological substrate of all the compounds tested and that the enzyme possessestwo separate sites, one for a glutamine residue bound in peptide linkage and a second for an amine. MATERIALS Carbobenzoxy-L-alanine was synthesized according to the procedure of Bergmann and Zervas (4). Carbobenzoxy-L-glutamine was synthesized according to the procedure of Neidle (5). L-Valine ethy ester was synthesized according to the procedure of Vaughan and Eichler (6). Carbobenzoxy-L-glutaminyl-L-valine-ethyl eater was prepared according to the mixed enhydride procedure of Boissonas (7). Carbobenzoxy-Lglutamine, (10.3 g, 0.04 moles) was dissolved in a mixture of dimethyl foramide (10 ml) and tetrahy-
1 Taken from the doctoral dissertation of Jack H. Pincus, Columbia University (1966). 2 Present address: Biochemistry Laboratory, Cardiac Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. All inquiries should be sent to this address. 3 Deceased March 22, 1966. 4 The term “amine” as used in this paper operationally refers to any compound containing a primary amino group. 44
SPECIFICITY
OF
TRANSGLUTAMINASE.
drofuran (10 ml) that contained one equivalent of tributylamine. The solution was cooled at 0” and 1 equivalent of ethyl chloroformate was added. The reaction mixture was kept at 0” in an ice bath for 15 min. A solution of valine ethyl ester hydrochloride (7.2 g, 0.04 moles) in dimethylformamide (10 ml) and tetrahydrofuran (10 ml) containing 1 equivalent of tributylamine was then added slowly with stirring to the mixed anhydride solution. The reaction mixture was allowed to stand at 0” for 30 min a,nd then at room temperature for 2 hours. It was reduced to small volume in vacua, ethyl acetate added, and the solution washed with 2 N hydrochloric acid, water, 5yo sodium carbonate, and water, in that order. After drying over anhydrous sodium sulfate the ethyl acetate was removed in vacua and the residue recrystallized from 50% aqueous ethanol. Yield Calculated
8 g (50%); melting for CzoNzsNaOs: C, 58.95; H, 7.17;
point
184-187”
N, 10.31
C,
N, 10.31
Found6: 58.99;
H,
7.15;
Carbobenzoxy-L-alanyl-L-glutaminyl-L-valineethyl ester was prepared from carbobenzoxyl-r,alanine and L-glutaminyl-L-valine-ethyl ester by the mixed anhydride procedure of Boissonas (7). Carbobeneoxy-L-glutaminyl-L-valine-ethyl ester (0.13 moles, 7.8 g) was suspended in 50 ml of ethanol and 1 equivalent of hydrochloric acid was added. Palladium oxide (400 mg) was suspended in the mixture and hydrogen gas bubbled through until carbon dioxide evolution had ceased, as determined by passing the effluent gases through barium hydroxide. After the catalyst had been removed by filtration the solution was evaporated in vacua, resulting in an oil that was coupled to carbobenaoxy-L-alanine. Carbobenzoxy-L-alanine (0.019 moles, 4.3 g) was dissolved in a mixture of tetrahydrofuran (10 ml) and dimethylformamide (10 ml) containing 1 equivalent of the tributylamine. The solution was cooled to O’, 0.019 moles of ethyl chloroformate added, and the reaction mixture was allowed to stand at 0” for 15 min. L-Glutaminyl-L-valineethyl ester in a. mixture of tetrahydrofuran (10 ml) and dimethylformamide (10 ml) containing 1 equivalent of tributylamine was added to the mixed anhydride slowly with stirring. The reaction mixture was allowed to stand at 0” for 30 min and then at room temperature for 2 hours. It was then reduced to a small volume in vacua and taken up 5 Carbon and hydrogen analyses formed by Schwarzkopf Microanalytical ratory, Woodside, New York. Nitrogen were performed by Mr. T. Zelmenis.
were
perLaboanalyses
II.
45
in ethyl acetate. The ethyl acetate was with 2 N hydrochloric acid, water, 5% carbonate and water, in that order. After over anhydrous sodium sulfate the ethyl was removed in vacua and the resulting recrystallized from 50% aqueous ethanol. Yield 7.0 g (81)‘%; melting Calculated for C23H34N407: C, 57.72; H, 7.16; Found : C, 57.64; H, 6.95;
point
washed sodium drying acetate residue
213-215”
N,
11.71
N,
11.73
Enzyme preparation. The preparation of transglutaminase is that which has been previously described (1). Glycine-l-l% ethyl ester hydrochloride, histamine-l%, methylamine-“C hydrochloride, ethanolamine-1,2-l% hydrochloride, tyramine-l-l% hydrobromide, e-amino-l-14C caproic acid, yamino-l-14C butyric acid, /3-1-14C alanine, taurine1 ,2J4C, and tryptamine-2J4C bisuccinate were obtained from New England Nuclear Corporation, Boston, Massachusetts. These were diluted with unlabeled amines (obtained from Nutritional Biochemicals, Inc., Cleveland, Ohio) to give a final specific activity of 0.05 pC! rmole. METHODS
Apparent
K, Determinations
Carbobenzoxy - L - alanyl- L - glutaminyl -Lvaline ethyl ester was freshly prepared as a 10 % solution in 60% aqueous ethanol at the time of its use. In all apparent K, determinations performed, the standard set of reaction components consisted of 80 pmoles of Tris buffer, pH 7.8; 40 bmoles of calcium chloride; 20 pmoles of glutathione; and 200 units of transglutaminase (0.051 mg protein). The following specific additions to this standard set of reaction components were used: For carbobenzoxy - L - glutaminyl - L -valin.eethyl ester. Either 80 pmoles of methylamine14C or 100 Fmoles of hydroxylamine (freshly prepared by neutralizing hydroxylamine hydrochloride with sodium hydroxide), and amounts of this tripeptide as shown by the points in the figures. For the amines. 10.3 kmoles of carbobenzoxy - L - alanyl - L - glutaminyl - L - valine ethyl ester (0.5 ml of a 10% solution of the tripeptide in 60 % ethanol) and amounts of radioactive amine as indicated in the figures. The final volume in all cases was 2 ml and
40
PINCUS
AND
contained sufficient ethanol to give a final ethanol concentration of 15%. The reaction was run for 3 min at 37” except when histamine and glycinc ethyl ester were used. When these compounds wcrc tested, the reaction was run for 1 minute at 37”. Zero time blanks were employed for each amine concentrat.ion and each determination was performed in duplicate. In all experiments, lessthan 10% of either substrate (tripeptide or amine) reacted during the incubat.ion. The incorporation of amines into the synthetic tripeptide was determined by methods described below. The apparent, K, values were calculated from Lineweaver-Rurk plots (8). Determindion
WAELSCH
scintillation counter (Model 314EX). An aliquot of the radioactive amine used in each experiment was used as a standard. Preliminary experiments indicated t.hat quantitative recovery of the modified tripeptide was achieved using t.he assay method as described above. Inhibition Studies The ability of various compounds to inhibit the initial rate of histamine incorporation into carbobenzoxy - L - alanyl - L - glutaminyl-L-valinc ethyl ester was tested as follows: A reaction mixture containing
of the Incorporated Amine
Hydroxylamine Incorporation was determined by the N-Z amine assay of Waelsch and Mycek (9) using carbobenzoxy-Lnlanyl-L-glut.aminyl-r>-valine ethyl ester as the amine acceptor and r-glutamyl hydroxan& acid as a standard. Details of t.he procedure are given in the legendsto the figures. The assumption is made that t.hc color equivalents of the tripeptide hydroxamate and -y-glut.amyl hydroxamate are the same. Radioactive Amine Incorporation was determined as follows. At t,he end of the incubation the reaction was stopped by the addition of 0.1 ml of 2 N hydrochloric acid. Two ml of ethyl acetate were added and the entire cont.ents of the tube stirred with a vibrating mixer, for 30 seconds. The emulsion that, formed was separated by centrifugation in a desk-top centrifuge for 5 min. The organic layer was carefully pipetted off and washed with 2 ml of 0.1 N HCl. When histamine was tested the reaction was stopped by directly adding 2 ml of ethyl acet,ate and immediately mixing t.he entire contents of the tube as described above. The organic layer was then washed with 2 ml of distilled water instead of 0.1 K hydrochloric acid. In all c:1scs0.5 ml of the ethyl acetate Inyer that contained the radioactive peptide was placed in a polyethylene scintillation vial with 10, ml of naphthalenc-dioxane scintillation mixt,ure (10). The radioactivity was determined in a Packard Tri-Carb
FIG. 1. Incorporation of hydroxylamine into carbobenzoxy - L - alanyl - L - glutaminyl - I, - valine ethyl ester as a function of time. The react,ion mixture contained SO rmoles of Tris buffer pH 7.8, 40 rmoles of calcium chloride, 20 rmoles of glutathione, 100 rmoles of hydroxylamine, 5.15 pmoles of carbobenzoxy-I.-alanyl-I,-glutaminylL-valine-ethyl ester (0.5 ml of a 1% solution in 60% ethanol), and 200 units of transglutaminase. The final volume was 2 ml. Each point on the curve represonti a separate experiment. At the times indicated on the curve the reaction was stopped by the addition of 1.5 ml of ferric: chlorideTCA (21). After centrifugation, the optical density at 525 * was determined using a Coleman Junior Speotrophotometer.
SPECIFICITY
OF TRASSGLUTAMINASE.
II.
47
Fro. 2. Apparent K, determination of carbobenzoxy-L-alanyl-L-glutaminyl-L-valine ethyl ester using methylamine and hydroxylamine as the replacing amines. For details see Methods. S = molar tripeptide concentration, \: = moles of amine incorporated per liter per minute using 100 units of enzyme per ml. 0, hydroxylamine; 0, methylamine. The apparent K, of the tripeptide is 7.i X IOWJu. The V,,, with met.hylamine is 11.5 X IO-” moles of amine incorporated per lit.er per minute. With hydroxylamine the V, is 23.6 X 10-S moles of amine incorporated per liter per minute.
calcium chloride; 20 pmoles of glutathione; 0.4 ctmoles of histamincJ4C, 200 units of transglutaminase (0.054 mg protein); 10.3 pmoles of carbobenzoxy-r,-alanyl-L-glutaminyl-L-valine et,hyl est.er (0.5 ml of a 10% solution in 60% ethanol) and amounts of compounds t.o be tested for inhibition as indicated by t.he points in t,he figures. The final volume was 2 ml. nftcr a 1-min incubation at. 37” the reaction was stopped and assayed as described above for histamine.
thereby altering t,he reaction kinetics in each instance. The apparent K,,, of the t,ripcptide was therefore determined using hydroxylamine and methylamine as the amine donors. The results of these experiments arc shown in Fig. 2, although t,he V’, in bot.h instances was different, the apparent K, of 7.7. X 10m9M for the tripcpt.ide substrate was the same. This is in agreement with the results of Folk and Cole (11) who found that t hc K, values for carbobenzoxy-L-glutaminyl glytine, carbobenzoxy-L-glutaminyl glycine ILESULTS ethyl ester and benzoyl-L-glut,ammyl glycine are the same in both amine-incorporation Figure 1 shows the course of hydroxylamine incorporation into carbobenzoxy-Lreaction and amide hydrolysis in the abalanyl-L-glutaminyl-L-valine ethyl ester as a senceof amine. The resu1t.sof experiments in which the function of time. The amine incorporation increases linearly for about 3 min and then apparent K, values of amines I\-ere deterdeviates from linearity. All incubations were mined are shown in Figs. 3 and d and summarized in Table I, together with the pK thcreforc performed for 3 min or Icss. Before atkmpting a determination of t.he values of some of the compounds. Since the apparent K, values of aminesit was essential I’,,, values of all the compounds arc in good to determine nhet,her different amines af- agreement, the apparent K, offers :I means fected the K, of the tripeptide subst.rate,6 of comparison. Of the compounds tested, the two best substrates appear to be glycine 6 Carbobenzoxy- L - alanyl -L - glutaminyl - I, -valine-ethyl ester. ethyl ester and histamine. Tyraminc and
4s
PINCUS
AND
WAELSCH
FIG. 3. Apparent K, determination of glycine ethyl ester, and histamine. For details see Methods. S = molar amine concentration, V = moles of amine incorporated per liter per minute using 100 units of enzyme per ml. 0, glycine ethyl ester; 0, histamine.
m6050s&40I>
FIG. 4. Apparent K, determination of c-amino caproic acid, tyramine, andethanolamine. see Methods. S = molar amine concentration, V = moles of amine incorporated per liter rising 100 units of enzyme per ml. 0, e-amino caproic acid; 0, tyramine; A, ethanolamine. tryptamine have apparent Km values that are greater than 100 fold higher than glycine ethyl ester or histamine. Compounds with a carboxyl group that have fewer than 5
For details per minute
methylene groups between the carboxyl and amino group do not participate in the transglutaminase reaction. (E-Amino caproic acid in which the carboxyl end is separated from
SPECIFICITY TABLE
OF TRANSGLUTAMINASE.
Compound
Km (maa)
pK of amino group
Reference for PK
Glycine ethyl ester Histamine Methylamine Ethanolamine Tyramine C-Amino caporic acid y-Amino butyric acid p-Alanine Taurine Tryptamineb Hydroxylamine”
0.186 0.178 1.42 8.6 23.8 50.0
7.73 9.8 10.54 9.44 10.75
(24) (25) (23) (23) -
-
10.6
(25)
-
10.2 9.1
(25) (25) -
a Values obtained b Value too high c Value too low
from plots to measure. to measure.
in Figs.
49
r
I
APPARENT K,,, VALUES OF ANNEX AND RELATED COMPOUNDS~
5.96
II.
(24)
(25) 3 and 4.
the amino end by 5 methylene groups has a very high apparent K,,,.) It may be, however, that any negatively charged group in close pro:ximity to the amino group is detrimental as seen from the fact that taurine is not a substrate. It can finally be seen that a relationship between the apparent K, of a compound and its pK is lacking. Several amines were not available in radioactive form and could therefore not be tested directly. These were therefore tested for their ability to inhibit histamine incorporation into t’he tripeptide substrate. Figures 5 and 6 illustrate the results of these experiments. Glycine ethyl ester is included here for comparative purposes. Taurine and y-amino butyric acid, compounds which are not substrates, as determined by the direct assay, are also not inhibitors. Thus, only compounds that are competitive substrates will inhibit histamine incorporation. The order of inhibition of the other compounds is hydroxylamine > putrescine > ammonium chloride > glycine ethyl ester > alanine ethyl ester > leucine ethyl ester. Glycine ethyl ester was of particular interest as a substrate, since it has been shown that it is an inhibitor of fibrin crosslinking in the thrombin-activated fibrinstabilizing factor system of Lorand (12).
INHIBITOR
CONCENTRATION (MxlO’)
FIG. 5. Effect of glycine ethyl ester, hydroxylamine, putrescine, and ammonium chloride on the initial rate of histamine incorporation into carbobenzoxy -L -alanyl -L - glutaminyl - L - valine ethyl ester. For details see Methods. The pK values of the compounds are: hydroxylamine 5.96 (25), ammonium chloride 9.2 (23). O---O, glycine ethyl ester; l - - - - -0, hydroxylamine; 0- - - - -0, putrescine; 0-0 ammonium chloride.
The inhibition takes place via the incorporation of this compound into fibrin in a manner similar to that which transglutaminase incorporates amines into proteins (13). Glycyl-glycine ethyl ester and several homo glycine peptides have also been shown to be good inhibitors of fibrin cross-linking (14). Thus, experiments were designed to see whether the specificity of transglutaminase and fibrin-stabilizing factor were the same toward these compounds. Figures 5 and 7 illustrate the results of experiments in which glycine ethyl ester and other homoglycine peptides were tested by the indirect assay described above. At a glycine ethyl ester concentration of 2 X low4 M, the initial rate of histamine incorporation decreasedto 79 % of the uninhibited value, while a 50% decrease was observed at a concentration of 8 X lop4 M. Significant inhibition with the larger peptides was only
PINCUS
AND WAELSCH
I a IN2”lBITOR4CONCENbN (MxlO~) FIG. 6. Effect of alanine ethyl ester, r-amino butyric acid, taurine and leucine ethyl ester on the initial rate of histamine incorporation into carbobenzoxy - L - alanyl -L - glutaminyl - L-valine ethyl ester. For details see Methods. The pK values of the amino groups of the compounds are: lysine 10.53 (24), r-amino butyric acid 10.6 (25), taurine 9.1 (25), leucine ethyl ester 7.63 (24), alanine ethyl ester 7.8 (24). A, alanine ethyl ester; 0, taurine; 0, r-amino butyric acid; A, leucine ethyl ester.
observed at concentrations of the order of 1OP M. The potency of these compounds as the inhibitors is a function of the distance between the amino and carboxyl group (tetraglycine > triglycine > diglycine). However, glycyl-glycine ethyl ester, which does not have a free carboxyl group, is only slightly better than triglycine. Thus, none of the longer glycine peptides tested inhibited as well as glycine ethyl ester. DISCUSSION
It is only through the use of a substrate with a single glutamine residue, such as the tripeptide used here as the amine acceptor, that meaningful initial rates of amine incorporation can be determined. The apparent K,,, values of the different amines can then be determined and these values used as a basis for comparison of the different com-
2
INHIBITOR
I 4
I
I
6
a
I
CONCENTRATION WlO’)
FIG. 7. Effect of glycyl-glycine, glycyl- glycine ethyl ester, triglycine, and tetraglycine on the initial rate of histamine incorporation into carbobenzoxy - L - alanyl -L - glutaminyl - L - valine ethyl ester. For details see Methods. The pK values of the amino groups of the compounds are: glycylglycine 8.13 (24), glycyl-glycine ethyl ester 7.75 (24) ,, triglycine 7.91 (25)) tetraglycine 7.75 (25).
pounds. The tripeptide carbobenxoxy-nalanyl-n-glutaminyl-n-valine ethyl ester was thus employed as the amine acceptor since its sequence is the same as the amino acid sequence around the single glutamine residue in the CYchain of hemoglobin, which was shown to be a substrate in the previous paper. It is necessary to block it at the amino and carboxyl ends since free di- and tripeptides are not substrates for transglutaminase (15). Enzyme reactions involving two substrates exhibit complex kinetic behavior. A detailed discussionof this is given by Dixon and Webb (16). Since the details of the mechanism of action of transglutaminase have not been worked out, the correct rate equation cannot be written and the exact meaning of Km is unclear. However, the Lineweaver-Burk plots in all the amine K,,, determinations intersect the l/V axis at approximately the same point. The values for Km determined from these plots can then
SPECIFICITY
OF TRANSGLUTAMINASE.
be used to compare amine reactivity if K,,, is given the classical operational definition of “the substrate concentration at which the reaction velocity is one-half the maximum velocity.” The Km values determined in this work are therefore referred to sts apparent K,,, values and do not have an absolute meaning but only a comparative meaning in relation to each other. When insulin was used as a substrate for transglutaminase it was observed that ammonia was enzymatically releasedin the absence of an amine (17). This same behavior has been observed by Folk and Cole using synthetic blocked glutamine depeptides (11) and in the caseof many proteolytic enzymes that catalyze both hydrolytic and transpeptidation reactions (18). These observations raise the question of whether the biological role of transglutaminase is one of hydrolysis or transamidation. If the mechanism of action of transglutaminase is such that the second substrate (water or an amine) reacts directly with the enzyme sub&ate complex, rather than first binding to the enzyme, then the transamidation reaction would represent a successful competition of an amine over water for the enzyme substrate complex when an amine is present in the reaction mixture. If this were the case, then the amount of ammonia released would be greater than the amount of amine incorporated since hydrolysis and transpeptidation could occur simultaneously as seen in the case of papain (19). However, using carbobenzoxy L-glutaminyl glycine and ethanolamine as substrates for transglutaminase, Folk and Cole (11) have shown that all of the ammonia released can be accounted for by transamidation. The data presented here also indicate that in the presence of amine a hydrolytic mechanism is not operating. The apparent Km values indicate that the enzyme showed a marked degree of specificity for the amine substrate. There is also a lack of correlation between the apparent Km and the pK values of the amines (seeTable I), and, compounds with similar pK values do not inhibit histamine incorporation into carbobenzoxy-L-alanylL-glutaminyl-.L-valine ethyl ester equally well. (Compare the inhibition curves for glycine ethyl ester, alanine ethyl ester, leu-
II.
51
tine ethyl ester, glycyl-glycine, triglycine, and tetraglycine.) If a mechanism were operating in which the amine directly attacked the enzyme substrate complex, without being bound to the enzyme, the reactivity of the amine would depend only upon the quantity of the unprotonated form present and therefore be proportional to its pK. Thus, the second substrate (amine) appears to be bound rather specifically to the enzyme and its biological role appears to be transamidation and not hydrolysis. From the data presented in this paper it would seemthat there are a minimum of two points of attachment for the amine relatively close to each other. One of these is for the primary amino group while the second for a free pair of electrons on the side chain. Thus, small compounds, such as hydroxylamine and ammonium chloride, are probably bound at the primary amino group site without interfering with the second site and are therefore good inhibitors of histamine incorporation. Of all the compounds with side chains that were tested, histamine and glycine ethyl ester appear to be the best substrates while putrescine inhibits histamine incorporation only slightly better than glycine ester. The feature common to all of these is the free pair of electrons on the imidazole nitrogen of histamine, the second amino nitrogen of putrescine, and the ester oxygen of glycine ethyl ester. The other compounds tested have higher apparent K,,, values or are poor inhibitors of histamine incorporation. This is probably due to the fact that they have a side chain that does does not fit well at the second point of attachment or is too large to fit the amine site, or that they possessa negative charge. The low apparent K, values of histamine and glycine ethyl ester are of interest since both of these are good inhibitors of fibrin polymerization by the enzyme thrombinactivated stabilizing factor (12). However, in the case of transglutaminase, glycine peptides and glycyl-glycine ethyl ester bind much more poorly than glycine ethyl ester (based on their inhibition of histamine incorporation). Although the underlying mechanism of both enzymes might be the same, the biological function of transglutaminase is probably not the cross-linking of proteins in
52
PINCUS
AND
the same manner as fibrin-stabilizing factor. The low apparent K,,, of histamine ties in with results obtained by Ginsburg et al. (20). These workers have demonstrated that the level of liver transglutaminase in mice increased between three and four fold when the animals are injected with bacterial endotoxins. They have also found that there is a small but significant incorporation of histamine into liver protein under these conditions. It is known that bacterial endotoxins increase the sensitivity of animals to histamine (21) and cause the histamine content of the liver to rise (22). Since transglutaminase shows a preference for histamine, the increase in enzyme activity when an animal receives bacterial endotoxins appears to be important. Although the exact role of the enzyme under these conditions is as yet uncertain, two possibilities are immediately apparent. The incorporatioil of histamine into a protein may be necessary for it to exert its biological effect, or, the amine incorporation into proteins may serve as a detoxification mechanism. Experiments to elucidate the biological role of transglutaminase are currently in progress in this laboratory. ACKNOWLEDGMENT This work was supported in part by grants from the National Institute of Neurological Diseases and Blindness, Public Health Service Research Grant NB 00226, and from the Supreme Council, 33” Scottish Rite Masons of the Northern Jurisdiction, United States of America. We wish to thank Dr. Amos Neidle for his help and advice concerning peptide synthesis and for his helpful suggestions during the preparation of this manuscript. REFERENCES 1. PINCUS, J. H., AND WAELSCH, H., Arch. Biothem. Biophys., 126, 34 (1968). 2. SARKAR, N. K., CLARKE, D. D., AND WAELSCH, H., Biochem. Biophys. Acta, 26, 451 (1957). 3. CLARKE, D. D., MYCEK, M. J., NEIDLE, A., WAELSCH, H., Arch. Biochem. Biophys., 79, 338 (1959).
WAELSCH 4. BERGMANN, F., AND ZERVAS, L., Chem. Ber.. 66, 1192 (1932). 5. NIUDLE, A., manuscript in preparation. 6. VAUGHAN, J. R., AND EICHLER, J. A., J. Am. Chem. Sot., 76,5536 (1953). 7. BOISSONAS, R. A., Helu. Chim. Acta, 34, 874 (1951). 8. LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66, 658 (1934). 9. WAF:LSCH, H., AND MYCEK, M. J., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. V, p. 833. Academic Press, New York (1962). 10. BRAY, G. A., Anal. Biochem., 1, 279 (1960). 11. FOLK, J. E., AND COLE, P. W., J. Biol. Chem., 240, 2951 (1965). 12. LORAND, L., AND JACOBSEN, A., Biochemistry. 3, 1939 (1964). 13. LORAND, L., Federation Proc., 24,784 (1965). 14. LORAND, L., DOOLITTLE, R. F., KONISHI, K., AND RIGGS, S. K., Arch. Biochem. Biophys., 102, 171 (1963). 15. NEIDLF:, A., AND Acs, G., Federation Proc., 20, 234 (1961). 16. DIXON, M., AND WEBB, E. C., in “The Enzymes” 2nd Edit., pp. 70-75; 100-102. ilcademic Press, New York (1964). 17. MYCEIC, M. J., CLARKE, D. D., NEIDLE, A., ANU WAF,LSCH, H., Arch. Biochem. Biophys., 84, 528 (1959). 18. FRUTON, J. S., Harvey Lectures, 61, 64 (1957). 19. JOHNSTON, R. J., MYCEK, M. J., AND FRUTON, J. S., J. Biol. Chem., 186, 629 (1950). 20. GINSBURG, M., WAJDA, I., AND WAELSCH, H., Biochem. Phamnacol., 12, 251 (1963). 21. MALKIEL, S., AND HARGIS, B. J., Proc. Sot. Exptl. Biol. Med., 30, 122 (1952). 22. KIM, E., Proc. Sot. Exptl. Biol. Med. 66, 197 (1947). 23. HODGMAN, C. D., WEAST, R. C., SHANKLAND, R. I)., AND SELBY, S. M., Eds., “Handbook of Chemistry and Physics” 44th Ed., pp. 1749-1751. Chem. Rubber Publ. Co., Cleveland, Ohio (1952). 24. COHN, E. J., AND EDSALL, J. T., “Proteins, Amino Acids and Peptides as Ions and Dipolar Ions,” pp. 680683. Reinhold, New York (1943). 25. LONG, C., “Biochemists Handbook,” pp. 43-52. Spon, London (1961).
OF
BIOCHEMISTRY
AND
The
BIOPHYSICS
Specificity
II. Structural JACK Department
of Biochemistry, Pharmacology,
Columbia New York
(1968)
of Transglutaminase
Requirements
H. PINCUS2
Received
126, 44-52
of the Amine HElNRICH
AND
WAELSCH3
University College of Physicians State Psychiatric Institute, New
December
11, 1967; accepted
Substrate’
January
and Surgeons, and Department York, New York 10032
of
22, 1968
The blocked tripeptide carbobenzoxy-L-alanyl-L-glutaminyl-L-valine-ethyl ester w&s used as an amine acceptor to determine the apparent K, values of some amines and related compounds in the transglutaminase reaction. A marked degree of specificity for the amine was observed; the two substrates with the lowest apparent K, of those test,ed were histamine and glycine ethyl ester. Larger homoglycine peptides did not inhibit histamine incorporation into the synthetic tripeptide as well as glycine ethyl es&-. The data presented here suggest that there are two sites on the enzyme, one that binds a glutamine residue within the protein substrate and a second that binds the amine.
In the previous paper (1) the factors affecting the reactivity of a protein substrate in the transglutaminase reaction were discussed.The second substrate involved in the enzyme reaction is an amine4 (2). The initial work on the amine specificity of the enzyme by Clarke e2 al. (3) showed that most compounds that possess a primary amino group could be incorporated into proteins, although to somewhat different degrees. However, each of these was tested at only one concentration and initial rates of amine incorporation were not determined. It, is therefore difficult to draw any definite conclusions concerning the relative reactivity ef the different compounds, nor can it be determined whether the enzyme possesses a binding site for the amine or whether
the amine competes with water in a nucleophilic displacement reaction. The present work describes the use of a single glutamine containing peptide, carbobenzoxy - L - alanyl - L - glutaminyl - L - valine ethyl ester, as the amine acceptor in order to determine the apparent K,,, values of several amines and thereby compare their relative reactivities in the transglutaminase reaction. The results indicate that histamine is the best biological substrate of all the compounds tested and that the enzyme possessestwo separate sites, one for a glutamine residue bound in peptide linkage and a second for an amine. MATERIALS Carbobenzoxy-L-alanine was synthesized according to the procedure of Bergmann and Zervas (4). Carbobenzoxy-L-glutamine was synthesized according to the procedure of Neidle (5). L-Valine ethy ester was synthesized according to the procedure of Vaughan and Eichler (6). Carbobenzoxy-L-glutaminyl-L-valine-ethyl eater was prepared according to the mixed enhydride procedure of Boissonas (7). Carbobenzoxy-Lglutamine, (10.3 g, 0.04 moles) was dissolved in a mixture of dimethyl foramide (10 ml) and tetrahy-
1 Taken from the doctoral dissertation of Jack H. Pincus, Columbia University (1966). 2 Present address: Biochemistry Laboratory, Cardiac Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. All inquiries should be sent to this address. 3 Deceased March 22, 1966. 4 The term “amine” as used in this paper operationally refers to any compound containing a primary amino group. 44
SPECIFICITY
OF
TRANSGLUTAMINASE.
drofuran (10 ml) that contained one equivalent of tributylamine. The solution was cooled at 0” and 1 equivalent of ethyl chloroformate was added. The reaction mixture was kept at 0” in an ice bath for 15 min. A solution of valine ethyl ester hydrochloride (7.2 g, 0.04 moles) in dimethylformamide (10 ml) and tetrahydrofuran (10 ml) containing 1 equivalent of tributylamine was then added slowly with stirring to the mixed anhydride solution. The reaction mixture was allowed to stand at 0” for 30 min a,nd then at room temperature for 2 hours. It was reduced to small volume in vacua, ethyl acetate added, and the solution washed with 2 N hydrochloric acid, water, 5yo sodium carbonate, and water, in that order. After drying over anhydrous sodium sulfate the ethyl acetate was removed in vacua and the residue recrystallized from 50% aqueous ethanol. Yield Calculated
8 g (50%); melting for CzoNzsNaOs: C, 58.95; H, 7.17;
point
184-187”
N, 10.31
C,
N, 10.31
Found6: 58.99;
H,
7.15;
Carbobenzoxy-L-alanyl-L-glutaminyl-L-valineethyl ester was prepared from carbobenzoxyl-r,alanine and L-glutaminyl-L-valine-ethyl ester by the mixed anhydride procedure of Boissonas (7). Carbobeneoxy-L-glutaminyl-L-valine-ethyl ester (0.13 moles, 7.8 g) was suspended in 50 ml of ethanol and 1 equivalent of hydrochloric acid was added. Palladium oxide (400 mg) was suspended in the mixture and hydrogen gas bubbled through until carbon dioxide evolution had ceased, as determined by passing the effluent gases through barium hydroxide. After the catalyst had been removed by filtration the solution was evaporated in vacua, resulting in an oil that was coupled to carbobenaoxy-L-alanine. Carbobenzoxy-L-alanine (0.019 moles, 4.3 g) was dissolved in a mixture of tetrahydrofuran (10 ml) and dimethylformamide (10 ml) containing 1 equivalent of the tributylamine. The solution was cooled to O’, 0.019 moles of ethyl chloroformate added, and the reaction mixture was allowed to stand at 0” for 15 min. L-Glutaminyl-L-valineethyl ester in a. mixture of tetrahydrofuran (10 ml) and dimethylformamide (10 ml) containing 1 equivalent of tributylamine was added to the mixed anhydride slowly with stirring. The reaction mixture was allowed to stand at 0” for 30 min and then at room temperature for 2 hours. It was then reduced to a small volume in vacua and taken up 5 Carbon and hydrogen analyses formed by Schwarzkopf Microanalytical ratory, Woodside, New York. Nitrogen were performed by Mr. T. Zelmenis.
were
perLaboanalyses
II.
45
in ethyl acetate. The ethyl acetate was with 2 N hydrochloric acid, water, 5% carbonate and water, in that order. After over anhydrous sodium sulfate the ethyl was removed in vacua and the resulting recrystallized from 50% aqueous ethanol. Yield 7.0 g (81)‘%; melting Calculated for C23H34N407: C, 57.72; H, 7.16; Found : C, 57.64; H, 6.95;
point
washed sodium drying acetate residue
213-215”
N,
11.71
N,
11.73
Enzyme preparation. The preparation of transglutaminase is that which has been previously described (1). Glycine-l-l% ethyl ester hydrochloride, histamine-l%, methylamine-“C hydrochloride, ethanolamine-1,2-l% hydrochloride, tyramine-l-l% hydrobromide, e-amino-l-14C caproic acid, yamino-l-14C butyric acid, /3-1-14C alanine, taurine1 ,2J4C, and tryptamine-2J4C bisuccinate were obtained from New England Nuclear Corporation, Boston, Massachusetts. These were diluted with unlabeled amines (obtained from Nutritional Biochemicals, Inc., Cleveland, Ohio) to give a final specific activity of 0.05 pC! rmole. METHODS
Apparent
K, Determinations
Carbobenzoxy - L - alanyl- L - glutaminyl -Lvaline ethyl ester was freshly prepared as a 10 % solution in 60% aqueous ethanol at the time of its use. In all apparent K, determinations performed, the standard set of reaction components consisted of 80 pmoles of Tris buffer, pH 7.8; 40 bmoles of calcium chloride; 20 pmoles of glutathione; and 200 units of transglutaminase (0.051 mg protein). The following specific additions to this standard set of reaction components were used: For carbobenzoxy - L - glutaminyl - L -valin.eethyl ester. Either 80 pmoles of methylamine14C or 100 Fmoles of hydroxylamine (freshly prepared by neutralizing hydroxylamine hydrochloride with sodium hydroxide), and amounts of this tripeptide as shown by the points in the figures. For the amines. 10.3 kmoles of carbobenzoxy - L - alanyl - L - glutaminyl - L - valine ethyl ester (0.5 ml of a 10% solution of the tripeptide in 60 % ethanol) and amounts of radioactive amine as indicated in the figures. The final volume in all cases was 2 ml and
40
PINCUS
AND
contained sufficient ethanol to give a final ethanol concentration of 15%. The reaction was run for 3 min at 37” except when histamine and glycinc ethyl ester were used. When these compounds wcrc tested, the reaction was run for 1 minute at 37”. Zero time blanks were employed for each amine concentrat.ion and each determination was performed in duplicate. In all experiments, lessthan 10% of either substrate (tripeptide or amine) reacted during the incubat.ion. The incorporation of amines into the synthetic tripeptide was determined by methods described below. The apparent, K, values were calculated from Lineweaver-Rurk plots (8). Determindion
WAELSCH
scintillation counter (Model 314EX). An aliquot of the radioactive amine used in each experiment was used as a standard. Preliminary experiments indicated t.hat quantitative recovery of the modified tripeptide was achieved using t.he assay method as described above. Inhibition Studies The ability of various compounds to inhibit the initial rate of histamine incorporation into carbobenzoxy - L - alanyl - L - glutaminyl-L-valinc ethyl ester was tested as follows: A reaction mixture containing
of the Incorporated Amine
Hydroxylamine Incorporation was determined by the N-Z amine assay of Waelsch and Mycek (9) using carbobenzoxy-Lnlanyl-L-glut.aminyl-r>-valine ethyl ester as the amine acceptor and r-glutamyl hydroxan& acid as a standard. Details of t.he procedure are given in the legendsto the figures. The assumption is made that t.hc color equivalents of the tripeptide hydroxamate and -y-glut.amyl hydroxamate are the same. Radioactive Amine Incorporation was determined as follows. At t,he end of the incubation the reaction was stopped by the addition of 0.1 ml of 2 N hydrochloric acid. Two ml of ethyl acetate were added and the entire cont.ents of the tube stirred with a vibrating mixer, for 30 seconds. The emulsion that, formed was separated by centrifugation in a desk-top centrifuge for 5 min. The organic layer was carefully pipetted off and washed with 2 ml of 0.1 N HCl. When histamine was tested the reaction was stopped by directly adding 2 ml of ethyl acet,ate and immediately mixing t.he entire contents of the tube as described above. The organic layer was then washed with 2 ml of distilled water instead of 0.1 K hydrochloric acid. In all c:1scs0.5 ml of the ethyl acetate Inyer that contained the radioactive peptide was placed in a polyethylene scintillation vial with 10, ml of naphthalenc-dioxane scintillation mixt,ure (10). The radioactivity was determined in a Packard Tri-Carb
FIG. 1. Incorporation of hydroxylamine into carbobenzoxy - L - alanyl - L - glutaminyl - I, - valine ethyl ester as a function of time. The react,ion mixture contained SO rmoles of Tris buffer pH 7.8, 40 rmoles of calcium chloride, 20 rmoles of glutathione, 100 rmoles of hydroxylamine, 5.15 pmoles of carbobenzoxy-I.-alanyl-I,-glutaminylL-valine-ethyl ester (0.5 ml of a 1% solution in 60% ethanol), and 200 units of transglutaminase. The final volume was 2 ml. Each point on the curve represonti a separate experiment. At the times indicated on the curve the reaction was stopped by the addition of 1.5 ml of ferric: chlorideTCA (21). After centrifugation, the optical density at 525 * was determined using a Coleman Junior Speotrophotometer.
SPECIFICITY
OF TRASSGLUTAMINASE.
II.
47
Fro. 2. Apparent K, determination of carbobenzoxy-L-alanyl-L-glutaminyl-L-valine ethyl ester using methylamine and hydroxylamine as the replacing amines. For details see Methods. S = molar tripeptide concentration, \: = moles of amine incorporated per liter per minute using 100 units of enzyme per ml. 0, hydroxylamine; 0, methylamine. The apparent K, of the tripeptide is 7.i X IOWJu. The V,,, with met.hylamine is 11.5 X IO-” moles of amine incorporated per lit.er per minute. With hydroxylamine the V, is 23.6 X 10-S moles of amine incorporated per liter per minute.
calcium chloride; 20 pmoles of glutathione; 0.4 ctmoles of histamincJ4C, 200 units of transglutaminase (0.054 mg protein); 10.3 pmoles of carbobenzoxy-r,-alanyl-L-glutaminyl-L-valine et,hyl est.er (0.5 ml of a 10% solution in 60% ethanol) and amounts of compounds t.o be tested for inhibition as indicated by t.he points in t,he figures. The final volume was 2 ml. nftcr a 1-min incubation at. 37” the reaction was stopped and assayed as described above for histamine.
thereby altering t,he reaction kinetics in each instance. The apparent K,,, of the t,ripcptide was therefore determined using hydroxylamine and methylamine as the amine donors. The results of these experiments arc shown in Fig. 2, although t,he V’, in bot.h instances was different, the apparent K, of 7.7. X 10m9M for the tripcpt.ide substrate was the same. This is in agreement with the results of Folk and Cole (11) who found that t hc K, values for carbobenzoxy-L-glutaminyl glytine, carbobenzoxy-L-glutaminyl glycine ILESULTS ethyl ester and benzoyl-L-glut,ammyl glycine are the same in both amine-incorporation Figure 1 shows the course of hydroxylamine incorporation into carbobenzoxy-Lreaction and amide hydrolysis in the abalanyl-L-glutaminyl-L-valine ethyl ester as a senceof amine. The resu1t.sof experiments in which the function of time. The amine incorporation increases linearly for about 3 min and then apparent K, values of amines I\-ere deterdeviates from linearity. All incubations were mined are shown in Figs. 3 and d and summarized in Table I, together with the pK thcreforc performed for 3 min or Icss. Before atkmpting a determination of t.he values of some of the compounds. Since the apparent K, values of aminesit was essential I’,,, values of all the compounds arc in good to determine nhet,her different amines af- agreement, the apparent K, offers :I means fected the K, of the tripeptide subst.rate,6 of comparison. Of the compounds tested, the two best substrates appear to be glycine 6 Carbobenzoxy- L - alanyl -L - glutaminyl - I, -valine-ethyl ester. ethyl ester and histamine. Tyraminc and
4s
PINCUS
AND
WAELSCH
FIG. 3. Apparent K, determination of glycine ethyl ester, and histamine. For details see Methods. S = molar amine concentration, V = moles of amine incorporated per liter per minute using 100 units of enzyme per ml. 0, glycine ethyl ester; 0, histamine.
m6050s&40I>
FIG. 4. Apparent K, determination of c-amino caproic acid, tyramine, andethanolamine. see Methods. S = molar amine concentration, V = moles of amine incorporated per liter rising 100 units of enzyme per ml. 0, e-amino caproic acid; 0, tyramine; A, ethanolamine. tryptamine have apparent Km values that are greater than 100 fold higher than glycine ethyl ester or histamine. Compounds with a carboxyl group that have fewer than 5
For details per minute
methylene groups between the carboxyl and amino group do not participate in the transglutaminase reaction. (E-Amino caproic acid in which the carboxyl end is separated from
SPECIFICITY TABLE
OF TRANSGLUTAMINASE.
Compound
Km (maa)
pK of amino group
Reference for PK
Glycine ethyl ester Histamine Methylamine Ethanolamine Tyramine C-Amino caporic acid y-Amino butyric acid p-Alanine Taurine Tryptamineb Hydroxylamine”
0.186 0.178 1.42 8.6 23.8 50.0
7.73 9.8 10.54 9.44 10.75
(24) (25) (23) (23) -
-
10.6
(25)
-
10.2 9.1
(25) (25) -
a Values obtained b Value too high c Value too low
from plots to measure. to measure.
in Figs.
49
r
I
APPARENT K,,, VALUES OF ANNEX AND RELATED COMPOUNDS~
5.96
II.
(24)
(25) 3 and 4.
the amino end by 5 methylene groups has a very high apparent K,,,.) It may be, however, that any negatively charged group in close pro:ximity to the amino group is detrimental as seen from the fact that taurine is not a substrate. It can finally be seen that a relationship between the apparent K, of a compound and its pK is lacking. Several amines were not available in radioactive form and could therefore not be tested directly. These were therefore tested for their ability to inhibit histamine incorporation into t’he tripeptide substrate. Figures 5 and 6 illustrate the results of these experiments. Glycine ethyl ester is included here for comparative purposes. Taurine and y-amino butyric acid, compounds which are not substrates, as determined by the direct assay, are also not inhibitors. Thus, only compounds that are competitive substrates will inhibit histamine incorporation. The order of inhibition of the other compounds is hydroxylamine > putrescine > ammonium chloride > glycine ethyl ester > alanine ethyl ester > leucine ethyl ester. Glycine ethyl ester was of particular interest as a substrate, since it has been shown that it is an inhibitor of fibrin crosslinking in the thrombin-activated fibrinstabilizing factor system of Lorand (12).
INHIBITOR
CONCENTRATION (MxlO’)
FIG. 5. Effect of glycine ethyl ester, hydroxylamine, putrescine, and ammonium chloride on the initial rate of histamine incorporation into carbobenzoxy -L -alanyl -L - glutaminyl - L - valine ethyl ester. For details see Methods. The pK values of the compounds are: hydroxylamine 5.96 (25), ammonium chloride 9.2 (23). O---O, glycine ethyl ester; l - - - - -0, hydroxylamine; 0- - - - -0, putrescine; 0-0 ammonium chloride.
The inhibition takes place via the incorporation of this compound into fibrin in a manner similar to that which transglutaminase incorporates amines into proteins (13). Glycyl-glycine ethyl ester and several homo glycine peptides have also been shown to be good inhibitors of fibrin cross-linking (14). Thus, experiments were designed to see whether the specificity of transglutaminase and fibrin-stabilizing factor were the same toward these compounds. Figures 5 and 7 illustrate the results of experiments in which glycine ethyl ester and other homoglycine peptides were tested by the indirect assay described above. At a glycine ethyl ester concentration of 2 X low4 M, the initial rate of histamine incorporation decreasedto 79 % of the uninhibited value, while a 50% decrease was observed at a concentration of 8 X lop4 M. Significant inhibition with the larger peptides was only
PINCUS
AND WAELSCH
I a IN2”lBITOR4CONCENbN (MxlO~) FIG. 6. Effect of alanine ethyl ester, r-amino butyric acid, taurine and leucine ethyl ester on the initial rate of histamine incorporation into carbobenzoxy - L - alanyl -L - glutaminyl - L-valine ethyl ester. For details see Methods. The pK values of the amino groups of the compounds are: lysine 10.53 (24), r-amino butyric acid 10.6 (25), taurine 9.1 (25), leucine ethyl ester 7.63 (24), alanine ethyl ester 7.8 (24). A, alanine ethyl ester; 0, taurine; 0, r-amino butyric acid; A, leucine ethyl ester.
observed at concentrations of the order of 1OP M. The potency of these compounds as the inhibitors is a function of the distance between the amino and carboxyl group (tetraglycine > triglycine > diglycine). However, glycyl-glycine ethyl ester, which does not have a free carboxyl group, is only slightly better than triglycine. Thus, none of the longer glycine peptides tested inhibited as well as glycine ethyl ester. DISCUSSION
It is only through the use of a substrate with a single glutamine residue, such as the tripeptide used here as the amine acceptor, that meaningful initial rates of amine incorporation can be determined. The apparent K,,, values of the different amines can then be determined and these values used as a basis for comparison of the different com-
2
INHIBITOR
I 4
I
I
6
a
I
CONCENTRATION WlO’)
FIG. 7. Effect of glycyl-glycine, glycyl- glycine ethyl ester, triglycine, and tetraglycine on the initial rate of histamine incorporation into carbobenzoxy - L - alanyl -L - glutaminyl - L - valine ethyl ester. For details see Methods. The pK values of the amino groups of the compounds are: glycylglycine 8.13 (24), glycyl-glycine ethyl ester 7.75 (24) ,, triglycine 7.91 (25)) tetraglycine 7.75 (25).
pounds. The tripeptide carbobenxoxy-nalanyl-n-glutaminyl-n-valine ethyl ester was thus employed as the amine acceptor since its sequence is the same as the amino acid sequence around the single glutamine residue in the CYchain of hemoglobin, which was shown to be a substrate in the previous paper. It is necessary to block it at the amino and carboxyl ends since free di- and tripeptides are not substrates for transglutaminase (15). Enzyme reactions involving two substrates exhibit complex kinetic behavior. A detailed discussionof this is given by Dixon and Webb (16). Since the details of the mechanism of action of transglutaminase have not been worked out, the correct rate equation cannot be written and the exact meaning of Km is unclear. However, the Lineweaver-Burk plots in all the amine K,,, determinations intersect the l/V axis at approximately the same point. The values for Km determined from these plots can then
SPECIFICITY
OF TRANSGLUTAMINASE.
be used to compare amine reactivity if K,,, is given the classical operational definition of “the substrate concentration at which the reaction velocity is one-half the maximum velocity.” The Km values determined in this work are therefore referred to sts apparent K,,, values and do not have an absolute meaning but only a comparative meaning in relation to each other. When insulin was used as a substrate for transglutaminase it was observed that ammonia was enzymatically releasedin the absence of an amine (17). This same behavior has been observed by Folk and Cole using synthetic blocked glutamine depeptides (11) and in the caseof many proteolytic enzymes that catalyze both hydrolytic and transpeptidation reactions (18). These observations raise the question of whether the biological role of transglutaminase is one of hydrolysis or transamidation. If the mechanism of action of transglutaminase is such that the second substrate (water or an amine) reacts directly with the enzyme sub&ate complex, rather than first binding to the enzyme, then the transamidation reaction would represent a successful competition of an amine over water for the enzyme substrate complex when an amine is present in the reaction mixture. If this were the case, then the amount of ammonia released would be greater than the amount of amine incorporated since hydrolysis and transpeptidation could occur simultaneously as seen in the case of papain (19). However, using carbobenzoxy L-glutaminyl glycine and ethanolamine as substrates for transglutaminase, Folk and Cole (11) have shown that all of the ammonia released can be accounted for by transamidation. The data presented here also indicate that in the presence of amine a hydrolytic mechanism is not operating. The apparent Km values indicate that the enzyme showed a marked degree of specificity for the amine substrate. There is also a lack of correlation between the apparent Km and the pK values of the amines (seeTable I), and, compounds with similar pK values do not inhibit histamine incorporation into carbobenzoxy-L-alanylL-glutaminyl-.L-valine ethyl ester equally well. (Compare the inhibition curves for glycine ethyl ester, alanine ethyl ester, leu-
II.
51
tine ethyl ester, glycyl-glycine, triglycine, and tetraglycine.) If a mechanism were operating in which the amine directly attacked the enzyme substrate complex, without being bound to the enzyme, the reactivity of the amine would depend only upon the quantity of the unprotonated form present and therefore be proportional to its pK. Thus, the second substrate (amine) appears to be bound rather specifically to the enzyme and its biological role appears to be transamidation and not hydrolysis. From the data presented in this paper it would seemthat there are a minimum of two points of attachment for the amine relatively close to each other. One of these is for the primary amino group while the second for a free pair of electrons on the side chain. Thus, small compounds, such as hydroxylamine and ammonium chloride, are probably bound at the primary amino group site without interfering with the second site and are therefore good inhibitors of histamine incorporation. Of all the compounds with side chains that were tested, histamine and glycine ethyl ester appear to be the best substrates while putrescine inhibits histamine incorporation only slightly better than glycine ester. The feature common to all of these is the free pair of electrons on the imidazole nitrogen of histamine, the second amino nitrogen of putrescine, and the ester oxygen of glycine ethyl ester. The other compounds tested have higher apparent K,,, values or are poor inhibitors of histamine incorporation. This is probably due to the fact that they have a side chain that does does not fit well at the second point of attachment or is too large to fit the amine site, or that they possessa negative charge. The low apparent K, values of histamine and glycine ethyl ester are of interest since both of these are good inhibitors of fibrin polymerization by the enzyme thrombinactivated stabilizing factor (12). However, in the case of transglutaminase, glycine peptides and glycyl-glycine ethyl ester bind much more poorly than glycine ethyl ester (based on their inhibition of histamine incorporation). Although the underlying mechanism of both enzymes might be the same, the biological function of transglutaminase is probably not the cross-linking of proteins in
52
PINCUS
AND
the same manner as fibrin-stabilizing factor. The low apparent K,,, of histamine ties in with results obtained by Ginsburg et al. (20). These workers have demonstrated that the level of liver transglutaminase in mice increased between three and four fold when the animals are injected with bacterial endotoxins. They have also found that there is a small but significant incorporation of histamine into liver protein under these conditions. It is known that bacterial endotoxins increase the sensitivity of animals to histamine (21) and cause the histamine content of the liver to rise (22). Since transglutaminase shows a preference for histamine, the increase in enzyme activity when an animal receives bacterial endotoxins appears to be important. Although the exact role of the enzyme under these conditions is as yet uncertain, two possibilities are immediately apparent. The incorporatioil of histamine into a protein may be necessary for it to exert its biological effect, or, the amine incorporation into proteins may serve as a detoxification mechanism. Experiments to elucidate the biological role of transglutaminase are currently in progress in this laboratory. ACKNOWLEDGMENT This work was supported in part by grants from the National Institute of Neurological Diseases and Blindness, Public Health Service Research Grant NB 00226, and from the Supreme Council, 33” Scottish Rite Masons of the Northern Jurisdiction, United States of America. We wish to thank Dr. Amos Neidle for his help and advice concerning peptide synthesis and for his helpful suggestions during the preparation of this manuscript. REFERENCES 1. PINCUS, J. H., AND WAELSCH, H., Arch. Biothem. Biophys., 126, 34 (1968). 2. SARKAR, N. K., CLARKE, D. D., AND WAELSCH, H., Biochem. Biophys. Acta, 26, 451 (1957). 3. CLARKE, D. D., MYCEK, M. J., NEIDLE, A., WAELSCH, H., Arch. Biochem. Biophys., 79, 338 (1959).
WAELSCH 4. BERGMANN, F., AND ZERVAS, L., Chem. Ber.. 66, 1192 (1932). 5. NIUDLE, A., manuscript in preparation. 6. VAUGHAN, J. R., AND EICHLER, J. A., J. Am. Chem. Sot., 76,5536 (1953). 7. BOISSONAS, R. A., Helu. Chim. Acta, 34, 874 (1951). 8. LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66, 658 (1934). 9. WAF:LSCH, H., AND MYCEK, M. J., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. V, p. 833. Academic Press, New York (1962). 10. BRAY, G. A., Anal. Biochem., 1, 279 (1960). 11. FOLK, J. E., AND COLE, P. W., J. Biol. Chem., 240, 2951 (1965). 12. LORAND, L., AND JACOBSEN, A., Biochemistry. 3, 1939 (1964). 13. LORAND, L., Federation Proc., 24,784 (1965). 14. LORAND, L., DOOLITTLE, R. F., KONISHI, K., AND RIGGS, S. K., Arch. Biochem. Biophys., 102, 171 (1963). 15. NEIDLF:, A., AND Acs, G., Federation Proc., 20, 234 (1961). 16. DIXON, M., AND WEBB, E. C., in “The Enzymes” 2nd Edit., pp. 70-75; 100-102. ilcademic Press, New York (1964). 17. MYCEIC, M. J., CLARKE, D. D., NEIDLE, A., ANU WAF,LSCH, H., Arch. Biochem. Biophys., 84, 528 (1959). 18. FRUTON, J. S., Harvey Lectures, 61, 64 (1957). 19. JOHNSTON, R. J., MYCEK, M. J., AND FRUTON, J. S., J. Biol. Chem., 186, 629 (1950). 20. GINSBURG, M., WAJDA, I., AND WAELSCH, H., Biochem. Phamnacol., 12, 251 (1963). 21. MALKIEL, S., AND HARGIS, B. J., Proc. Sot. Exptl. Biol. Med., 30, 122 (1952). 22. KIM, E., Proc. Sot. Exptl. Biol. Med. 66, 197 (1947). 23. HODGMAN, C. D., WEAST, R. C., SHANKLAND, R. I)., AND SELBY, S. M., Eds., “Handbook of Chemistry and Physics” 44th Ed., pp. 1749-1751. Chem. Rubber Publ. Co., Cleveland, Ohio (1952). 24. COHN, E. J., AND EDSALL, J. T., “Proteins, Amino Acids and Peptides as Ions and Dipolar Ions,” pp. 680683. Reinhold, New York (1943). 25. LONG, C., “Biochemists Handbook,” pp. 43-52. Spon, London (1961).