Mechanism of Action of Guinea Pig Liver Transglutaminase SITE STUDIES WITH GROUP-LABELED HALOMETHYL

SUMMARY The reaction of cr-bromo-4-hydroxy-3-nitroacetophenone (BHNA) with transglutaminase in the presence of CaClz (25 mM) produces a catalytically inactive labeled protein in which the phenacyl group is covalently attached to the active site --SH. The spectral properties of this group attached to the enzyme are consistent with that of the group in a hydrophobic region of the molecule. Addition of ethylene-diaminetetraacetate results in a shift of the spectrum toward shorter wave lengths, indicative of a more polar environment for this -SH in the absence of Ca++. Attachment of the phenacyl group to positions in the enzyme other than the active --SH by reaction with BHNA in the absence of Ca++ results in losses in transferase activity, but essentially no loss in esterase activity. The spectrum of the groups bound to enzyme in the absence of Ca++ is identical with that of the phenacyl group in water. This spectrum is unchanged by subsequent addition of Ca++. In the catalytically inactive forms of transglutaminase, pro-duced by the reaction of D and L forms of methyl N-(2-hy-droxyd-nitrophenylacetyl)-2-amino-4-0x0-5 - chloropentano ate (PACK) and 1-chloro-4-(2-hydroxy-5-nitrophenylacetyl)-amidobutan-2-one


SUMMARY
The reaction of cr-bromo-4-hydroxy-  with transglutaminase in the presence of CaClz (25 mM) produces a catalytically inactive labeled protein in which the phenacyl group is covalently attached to the active site --SH.
The spectral properties of this group attached to the enzyme are consistent with that of the group in a hydrophobic region of the molecule. Addition of ethylenediaminetetraacetate results in a shift of the spectrum toward shorter wave lengths, indicative of a more polar environment for this -SH in the absence of Ca++. Attachment of the phenacyl group to positions in the enzyme other than the active --SH by reaction with BHNA in the absence of Ca++ results in losses in transferase activity, but essentially no loss in esterase activity.
The spectrum of the groups bound to enzyme in the absence of Ca++ is identical with that of the phenacyl group in water. This spectrum is unchanged by subsequent addition of Ca++. In the catalytically inactive forms of transglutaminase, produced by the reaction of D and L forms of methyl N-(2-hydroxyd-nitrophenylacetyl)-2-amino-4-0x0-5 -chloropentano ate (PACK) and 1-chloro-4-(2-hydroxy-5-nitrophenylacetyl)amidobutan-2-one (PBCK) with enzyme in the presence of Ca++, the phenolic reporter group is attached covalently to the enzyme's active -SH. The rapid rate of inactivation by L-PACK compared to D-PACK and PBCK implies that L-PACK, by virtue of its structural similarity to transglutaminase substrates, is properly oriented at the substrate-binding site of enzyme prior to the covalent reaction. The pK, of the phenolic group in the acyl portion of each of these inactivators is shifted toward that of a weaker acid in the reporterlabeled enzyme proteins. The identical changes in pK, with each inactivator suggest that the phenylacetyl side chain in each case is positioned in the same manner within the matrix of the calcium-activated enzyme derivative. Addition of EDTA results in a shift in pKa of the phenolic group in each enzyme derivative back to that of the parent inactivator. This, together with the findings with BHNA, forms the basis for a suggestion that the active -SH is located at or near the surface in the unactivated enzyme, i.e., in the absence of Ca++. Active site titration procedures for transglutaminase are described.
A rate assay procedure utilizing either BHNA or L-PACK was found to give results in excellent agreement with those of a direct spectrophotometric method carried out with the use of BHNA. The latter titration is based on the dilTerences in the absorption spectrum of the phenacyl group bound to the enzyme's active -SH and that of this group attached to other positions on the enzyme molecule.
The sulfhydryl group of a single cysteine residue in transglutaminase has been identified as essential for the catalytic activities of the enzyme (1, 2). Although transglutaminase contains 17 or 18 free -SH groups (2), rapid selective alkylation of the essential -SH by iodoacetamide occurs between pH 6 and 7 in the presence of calcium ion, which is necessary for enzymatic activity (1, 2). A sequence of amino acids surrounding this cysteine has been reported (1). Identification of the same cysteine -SH as the site of acylation during the course of hydrolysis of p-nitrophenyl trimethylacetate has supplied strong evidence for participation of this group in the intermediate formation of acyl enzyme through thioester linkage (3). Kinetic findings for hydrolysis and transfer of both active ester and amide substrates are in accord with the theory of acyl enzyme formation in the transglutaminase mechanism (4, 5). The selective reactivity of the essential -SH, evidenced as the center of the active site of transglutaminase, together with the Ca++ requirement for its reactivity, singles out this enzyme protein as an especially attractive model for study with the use of covalently attached environmentally sensitive groups. Several other features of transglutaminase specificity and catalysis stinlulated and directed approaches to this study.
These include a reversible conformational alteration in the enzyme protein induced by Ca++ (6), the requirement that glutamine substrate residues be peptide or protein bound (7,8), and evidence that glutamine substrate is the first to add to enzyme in the catalytic reaction (4, 5). Thus, the chemical agents employed for the study reported here were selected with a design to explore the environment in several regions of the enzymatic center of transglutaminase.
Early attempts to obtain knowledge of the area in the vicinity of the active site SH of transglutaminase made use of the "reporter" group-containing agent, 2-bromoacetamido-4-nitrophenol (9) .l These proved unfruitful because of the tendency of the labeled protein derivative to precipitate rapidly from solution.

Materials
'rransglutslniaase was prepared from fresh guinea pig liver by :I published procedure (I 0). The enzyme showed 95 + 5% of the reported specific activity when assayed by hydroxamate forma tion with the specific substrate bcllzylosycarbonyl-Lglutamill~lglycine (2). Enzyme concentration was determined by the use of the Eiz of 15.8 and a molecular weight of 90,000 (2).
A sample of a-bromo-4-hydroxy-3-nitroacetophenone was kindly supplied by Dr. E. T. Kaiser. 1311NA2 was also prepared by the method of Sipos and Szabo (11).
Other materials and reagents have been described in previous publications (2-5).
a\fter stirring for 15 min an additioiinl I ml of N NaOI-I was added and stirring was continued for 5 min.
The solvents were removed under vacuum, and to the residue were added 25 ml of ethyl acetate and 25 ml of 0.5 x H('1. The ethyl acetate layer was separated, washed successively with lvater, dilute NaHCOs, wat,er, dilute HCl, and water, and dried over NasS04.
The residue obtained upon removal of solvent was warmed with 5 ml of water while ethanol was added dropwise un til it dissolved.

Synthesis of PHCR
~-Chloro-4-Z-amidobu~u~-Z-one-Z-p-alnlli~ie (4.1 g) (Clyclo Chemical Company) was dissolved in 20 ml of thionyl chloride and heated at 40" for 30 min under anhydrous conditions. The unreacted thionyl chloride was rernoved under high vacuum, and to the residue was added excess diazornethane in ether. After standing overnight the mixture was reduced to an oil under vacuurn.
This was dissolved in 50 ml of ether and a stream of dry HCl gas was passed through the solution for 30 min.
The oil obtained upon removal of the solvent formed crystals under pentane in the cold.
The acid was removed under vacuum and the resultant oil was washed three times by tritration with ether. This was dissolved in 2 ml of 0.5 N NaOH and added to a solution of 179 mg of 5.nitro-2-coumaranone in 10 ml of dioxane.
After stirring for 15 min the solvents were removed under vacuum and the oil dissolved in ethyl acetate.
The compound showed single areas in the two chromatography systems used for PACK. The findings by mass spectral analysis were in accord with the theoretical molecular weight; the molecular ion showed the expected chlorine isotope cluster. Synthesis oJ PG Z-r-glutarnine methyl ester (300 mg) (15) was decarbobenzosylated by hydrogenation in a methanol-water rnixture using Pd black catalyst.
The solvents were removed under vacuum after removal of the catalyst by filtration.
The resultant oil was dissolved in I ml of water and a solution of 179 mg of 5-nitro-2coumarnnone in 7.5 ml of dioxane was added.
After stirring for 1 hour the solvents were removed under vacuum and the residue was dissolved in 20 ml of ethyl acetate.
The ethyl acetate solution was washed with dilute HCl and water and dried over Nag-SC1. The compound was crystallized by the addition of pentane; yield 114 mg (34oj,), m.p. 159-160".
A sample was recrystallized from water for analysis.
The melting point was unchanged. This compound showed single areas in the two chromatography systems described above. PSBA-Z-y-aminobutyramide was decarbobenzoxylated by hydrogenation in methanol and coupled with 5-nit,ro-2-coumaranone as outlined above for the preparation of PG. The product was obtained in 40% yield after crystallization from absolute ethanol.
It showed single areas in the two chromatography systems described above.
The esterase assay was carried out in 0.1 M Tris-HCl containing 0.5 mM p-nitrophenyl acetate, 50 PM EDTA, 10 mM CaCle, and 5% n-propyl alcohol, at pH 7.0 and 25" (I 6). Rates of liberation of p-nitrophenol were measured at 400 m,u within the first 20 to 40 s of hydrolysis.
The transglutaminase-catalyzed incorporation of [14C]glycine ethyl ester in place of -NH:! at the carboxamide groups of PG and PABA was measured by a paper strip ion exchange procedure similar in principle to that outlined by Sherman (17). Aliquots of incubation mixtures (10 to 30 ~1) were applied to strips (1.5 X 14 cm) of cellulose phosphate ion exchange paper (P81; capacity, 18 peq per cm2; basis weight 85 g per m2 (Whatman)), enzymatic: reactions were stopped by the immediate application of approximately 100 ~1 of absolute ethanol, and the strips were eluted with water in a manner similar to that outlined by Sherman (17). The Y-labeled amine, glycine ethyl ester, remained at the position of application of the reaction mixture, while PG, PAUA, and the labeled products of the transfer reaction were washed toward the bottom of the strips. After drying, the lower 5 cm of the strips were removed, placed in counting vials containing 10 ml of Liquifluor-toluene counting fluid (New England Nuclear), and the radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrometer. Experiments in which PG or PABA and the reaction products were eluted from the lower 5 cm of the paper strips with NaHC03 solution showed quantitative recovery of these materials as measured by absorbance at 410 mp.
The substrates (PG, RF 0.18; PAUA, RF 0.15) and the products (RF values of 0.47 and 0.40, respectively) were visualized by exposure of the chromatograms to NH3 vapor. Stock solutions of BHNA (1 mM) were prepared in 0.01 M Tris-HCl, pH 7.0; those of PACK and PBCK (1 to 2 mM) in HzO; those of PG (0.1 M) in water and PhlU (0.02 M) in He0 at 50" with 1 eq of NaOH.
The concentrations of halomethyl ketones Mechanism of Transglutaminase. VIII Vol. 246,No. 21  titrations, small portions of appropriate concentration of acid, base, or reagent were added and the solutions were rapidly mixed.
In all cases the pH levels of solutions were determined before and after measurement of spectra.

Inactivation of Transglutaminase by Halomethyl
Ketones-In Table I where Ebt is the total enzyme concentration, Z(0) is the initial concentration of inactivator, and e(t) is the total active enzyme at time t. The level of Ca+f (25 mM) used for these inactivation studies and for the other studies reported here was chosen because the enzyme is stable for periods of at least 30 min at pH 7 at this level of metal ion. The rate of inactivation of transglutaminase by iodoacetamide is a function of the Ca++ concentration (19). This is probably also true for the halomethyl ketones listed in Table I, e.g., no inactivation by L-PACK was observed in the absence of Ca++.
Since the dissociation constant for Caf+ is 6 to 8 x lop3 M (16,19), one may assume that the rate constants given in Table I are 70 to 75% of those at saturating levels of Ca++. There are pronounced differences in the rates of inactivation of transglutaminase by L-PACK, D-PACK, and PBCK. It was found, however, that each of these chloromethyl ketones reacted with the -SH of GSH at the same rate under the conditions of  In the presence of Ca++, as in the case with PACK and PBCK, equivalent losses in transferase and esterase activities occurred.
However, without Ca++, no pronounced loss in esterase activity was found with up to 3 moles of BHNA per mole of enzyme, whereas significant losses in hydroxylamine-incorporating activity were observed. Site of Chemical Modi$cation with L-PACK and BIINA-L-PACK-inactivated transglutaminase, prepared from 0.2 pmole of enzyme essentially as outlined in Table I (1.1 mole of L-PACK per mole of enzyme, BO-min reaction time), was freed of reagents by dialysis and dried by lyophilization.
The labeled protein was dissolved in 2 ml of 0.2 M NH4HC03 containing 5 mM CaClz and digested for 18 hours at 37" with 1 mg of chymotrypsin A. The digest was applied to a column of Sephadex G-25 (fine) and eluted as outlined earlier for the trypsin-chromotrypsin digests of [W]iodoacetamide-labeled transglutaminase (1). All of the yellow material was eluted immediately following the salt fraction. The eluate containing the yellow material was taken to dryness in a stream of air. The residue was dissolved in a small amount of water and subjected to further purification using a peptide map ping procedure (21). Exposure of the map to NH3 vapors showed a single area (RF 0.21 in chromatography system, 2 cm toward cathode in electrophoresis system). The labeled peptide was eluted from the paper with dilute NH4HC03 and taken to dryness in a stream of air. This material appeared to be a single peptide as evidenced by a single ninhydrin-positive area in several thin layer chromatography systems. The COOH-terminal residue of this peptide was identified as by guest on March 23, 2020 http://www.jbc.org/ Downloaded from tryptophan by treatment with carboxypeptidase A followed by thin layer chromatography.
The acid hydrolysate was dansylated, and the derivatives were identified as those of glycine and glutamic acid by twodimensional thin layer chromatography using chloroform-t-butyl alcohol-acetic acid (6 : 3 : 1) and chloroform-ethanol-acetic acid (38 : 4 : 3) on silica gel. A third dansyl derivative remained at the origin in both chromatographic systems, as did the dansylated product of the reaction of L-2-amino-4-oxo-5-chloropentanoic acid with cysteine (equimolar quantities of each at pH 8 for 1 hour). Essentially the same isolation and identification procedure was carried out with a sample of transglutaminase that had been inactivated with BHNA in the presence of Ca++. The findings were the same with the exception that a chromatogram of an acid hydrolysate of the chromophoric chymotryptic peptide showed a single yellow area (RF 0.63 in 1-butanol-acetic acid-Hz0 (4: 1:2)) identical with that formed by the reaction of BHNA with cysteine (equimolar quantities of each at pH 7 for 10 min). The dansyl derivatives of glycine and glutamic acid, together with a yellow derivative which remained at the origin in both chromatographic systems, as did the dansylated product of reaction of BHNA with cysteine, were found as components of the dansylated acid hydrolysate of this peptide. Thus, the peptide liberated by chymotrypsin A from L-PACKinactivated transglutaminase appears to be identical in amino acid sequence with that obtained by chymotrypsin A digestion of the BHNA-inactivated enzyme.
These peptides have an NHZterminal glycine, a COOH-terminal tryptophan, and contain glutamic acid or glutamine and a derivative of cysteine. These observations support the conclusions that both PACK and BHNA are incorporated into calcium-activated transglutaminase by alkylation of the -SH group of a single cysteine residue, that this -SH is the same in each case, and that this is the same essential -SH group that is alkylated by iodoacetamide under similar experimental conditions. A portion of the amino acid sequence surrounding the cysteine that is alkylated by iodoacetamide has been identified as Gly-Gln-Cys-Trp (2).
Substrate Properties of PG and PABA-Z-r-glutamine methyl ester has been found to be a substrate for transglutaminase (8). It was anticipated that PG, the glutamine analogue of L-PACK, would also act as a transglutaminasc substrate, This proved to be the case. Using the SEQUEN computer program of Cleland (22) the following estimates for constants were obtained from reactions carried out in 0. These kinetic constants have been defined elsewhere in terms of rate constants (4).
Transglutaminase displays an almost absolute stereospecificity toward the L isomer of a glutamine substrate (5). The inactivators, D-and L-PACK, weredesigned to act in a substrate-like manner. As expected, the L form of PACK was the more effective inactivator (Table I). PBCK, in which the -COOHa group of PACK was replaced by hydrogen and, consequently, in which there is no asymmetric carbon atom, was a more efficient inactivator than D-PACK (Table I).
It was of prime interest to deter-mine if the carboxamide analogue of PBCK, PABA, would function as a transglutaminase substrate. Indeed, PABA did serve as a substrate, evidence that the cr-carboxyl portion of the glutamine moiety is not an essential part of substrates for transglutaminase.
The time-dependent accumulation of product in the PABA-glycine ethyl ester reaction was observed by thin layer chromatography.
The limiting solubility of PABA precluded estimation of meaningful kinetic constants. However, at 7.4 mM PABA and 1.2 mrvr [Wlglycine ethyl ester, the rate of transfer product formation was found to be 1.6 pmoles per min (per pmole of enzyme) under the experimental conditions used with PG. This rate is in the range of those observed with PG.
Spectral Properties of Q-Hyclroxy-S-nitrophenacyl Group in BHNA-modi$ed Transglutaminase-Figs. 1 and 2 show and compare the spectra of BHNA and of the 4-hydroxy-3-nitrophenacyl ("reporter") group that was attached to transglutaminase in the presence and in the absence of Ca++.
Initial attempts to prepare reporter-labeled enzyme free of excess reagents were unsuccessful.
In all cases the labeled enzyme tended to precipitate from solution.
Therefore, labeling was carried out with the use of stoichiometric amounts of enzyme and reagent directly in the spectrophotometer cells.
Labeled enzyme prepared in this man ner remained soluble at pH 7 and above but precipitated rapidly below pH 7.
Comparison of the findings in Figs. 1 and 2 show pronounced differences in the spectra of the reporter group attached to en zyme in the presence and in the absence of Ca+f.
The peak of absorbance for the phenacyl group attached to enzyme in the presence of Ca++ (observed in the presence of Ca++) is at 334 mu (Curve 2, Fig. I).
This peak is significantly higher in intensity and in wave length than that of BHNA (323 mp, Curve 1, Fig. 1) and that of the phenacyl group attached to enzyme in the absence of Ca++ (316 rnp, Curve d, Fig. 2). Addition of CaC& (25 mM) to solutions of BHNA or to enzyme to which phenacyl group was attached in the absence of Ca++ caused no change in their spectra.
The identity of the spectrum of reporter group bound to enzyme in the absence of Ca++ and that of this group attached to the -SH of GSH (Curve I, Fig. 2 in each case) suggests that this group on the enzyme is in an aqueous environment and remains so upon addition of Ca++. A 2nd and 3rd mole of BHNA added per mole of enzyme protein in the absence of Ca++ resulted in increases in the intensity of the spectrum with a peak at 316 rnp (Curve d, Fig. 2). Again, addition of Ca++ to these solutions caused no change in the observed curves.
The spectrum of reporter group bound to enzyme in the presence of Ca++ (Curve 2, Fig. 1) was unchanged by raising the pH of the solution to 8. This suggests that the pronounced shift in the spectrum toward longer wave lengths reflects a change in the environment of the group rather than a shift in the pK, of the phenolic portion of the reporter group.
A pK, value of 4.7 has been reported for ionization of the phenolic group in BHNA (23). The pK, of this group in the phenacyl-labeled species of papain was measured as 5.0 (23). The shift toward longer wave lengths, that results from decreasing the dielectric constant of the medium by addition of dioxane to the model system (GSH + BIINA), is in line with this interpretation (compare Curves 2 and S, Fig. 2). The spectrum with the 334 m,u peak (Curve 2, Fig. 1) is apparently unique for the reporter group attached to the single essential -SH group of transglutaminase. This was evidenced by the following.
Equal volume portions of a solution of enzyme, that was labeled with 1 mole of BHNA per mole of enzyme in the pres-  The enzyme retained less than 27, of its initial hydroxylamine-incorporating and esterase activities.
ence of Ca++ and that displayed the typical 334 rnp peak absorbance (Curve 9, Fig. I), were placed in matched cuvettes in the blank compartment and the sample compartment of the spectrophotometer.
Portions of BHNA solution and equal volume portions of dilute Tris buffer, pH 7, were added to the solutions in the sample and blank compartments, respectively. A spectrum was obtained after each addition.
These spectra, observed up to the level of 2.5 moles of BHNA per mole of enzyme, were typical of The enzyme retained 60% of its init,ial hydroxylamine-incorporating activit,y and 100% of its esterase activity (see Table II). that found for enzyme labeled with 13HNA in the absence of <:a++ (Curve d, Fig. 2). The solution in the sample compartment showed increasing slight turbidity after each addition of BHNA. Addition of more than 2.5 moles of reagent per mole of enzyme resulted in visible precipitation. " EDTA, added in excess of the La ++ to a solution of phenacyllabeled enzyme that was prepared in the presence of Ca++, caused an apparent shift in the spectrum toward shorter wave lengths (compare Curves d and S, Fig. 1). The broadness of the spectrum with added EDTA (Curve 3, Fig. 1) compared to that observed without EDTA (Curve 8, Fig. 1) suggests contribution of more than a single component.
The spectrum was shifted back to that observed without EDTA (Curve 6, Fig. 1) upon addition of CaCl:! in large excess over EDTA.
Addition of the substrates, Z-n-glutaminylglycine or glgcine ethyl ester, to solutions of transglutaminase that had been inactivated with RHNA in the presence of Ca++ caused no change in the spectrum shown in Fig. 1, Curve d.
Spectral Properties of Chromophoric Acyl Groups in PACKand PBCK-inactivated Transglutaminase-Spectral titration of the phenolic group of L-PACK showed it to have a pK, of 6.7. Reaction of L-PACK with excess GSH did not change this value.
The identical pK, values were found for the phenolic groups in n-PACK and PECK. These compounds showed an absorption maximum at 318 to 320 nm in the unionized state and one at 410 rnp in the ionized state with an isosbcstic point at 353 rnp. A portion of the absorption spectrum of L-PACK at pH 7 is shown in Fig. 3 The experimental conditions were those of Figs. 1 and 3. Aliquots of the reaction mixture were removed at the indicated times for assay by the hydroxylamine incorporation method.
Active site titration of transglutaminase by t,he rate assay method using the inactivator L-PACK. The esterase activity of enzyme inhibited by varying amounts of L-PACK (conditions of Fig. 1, 30.min incubation with inact,ivator) is plotted relative t,o that of enzyme incubated without inactivator. The enzyme concentration was 4.7 X 10e6 M. Essentially the same results were obtained by using the hydroxylamine incorporation assay.
phrnolic group of L-I'XCK in 33% (v/v) dioxanc showed that decreasing the dielectric const.ant of the medium shifts the pK, of this group to that of a weaker acid (pK, of 7.1).
Preliminary examination of the visual changes that accompany the inactivation of transglutaminase through alkylation by I,-and o-PACK and PBCK in the presence of Ca+f showed a significant decrease in the absorbance at 410 my with no change in the position of peak absorbance.
The visual spectrum of L-PACK-inactivat,ed transglutamine at pH 7 is shown in Fig. 3, Curve 6. That this change is a result of a shift in the ph', of the l)hcnolic group of the inactivator was evident from titration above $1 6.9 of this group in the labeled species of enzyme (pK, of 7.1 to 7.2).
As in the case of the BHNA-modified forms of transglutaminase, the PACK-and PBCK-inactivated enzymes could not be prepared free of reagents in a satisfactorily soluble form for spectral studies. As before, the labeled enzyme solutions, prepared by the use of stoichiometric amounts of enzyme and inactivators, were studied without further treatment.
Solutions of inactivated enzyme rap idly became cloudy below pH 6.9. Fig. 4 shows that the reduction in absorbance at 410 mu that results from reaction of L-PACK with transglutaminase occurs concomitantly with the loss in catalytic activity of the enzyme. Essentially the same quantitative changes in absorbance with relat'ion to loss in enzymatic activities were observed during the early stages of inactivation by II-PACK and PBCK. That no change in absorbance at 353 rnp, the isosbestic point, occurred during enzyme inactivation by these agents further suggests that only ionization of the phenolic group is effected by attachment of the acyl portion of the inactivators to enzyme. Evidence that removal of Ca+f from L-PACK-inactivated enzyme solutions resulted in a shift in the pK, of the phenolic group back to that of L-PACK (pK, of 6.7) was obtained by spectral titration of this group in t'he enzyme after addition of EDTA in excess of the (:a++. l&king the C 'L "* ++ level above that of EIYI'A again shifted the pK, of this group to that of a weaker acid (pK, of 7.1 to 7.2).
Addition of the substrates, %~I,-glutaniiliylglgcillc or glycine ethyl ester, to solutions of enzyme that had been inactivated with L-PACK at pH 7.0 caused no change in the observed spectrum. Active Site Titrations of Transglutaminase-Titration of the active site of transglutaminase was carried out by the use of BHNA and L-PACK.
With L-PACK, titration was accomplished by measuring the degree of inactivation using rate assays employing both the hydroxylamine incorporation and esterase procedures. The results of such a titration arc shown in Fig. 5. The same procedure, using BHNA in place of L-PACK, gave identical resuits.
A second approach using BHNA involved a direct spectrophotometric titration. This procedure utilizes the differences in the absorption spectrum of the phrnacyl group bound to the en-Mechanism of Transglutarninase. Group in BHNA-modified Transglutaminase"). The results of a direct spectrophotometric titration are shown in Fig. 6. Findings obtained by this procedure were in excellent agreement with those obtained using the rate assay method of Fig. 5.
Further evidence that L-PACK and BHNA titrate the same essential -SH group of transglutaminase is as follows. Reaction of enzyme, first inactivated by 1 mole of L-PACK per mole, with BHNA resulted in the appearance of a spectrum typical of reaction of groups in the enzyme other than the active site one (Curue 2, Fig. 2).

DISCUSSION
The findings reported here were forthcoming from an effort to define certain environmental features of the active center of transglutaminase.
This effort, although limited by the insoluble nature of the enzyme derivatives at levels of pH below neutrality, was enhanced by the functional role of Ca++.
There is strong evidence that, in the presence of Ca++, reaction with each of the halomethyl ketones results in formation of a catalytically inactive enzyme derivative in which reporter group is covalently bound t,hrough the same essential group in the enzyme, presumably the active site -SH.
The observed shift in the spectrum of the 4-hydroxy&nitrophenacyl group upon attachment at the active center of transglutaminase (reaction with BHNA in the presence of Ca++, Curve d, Fig. 1) is congruent with orientation of this group in a hydrophobic region of the molecule.
The tendency of this spectrum to shift to shorter wave lengths with the addition of EDTA (Curve S, Fig. 1) suggests a more polar environment for this -SH group in the catalytically inactive form of the enzyme, i.e., in the absence of Ca++.
Both hydrophobic (24) and polar (9) regions have been identified at the active center of chymotrypsin A. The specificity pattern of chymotrypsin A, i.e., preference for aromatic derivatives over short chain aliphatic ones, suggests that hydrophobic bonding plays an important role in substrate attraction. This is the basis for speculations that the hydrophobic portion of the active center is the substrate recognition site, while the catalytic site requires a high water concentration for mediation of the hydrolytic process (9, 24). Transglutaminase catalyzes hydrolysis with glutamine substrates (7, 8) and with active esters (16). There is substantial kinetic evidence for the formation of a common intermediate acyl enzyme in the hydrolysis and transfer reactions (4, 5).  Fig. 2) has a pronounced influence on the hydroxylamine incorporation activity of the enzyme, but does not affect its esterase activity ( Table  II).
The spectral property of the chromophoric groups bound in these positions on the enzyme is characteristic of this group in a hydrophilic environment both in the presence and absence of Ca++ (Curve 2, Fig. 2). Structural analogies between BHNA and DTNB, e.g., the nitro group ortho to a negatively charged group and approximately the same distance from the reactive portion, suggest that the striking similarity in the effects of these two agents on the catalytic activities of transglutaminase (compare Tables II and III) results from reaction with the same enzyme groups in each case. With 1 mole of DTNB per mole of enzyme in the absence of Ca++, a single intramolecular disulfide bond is formed in the enzyme (25).
Inhibition studies suggest that this molecular change causes a loss in binding properties for glutamine substrate (25).
If one or both of these -SH groups participate directly in glutamine substrate binding, it seems possible, on the basis of the spectral finding and the similarity in effects of DTNB and BHNA, that a portion of the binding site of the enzyme is polar in nature.
In enzyme reactions, where intermediate acyl derivatives are formed from enzyme and the acyl portion of substrate, the specificity of the enzyme may be directed toward a single configuration of the acyl portion of substrate, e.g., chymotrypsin's specific action on peptide bonds in which the carboxyl part is contributed by amino acids of the L form and the almost absolute specificity of transglutaminase toward L-glutamine peptide (5). The question arises as to whether this is important only for proper noncovalent binding of substrate or whether the acyl portion of intermediate acyl enzyme remains or becomes aligned in a spatial arrangement especially suited for efficient deacylation. The chloromethyl ketones, D-and L-PACK, analogues of the transglutaminase substrates, were conceived with the intent of forming stable pseudo acyl enzymes in which similarities or differences in the orientation of the acyl group could be visualized.
In this regard, it was anticipated that each of the inactivators, PBCK and the D and I, forms of PACK, would react selectively with the enzyme's active -~--SH by virtue of t,heir halomethyl ketone feature. The total loss in enzyme act,ivities upon reaction of each of the inactivators at the level of 1 rnole per mole of enzyme is strong evidence for this.
It seems evident from the more rapid rate of  (Table I), that this isomer is acting in a substrate-like manner, i.e., is complexing with enzyme through the sul,strate-binding site. That L-PACK is a more efficient inactivat,or than T'BCK supports this conclusion, as does the fact t,hat each of these chloromet,hyl ketones reacts at the same rate with GSII.
The identical change in the pK, of the phetlolic group in each of these illactivators up011 covalent attach-melLt to enzyme, as reflected in the spectral changes (example Fig. 3), may be evidence for the same positioning in the acyl enzyme of the side chain attached to the glutamine residue. To draw any firm conclusion from these findings would be presumptuous since the inactive pseudo acyl enzymes may not manifest the properties of intermediate acyl enzyme formed during catalysis.
The shift in the pK, of the phenolic group back to that of a stronger acid as a result of addition of EDTA to the PACK-or PBCK-inactivated enzymes is compatible with evidence from the BHNA studies that the active site -SH is in a more polar environment in the absence of Ca++ than in the presence of this activating metal ion.
The enzyme protein would appear to have no influence on the ionization of this phenolic group in the absence of Ca++ since under this condition the pK, of the group is the same as that in the parent inactivators and in their reaction products with GSH.
We speculate on this basis that the active site -SH is on or close to the enzyme surface in the unactivated enzyme. PABA acts as a transglutaminase substrate. This was suggested by the finding that PBCK functioned as a more efficient inactivator of the enzyme than II-PACK (Table I). Z-L-glutaminylglpcine and Z-L-glutamine are substrates for transglutaminase, whereas L-glutamine, L-glutaminylglycine, and n-valeramide are neither substrates or inhibitors (8,26). It has been concluded from these findings that a peptide bond involving the CLamino group of glutamine is essential, while one at the carboxyl residue of glutamine is not. The present data show that the car-boxy1 of glutamine may be replaced by hydrogen. This is in accord with a suggestion that the single peptide bond through the amino group of glutamine participates directly in the binding of substrate to enzyme.
The nitro-substituted lactone, 5-nitro-2-coumaranone (13), proved invaluable in the preparation of the inactivators, PACK and PBCK, and the analogue substrates, PG and PABA, used in a portion of this work.
Synthesis in each case was accomplished by lactone ring opening of this internal active ester mediated by nucleophilic attack by the amino group of the esterified amino acid.