Catalytic and Structural Properties of Trypsin-treated 4-Aminobutyrate Aminotransferase*

sedimentation coefficients were corrected for the density and viscosity of water at 20 "C. Samples of enzyme at concen- trations varying between 3 and 10 pM in 0.1 M potassium phosphate (pH 7.0) were studied in the analytical ultracentifuge equipped with a photoelectric scanner (13). Spectroscopy-Absorption spectra were recorded in a Cary Model 15 spectrophotometer. Fluorescence emission spectra were recorded on a spectrofluorimeter equipped with two Bausch and Lomb mono- chromators. The slits of the monochromators were set to give a band width of 3 nm. Polarization of fluorescence measurements were performed on a SLM 4800 spectrofluorimeter. The excitation was set at 440, and fluorescence polarized light emitted by the was passed Corning Glass filter C.S.-3-69.

It is the purpose of this work to report the effect of trypsin digestion on the structural and catalytic properties of 4aminobutyrate aminotransferase.

EXPERIMENTAL PROCEDURES
Purification of Enzymes-4-Aminobutyrate aminotransferase from pig brain was purified according to a procedure previously described (1). This preparation has a specific activity of 20 units/mg at 25 "C, and it migrates as a single protein band on polyacrylamide gel electrophoresis.
Protein concentration was determined by the colorimetric method of Lowry et al. (4). The pyridoxal-5-P content of the purified aminotransferase was determined by the method of Wada and Snell (5). The enzyme succinic semialdehyde dehydrogenase from pig brain was purified by a method already described (6).
Enzymatic Assays-A coupled assay system consisting of two purified enzymes, i.e. 4-aminobutyrate aminotransferase and succinic semialdehyde dehydrogenase, was used to study the catalytic conversion of 4-aminobutyrate to succinic semialdehyde. Enzymatic assays were performed in 0.1 M sodium pyrophosphate (pH 8.4) containing 5 mM NAD+, 30 mM 4-aminobutyrate, and 10 mM 2-oxoglutarate. Initial rate measurements were carried out by monitoring the changes in absorbance a t 340 nm for at least 2 min. A unit of enzyme activity is defined as that amount of enzyme which produces 1 pmol/min of succininc semialdehyde a t 25 "C.
Titration of Thiol Groups-The number of reactive thiol groups was determined by reaction with DTNB' using the procedure of Ellman (7). A molar extinction coefficient of 13,600 M" cm" at 412 nm was used for the determination of the concentration of the anion 2-nitro-5-mercaptobenzoate.
Labeling of the Enzyme-4-Aminobutyrate aminotransferase at a concentration of 5 mg/ml was allowed to react with 1 mM IAF in 0.1 M potassium phosphate (pH 7) a t 4 "C. The reaction was allowed to proceed for 5 h at 4 "C. Excess of free reagent was removed by gel filtration through Sephadex G-25 equilibrated with 0.1 M potassium phosphate (pH 7) containing 0.1 mM 2-mercaptoethanol. The degree of labeling of the enzyme was determined spectrophotometrically using an extinction coefficient of 3.4 X 10' M" cm" a t 490 nm. The incorporation of 0.92 mol of dye/mol of enzyme does not affect the catalytic activity. Polyacrylamide Gel Electrophoresis-Polyacrylamide gel electrophoresis was performed according to the procedure of Davis (8). Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out at 25 "C as described by Laemmli (9). The gel (15% acrylamide) contained 0.1% sodium dodecyl sulfate.
Polyacrylamide gradient gel electrophoresis (4-30%) was performed at 10 "C using a voltage of 125 V for 15 h. Standards of known molecular weight, bovine serum albumin, catalase, and ceruloplasmin, were used in the plots of 1/XL (where XL is the distance (millimeter) of the protein from the origin) uerstu molecular weight 1/3 following the procedure of Manwell (10).
Protein bands were detected by staining with Coomassie blue dye for 1 h and subsequently destained overnight in a solution containing 10% methanol and 7% acetic acid in water.
Tryptic Digestion of 4-Aminobutyrate Aminotransferase-4-Aminobutyrate aminotransferase (5 mg/ml) was incubated with trypsin (0.025 mg/ml) in 50 mM ammonium bicarbonate (pH 7.6) at 37 "c for 2 h. At the end of the incubation, the mixture was filtered through
The fractions eluted from the column were monitored by absorbance measurements a t 280 nm. The protein, which eluted in the void volume of the column, was dialyzed against 0.1 M phosphate buffer (pH 7) containing 0.1 mM 2-mercaptoethanol and used for further characterization.
All the fractions eluted between the excluded and included volumes of the column were combined and lyophilized.
Amino Acid Analysis-Acid hydrolysis was carried out in 4 N methanesulfonic (11) acid and in 6 N HCI a t 110 "C in sealed evacuated tubes for 24 h. The amino acid composition of the peptide mixture before and after acid hydrolysis was determined in a Jeol-6-AH autoanalyzer using the expansion scale of 0.15 absorbance as full scale.
Fingerprint Analysis-This procedure was performed using the method of Schiltz and Reimbolt (12). The sample was applied to a microcrystalline cellulose plate (Polygram-cel 400). and electrophoresis in the first dimension was conducted at, 600 V using the solvent system pyridine/acetic acid/H20/acetone (2:4:7915, v/v) (pH 4.4). Chromatography in the second dimension was performed using the solvent system n-butyl alcohol/pyridine/acetic acid/H20 (15:103:12). The thin layer sheets were sprayed with fluorescamine (0.0176, w/v) in acetone and then sprayed with 10% (v/v) triethylamine in dichloromethane.
Analytical UltracentrifuRation-Sedimentation velocity experiments were conducted in the Spinco Model E analytical ultracentrifuge a t constant temperature in the range of 8-10 "C. For sedimentation velocity experiments, the ultracentrifuge was operated a t 60,000 rpm, and the sedimentation coefficients were corrected for the density and viscosity of water a t 20 "C. Samples of enzyme a t concentrations varying between 3 and 10 p M in 0.1 M potassium phosphate (pH 7.0) were studied in the analytical ultracentifuge equipped with a photoelectric scanner (13).
Spectroscopy-Absorption spectra were recorded in a Cary Model 15 spectrophotometer. Fluorescence emission spectra were recorded on a spectrofluorimeter equipped with two Bausch and Lomb monochromators. The slits of the monochromators were set to give a band width of 3 nm.
Polarization of fluorescence measurements were performed on a SLM 4800 spectrofluorimeter. The excitation was set a t 440, and fluorescence polarized light emitted by the sample was passed through a Corning Glass filter C.S.-3-69.
Fluorescence decay measurements were made using the monophoton technique with an Ortec Model 9200 ns spectrofluorimeter. A free running lamp operating in air a t 1 atm pressure was the exciting light source. The lamp was pulsed a t 10 kHz. Excitation was set a t 440 nm and the emission was filtered through a Corning Glass filter C.S.-3-60. Deconvolution of the data was performed with a computer program based on the least square method of Ware et al. (14).
Materials-Pig brains were obtained from East Tennessee Packing Co., Knoxville, TN. Trypsin was purchased from Worthington. Sephadex G-25, G-200, and G-75, DEAE-Sephadex, CM-Sephadex, and polyacrylamide gradient gels (PAA 4/30) were purchased from Pharmacia Fine Chemicals. Acrylamide, N-N'-methylenebisacrylamide, and standards for protein molecular weight were from Bethesda Research Laboratories, DTNB from Aldrich, and IAF from Molecular Probes.

RESULTS
Digestion of 4-Aminobutyrate Aminotransferase-4-Aminobutyrate aminotransferase (2 mg/ml) was incubated with trypsin (10 pg/ml) in 50 mM ammonium bicarbonate (pH 7.6) a t 37 "C. After various incubation times, samples were removed for enzyme assay and polyacrylamide gel electrophoresis. The overall catalytic activity of 4-aminobutyrate aminotransferase after 2 h of incubation a t 37 "C was observed to be unaffected by trypsin addition, whereas the polyacrylamide gel electrophoresis patterns revealed the presence of mainly one band displaying different mobility than the native enzyme ( Fig. 1). If digestion with trypsin had taken place, the peptides produced would be of small molecular weight and they would have migrated rapidly in the gel electrophoresis system used and therefore would be undetectable.
When the experiment was repeated with samples of enzyme were tested for enzymatic activity. Polyacrylamide (7.5%) gel electrophoresis pattern of native and enzyme treated with trypsin. The sample was digested for 2 h a t 37 "C.
(2 mg/ml) incubated with trypsin concentrations of 20 and 30 pg/ml for 2 h, the same results were obtained, i.e. the overall catalytic activity remained constant. No change in the catalytic behavior of 4-aminobutyrate aminotransferase was detected when the incubation with trypsin was conducted at pH 7 or 7.4 ( Fig. 1). At pH 8, the aminotransferase loses a good deal of its catalytic activity when maintained a t 37 "C for 1 h.
Separation of Peptides-For characterization of the action of trypsin on 4-aminobutyrate aminotransferase, the native enzyme in 0.1 M potassium phosphate buffer (pH 7 ) containing 0.1 mM 2-mercaptoethanol was dialyzed against 50 m M ammonium bicarbonate (pH 7.6). The dialyzed enzyme (100 mg/20 ml) was incubated with 0.5 mg of trypsin of 37 "C for 2 h.
At the end of the incubation, the trypsin-treated enzyme was applied to Sephadex G-75 as described under "Experimental Procedures." The fractions eluted between the excluded and included volumes of the column were combined and lyophilized. Approximately 4.6 mg of material were recovered in the lyophilized fractions. The void volume of the column (93 mg) was dialyzed against 0.1 M potassium phosphate (pH 7 ) containing 1 mM 2-mercaptoethanol and used for further experiments.
The lyophilized material (4.6 mg) was used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fingerprint analysis, and quantitative determination of the amino acid content.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, performed on gels containing 15% acrylamide, failed to detect the presence of peptides of molecular weight greater than 3000. Fingerprint analysis showed the presence of a t least 10 spots after the cellulose sheets were sprayed with fluorescamine (Fig. 2).
The amino acid composition of the peptides released by tryptic cleavage from 4-aminobutyrate aminotransferase was determined on samples hydrolyzed in 6 N HCl or 4 N methanesulfonic acid. The results of eight independent measure-  Determined from the amount of cysteic acid found in the performic acid oxidized sample, and from titrations of the unhydrolyzed material by DTNB.
ments are summarized in Table I. Basic amino acids (5 lysine, 4 arginine, 1 histidine) predominate over acidic ones (3 aspartic, 4 glutamic). The aromatic acids, 2 phenylalanine, 1 tyrosine, and 1 tryptophan, which were detected in unhydrolyzed peptides by absorption and fluorescence spectroscopy, are also present in the hydrolyzed samples. Cysteinyl residues were not detected when unhydrolyzed samples of peptides were titrated with DTNB in the presence and absence of 6 M guanidinium HC1. No free amino acid could be detected on unhydrolyzed samples to amino acid analysis. Characterization of the Shortened Enzyme Deriuatiues-The molecular weight of the shortened enzyme derivative was determined by polyacrylamide gradient gel electrophoresis using standards of known molecular weight, i.e. bovine serum albumin, ceruloplasmin, and catalase. Fig. 3 shows the plot of the reciprocal of the migration distance (millimeter) of the proteins from the origin (1/XL) versus the cubic root (molecular weight 1/3) of the molecular weight.
The plot is linear, and from this plot the molecular weight of the shortened enzyme derivative (Mr = 95,000) was found to be slightly smaller than the molecular weight of the native enzyme ( M , = 100,000).

Aminotransferase
The concentration dependence of the sedimentation coefficients of native and modified enzymes was examined in the analytical ultracentrifuge over the concentration ranges 8-3 ~L M (Fig. 4).
At a concentration of 8 p~, the sedimentation coefficient of the native enzyme (5.15 S) is decreased to a value of 5.0 S as a result of tryptic digestion. This reduction in sedimentation coefficient was also detected a t protein concentrations around 3 PM, the lowest concentration studied in the analytical ultracentrifuge equipped with optical scanner. As illustrated in Fig. 4, there is little variation in the sedimentation values of both species when the concentration is decreased, indicating that none of the enzyme species tend to dissociate into subunits of smaller molecular weight at pH 7 . Catalytic Properties-The absorption spectra of the shortened enzyme derivative displayed the characteristic features of the cofactor pyridoxal-5-P covalently bound to the catalytic site. Two absorption bands centered at 330 and 415 nm were detected over the spectral range 300-500 nm at pH 5.8,6, and 7.0. The intensity of the absorption bands are not affected by variations in the pH of the solution.
The specific activity of the modified enzyme is, within experimental error, identical with that of the native enzyme.
Addition of pyridoxal-5-P, followed by preincubation at 25 "C for 1 h prior to enzymatic assays, did not enhance the specific activity of the shortened enzyme derivative. Hence, the modified enzyme behaves as the native aminotransferase, and limited proteolysis did not abolish the apparent negative cooperativity between subunits. The results of these experiments are summarized in Table 11.
Reactivity of Sulfhydryl Groups-4-Aminobutyrate aminotransferase is easily inactivated by DTNB; the extent of chemical modification was measured by monitoring the release of 2-nitro-5-mercaptobenzoate at 412 nm. Under denaturing conditions, five " S H groups/dimer are titrable with DTNB (15). The time course for a typical reaction under pseudo-first order conditions is given in Fig. 5.
The reaction of approximately 1.2 sulfhydryl groups/dimer proceeds with an observed rate constant of 0.05 min" leading to 90% loss of catalytic activity (Fig. 5 ) . Addition of 2mercaptoethanol (1 mM), followed by dialysis against 0.1 M potassium phosphate (pH 7) containing 1 I~IM 2-mercaptoethanol, readily restores 90% of the original enzymatic activity (results not shown). This well behaved kinetic system can be  exploited to probe the reactivity of the sulfhydryl groups of the shortened enzyme derivative.
When the trypsin-treated enzyme was allowed to react with DTNB under pseudo-first order conditions, the reaction of approximately 2.5 sulfhydryl groups/mol of enzyme has taken place with an observed rate constant of 0.08 min". This increased accessibility of -SH groups to the attacking reagent DTNB reflects local conformational changes of the enzyme elicited by tryptic cleavage.
Fluorescence Polarization-Protein conformational changes elicited by trypsin action were examined using a fluorescent probe covalently linked to the protein.
IAF was selected for these studies for two reasons. First, because it displays absorption and emission properties distinct from the cofactor pyridoxal-5-P; and second, because IAF is a potential modifier of sulfhydryl groups which are not released during proteolytic cleavage (Table I).
In marked contrast to DTNB, IAF and iodoacetamide have no effect on the catalytic activity of the aminotransferase. The enzyme was reacted with IAF under the conditions described under "Experimental Procedures." The labeled enzyme exhibits an absorption band a t 495 nm and emission band centered at 515 nm which can be unambigously assigned to fluorescein (Fig. 6). Using an extinction coefficient of 3.4 X IO4 M" cm" for bound fluorescein, a degree of labeling of 0.9 chromophores/dimer was determined for the reacted enzyme.
In order to ascertain whether IAF has blocked sulfhydryl groups, samples of native and IAF aminotransferase were allowed to react with DTNB under denaturing conditions (6 M guanidinium HC1).
The results of those measurements indicated that 5 -SH groupddimer of native enzyme are blocked by DTNB, whereas only 3.9 -SH/dimer of IAF-aminotransferase have reacted with DTNB (Table 111). Thus, these results support the concept that there is one class of sulfhydryl groups in the enzyme which can be modified by the akylating reagent IAF without any concomitant loss of catalytic activity. The spectroscopic properties of bound fluorescein can be used conveniently to detect large structural fluctuations induced by reaction with trypsin. When IAF-aminotransferase (2 mg/ml) was incubated with trypsin (40 g/ml) at pH 7, and the polar- IAF-aminotransferase (2 mg/ml) incubated with trypsin (40 pg/ mi) for 1 h a t 25 "C in 0.1 M potassium phosphate (pH 7). At the end of the incubation, the digested sample was run through Sephadex G -25 equilibrated with 0.1 M potassium phosphate (pH 7 ) . The protein eluted in the void volume of the column was used for-SH titration and polarization measurements.
Enzymatic activity determined on the shortened enzyme derivative after passage through a Sephadex column G-25 (1 X 15 cm) equilibrated with 0.1 M potassium phosphate (pH 7) containing 1 mM 2-mercaptoethanol. ization values recorded as a function of time, the results included in Fig. 6 were obtained. The initial polarization of IAF-aminotransferase ( p = 0.22) is decreased to a value of p = 0.15 after 60 min of incubation at 25 "C. As shown in Fig.  6, an increase in the fluorescence intensity was also observed as a function of time.
Both parameters, i.e. fluorescence intensity and polarization of fluorescence, reach constant values after 1 h of incubation at 25 "C.
The changes in polarization and fluorescence intensity might be due to the release of either free dye or small peptides tagged with fluorescein. To examine this possibility, the mixture (2.5 ml) containing IAF-aminotransferase and trypsin was passed through a small Sephadex G-25 column (1 X 15 cm) equilibrated with 0.1 M potassium phosphate (pH 7) containing 1 mM 2-mercaptoethanol. The protein eluted in the void volume of the column was labeled with fluorescein and displayed a specific activity of 19.2 units/mg of protein (Table 111).
No free dye or small peptide tagged with fluorescein could be detected on the fractions collected after elution of the protein by fluorescence measurements at 530 nm (excitation 490 nm). These results demonstrate that changes in the luminescence parameters cannot be related to the release of small molecular weight peptides tagged with fluorescein.
When the polarization values of IAF-aminotransferase and trypsin-digested IAF-aminotransferase were recorded as a function of temperature, the plots of 1/P -1/3 versus T/v were found to be linear in the temperature range 6-25 "C ( Fig.   7).
Taking 7, the fluorescence lifetimes of bound fluorescein to be 2.7 and 4 ns (Table 111) for IAF-aminotransferase and trypsin-digested IAF-aminotransferase, respectively, the rotational relaxation time of IAF-aminotransferase, respectively, the rotational relaxation time of IAF-amminotransferase is &fold greater than the corresponding value of the digested protein (Table 111). A rotational relaxation time ratio ( p N / p T ) of 3, as obtained from steady polarization measurements, indicated flexibility of the polypeptide chains with enhanced rotational freedom of the probe covalently attached with sulfhydryl groups of the protein.

DISCUSSION
The results reported in this paper have direct bearing on the effect of conformational fluctuations on the catalytic parameters of the enzyme, 4-aminobutyrate aminotransferase.
Recently, the subject of limited proteolysis of aminotransferase has attracted the attention of two independent laboratories. Hargrove et al. (16) have shown that endopeptidase cathepsin T generates multiple forms of tyrosine aminotransferase. Upon incubation of tyrosine aminotransferase I with cathepsin, tyrosine aminotransferease I1 and a peptide of about 4500 D are generated without release of single amino acids.
On the other hand, Sandmeier and Christen (17) have reported that mitochondrial aspartate aminotransferase is cleaved selectively by trypsin at two peptide bonds, yielding enzyme derivatives devoid of catalytic activity.
The studies reported in this paper have demonstrated that limited proteolysis of 4-aminobutyrate aminotransferase does not impair its catalytic function. The shortened enzyme derivative generated by trypsin action is not only catalytically competent, but also preserves negative cooperativity between subunits.
As a consequence of tryptic digestion, a heterogeneous population of small molecular weight peptides are released from the native enzyme. The mechanism by which trypsin cleaves the aminotransferase remains unclear on the basis of the present data, and it is the subject of further investigations aimed at the elucidation of the sequence of COOH and NH2 terminus of the enzyme.
Research conducted in several laboratories indicate that protein molecules display conformational fluctions in the nanosceond, millisecond, and second time ranges (18)(19). More frequently considered are those fluctuations where a given structural part of the macromolecule is destroyed and a new configuration is built up. The shortened enzyme derivative of 4-aminobutyrate aminotransferase can be used to investigate the effect of large conformational fluctuations on the catalytic power of an enzyme.
The results obtained using chemical and biophysical methods indicate that conformational changes have taken place in 4-aminobutyrate aminotransferase as a result of tryptic action. Thus, the increased reactivity of thiol groups toward DTNB reflects structural fluctuations in the microenvironment surrounding sulfhydryl residues critically connected with catalytic activity.
On the other hand, the spectroscopic properties of a fluorescent probe covalently linked to another class of sulfhydryl groups are influenced by local conformational changes in the protein.
The polypeptide chains of the shortened enzyme derivative display a high degree of flexibility as revealed by polarization of fluorescence measurements. Indeed, the decrease in rotational relaxation time from 106 to 33 ns, the values corresponding to native and trypsin-digested enzymes, respectively, is more than can be expected for macromolecules exhibiting such small differences in molecular weight.
These conformational changes, however, did not prevent the development of the steps required for activation of the enzyme substrate complex, suggesting that structural fluctuations detected at the level of sulfhydryl groups did not perturb the catalytic site domain.