- Email: [email protected]
Indoleacetaldoxime hydro-lyase (4.2.1.29)
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
166-174 (1976)
13,
lndoleacetaldoxime
Hydro-Lyase
III. Further Studies on the Nature I’. S. SHUKLA Department
and Mode of Action of the Enzyme
AND
S. MAHADEVAN
of Biochemistry,
Indian
Received
21,1969; accepted
August
(4.2.1.29)
Institute
of Science, Bangalore-12, December
India
15,1969
Further purification of indoleacetaldoxime (IAOX) hydro-lyase from Gibberella fujikuroi by DEAE-cellulose chromatography is described. The purified enzyme was activated by dehydroascorbic acid (DHA), ascorbic acid (AA), and pyridoxal phosphate (PALP) and was inhibited by thiol compounds and thiol reagents including phenylthiocyanate. Ferrous ions but not ferric ions activated the purified enzyme. The enzyme was activated by dihydrofolic acid but inhibited by tetrahydrofolic acid. Phenylacetaldoxime, a competitive inhibitor, afforded partial protection of the enzyme from the action of N-ethylmaleimide suggesting the involvement of a thiol function at the active site or substrate-binding site. The inhibition of the enzyme by 2,3-dimercaptopropanol was reversed by DHA, PALP, or frozen storage. KCN inhibition of the enzyme was reversed by PALP. NaBHd reduction of the purified enzyme in the presence of PALP gave an active enzyme which was further activated by PALP or DHA but not by ferrous ions. These results suggested a “structural” role for PALP in the activity of IAOX hydro-lyase. Dilute solutions of the purified enzyme, obtained during DEAE-cellulose chromatography and concentrated using sucrose, showed enhanced activity upon frozen storage and thawing. The increase in activity of the enzyme during certain culture conditions, the activation and inhibition of the enzyme by several unrelated compounds, and the effect of freezing indicate that IAOX hydro-lyase is probably a metabolically regulated enzyme with a structure composed of subunits.
We have recently reported the partial purification of indoleacetaldoxime hydrolyase (EC4.2.1.29) from Gibberellafujikuroi, which catalyzes the conversion of indoleacetaldoxime (IAOX)’ to indoleacetonitrile (IAN) (1). The enzyme was found to be activated by dehydroascorbic acid or ascorbic acid, pyridoxal phosphate, and ferric ions. The sulfhydryl nature of the enzyme was indicated by its inhibition, heavy metal 1 Abbreviations used: IAOX, indoleacetaldoxime; PAOX, phenylacetaldoxime; IAN, indoleacetonitrile; DHA, dehydroascorbic acid; AA, ascorbic acid; PALP, pyridoxal phosphate; FA, folic acid; DHFA, dihydrofolic acid; THFA, tetrahydrofolic acid; PHMB, p-hydroxymercuribenzoate; NEM, N-ethylmaleimide; PTC, phenylthiocyanate; BAL, 2,3-dimercaptopropanol; BME, p-mercaptoethanol; GSH, reduced glutathione.
ions, and sulfhydryl reagents. Surprisingly, the enzyme was also inhibited by several thiol compounds such as 2,3-dimercaptopropanol (BAL), /3-mercaptoethanol (BME), cysteine, etc., suggesting a role for an oxidized function in enzyme activity. In this paper further studies on the properties of the enzyme are reported in an effort to elucidate the nature of the enzyme and its mode of action. MATERIALS
AND
METHODS
Chemicals Indoleacetaldoxime (IAOX), phenylacetaldoxime (PAOX), n-dehydroascorbic acid (DHA), recrystallized ammonium sulfate, and peroxidefree ether were prepared as described earlier (1). Dihydrofolic acid (DHFA) was prepared by dithionite reduction of folic acid in the presence of 166
INDOLEACETALDOXIME ascorbic acid (2). Phenylthiocyanate sytlthesized as reported earlier (3). folic acid (THFA) was a gift from Huennekens. All other chemicals were products of high purity.
(PTC) was TetrahydroI)r. F. M. commercial
Determinations IAN was estimated after conversion to indoleacetic acid (IAA) by the procedure outlined earlier (4). Protein was estimated by the method of Lowry et al. (5).
Gel Filtration
of the Enzyme on Xephadex G-25 Column:
Removal of small molecular weight compounds from the enzyme in several experiments reported in this paper was achieved by gel filtration through a Sephadex G-25 column. Coarse grade Sephadex G-25 was soaked in water and deaerated before being packed into a column of 2.5 cm diamet,er to a height of 35 cm. Void volume as determined by using blue dextran 2000 (Pharmacia) was found to be 60 ml. Ammonium sulfate was eluted after 95 ml. For desalting enzyme solutions, 1 or 2 ml volume of the enzyme solution was placed on the column and 15 or 25 ml of the eluate was collected aft,er t,he void volume. This eluate contained over 90y0 of the total protein and enzyme activity.
Culture of Gibberella fujikuroi: The requirement for a cold exposure (g-10”) for several days of the Gibberella fujikuroi (C.B.S., Baarn) mats for enhanced IAOX hydro-lyase act,ivity has already been reported (1). A screening of the following G. fujikuroi cultures, C.B.S. (Baarn), I.A.R.I., N.R.R.L.2633, N.R.R.L.2278, I.M.I. 45545, I.M.I. 112801, Sawada 6604 and Sawada 6349, indicated that Sawada 6604 showed maximum enzyme activity. This strain has been used in all experiment,s reported in this paper. Cultures were grown routinely between 19 and 22’ for 7 days on a potato-sucrose-yeast extract medium (4) and subsequently transferred to a cold chamber (g-10”) for 3-4 days. Mats from such cultures consistently showed high enzyme activity. The enzyme used for further purification by DEAE-cellulose chromatography was the 45p70”i0 ammonium sulfate fraction described earlier (1). This ammonium sulfate fract,ion will be referred to as enzyme I.
PuriJication of the Enzyme by DEAECellulose Column Chromatography: l>EAE-cellulose (Schleicher and Schull, 0.84 meq/g) was regenerated by soaking in 2 M K2HPOa
HYDRO-LYBSE
167
overnight and washed exhaustively with distilled water until free of phosphate. The washed DEAEcellulose was packed to a height of 10 cm in a small jacketed column of a l-cm diameter. The packed column was equilibrated with 1 X 1O-4 111phosphate buffer, pH 7.0, containing ascorbic acid phosphate (l@* M), EDTA (1e5 M), and pyridoxal (lOme M); the pH was adjusted to 7.0. Ice-cold water was circulated around the column. 911 operations were carried out in a cold room (4-8”). One to two milliliters of enzyme I containing 30-60 mg of protein was desalted by passing through the Sephadex G-25 column as described above. Phosphate buffer (1 X IF4 M, pH 7.0) containing PALP (10-O M), EDTA (10-j .II), and ascorbic acid (10m4 M) was used for eluting the protein during gel filtration, as these additives were found to stabilize the enzyme during gel filtration and subsequent fractionation on l>EAEcellulose. The Sephadex eluate was loaded on the l)EAE-cellulose column under slight positive pressure using an inflated rubber bladder. The enzyme was eluted wit,h phosphate buffer, pH 7.0, using a linear gradient between 1 X 10m4M (250 ml) and 2.5 X lo* M (250 ml) phosphate buffer, pH 7.0. The initial buffer contained the above additives . Three-milliliter fractions were collected in an automatic fraction collector. IAOX hydro-lyase activity and the protein content in the fractions were estimated. A typical elution pattern is given in Fig. 1. The enzyme act,ivity was eluted as a sharp peak between tubes 30 and 45, whereas the protein was distributed in all tubes. When these fractions were pooled (enzyme II), a twofold increase in specific activity over enzyme I was obtained. The purification achieved was low, due mainly to a loss of 65y0 in total activity. However, four-fifths of the extraneous proteins present in enzyme I were removed by DEAE-cellulose chromatography. Isnzyme II was either used immediately or was concentrated and stored in the following manner. Excess buffer salts in the enzyme solution could not be removed by dialysis owing to the instability of the enzyme during dialysis. The pooled samples were lyophilized, therefore, to a small volume (2 to 4 ml), and this solution was chilled to 0” in an ice bath until most of the buffer salts had crystallized; the latter were removed by centrifugation. It was found, however, that this method of concentration resulted in an appreciable loss (50@80y0) of enzyme activity. In later experiments, the enzyme II solution containing EDTA (1w4 M) and PALP (10-h M) was concentrated by dehydration using sucrose. For dehydration, the dialysis tubing was pretreated as follows: Visking dialysis tubing was boiled for
168
SHUKLA
AND
MAHADEVAN
1920 Y 5
::
1660
- 700 i - 600
-500
I$
-400
5 a
2
z i2 -300
6 d a 2
-200
-100
I
0
10
20
a
I
30
50 TUBE
FIG. 1. DEAE-cellulose chromatography hydro-lyase activity; Broken line, Protein
RESULTS
on the
The activation of enzyme I by DHA, AA, and PALP and the inhibition by thiol reagents and thiol compounds has been reported (1). These findings have been con-
60
I
60
90
I
Continuous
line,
100
NUMBER
of IAOX content.
20 min in 0.1 N NaOH, washed with distilled water, boiled again for 20 min in distilled water, soaked overnight in lOma M EDTA solution, pH 7.0, and finally washed in water. Sucrose (commercial grade) was stirred in 90% ethanol containing EDTA (200 mg/l), filtered on a Buchner funnel, and air-dried before use. The enzyme solution was placed in the treated dialysis tubing and buried in a tray containing chilled sucrose. By this procedure enzyme II was concentrated to one-tenth its volume in about 8 hr at 4-8”. The concentrated enzyme retained over 40y0 activity and could be stored frozen for several days.
A. Effects of Activators and Inhibitors PuriJied Enzyme II:
I
70
I
40
hydro-lyase.
IAOX
firmed with the purified enzyme II preparation
described
here.
Ferric
ions
(ferric
citrate), which were found to activate several preparations of enzyme I (l), had surprisingly no effect on enzyme II, whereas ferrous ions (ferrous sulfate) at 10e3 M activated enzyme II two- to threefold over controls in several experiments. B. Involvement of Sulfhydryl Group in the Activity of IAOX Hydro-lyase: It has been shown that PHMB at 10e5 M totally inhibited the enzyme, and the inhibition is partially reversed (20%) by the addition of 10B3 M glutathione (GSH) (1). In order to examine whether the enzyme could be protected against PHMB-inhibition by its substrate, IAOX, the enzyme was treated with PHMB in the presence of IAOX, and passed through Sephadex G-25
INDOLEACETALDOXIME
HYDRO-LYASE
169
Protection of IAOX hydro-lyase by PAOX from NEN inhibition. Since the affinity of PHMB to sulfhydryl groups is very high, it is not always possible to demonstrate the protection of an active -SH group by the substrate from the action of PHMB. On the other hand, NE11 reacts much more slowly with -SH groups, forming stable covalent bonds. Under these conditions it may be TABLE I possible to demonstrate the protection of an active -SH group from NEM action by the PROTECTION OF IAOX HYDRO-LYASE FROM NEM INHIBITION BY PAOXa substrate or its analog. Such an experiment was carried out, and the results are preIAN formed (qmoles) sented in Table I. The enzyme was treated Additive during incubation with NEM Activity of enzyme with NEM both in the presence and the without NEM PAOX NOW treatment (control) absence of PAOX, a competitive inhibitor of the enzyme (Ki = 2.2 X lo-* 11) (1). 0 58 460 The enzyme sample treated with NE_\1 alone a Two lots of 1 ml each of enzyme I in 0.1 M was totally inactivated, whereas the enzyme phosphate buffer, pH 7.0, were taken, and to one treated with ?JEM in the presence of PAOX lot 0.1 rmole of PAOX was added. After 2 min, retained some enzyme activity. This exNEM was added to both lots to give a final con periment shows that an -SH group is centration of 1OV M. The tubes were placed at present at the active or substrate-binding room temperature (25”) for 30 min for NEM to site of the enzyme. react with the enzyme. The contents of each tube Effect of sodium arsenite on IAOX hyclrowere filtered separately through a column of Sephlyase: Sodium arsenite is shown to inhibit adex G-25 using 0.01 M phosphate buffer, pH 7.0, sulfhydryl enzymes, particularly those conas the eluant to remove unreacted NEM and PAOX. Fifteen milliliters of the eluate following taining vicinal dithiols (6). Eighty-five and the void volume was collected in each case. Alisixty-five per cent inhibitions were obtained quots of the eluates were tested for activity with by sodium arsenite at lop3 and lo-* WI, IAOX (1 pmole). Incubation was for 30 min at 30”. respectively. An equivalent amount of enzyme not treated with Effect of phenylthiocyanate (PTC) on NEM was also tested for determining the original IAOX hydra-lyase activity: PTC has been activity of the enzyme. shown to interact specifically with -SH groups of simple thiol compounds and also TABLE II to inhibit several -SH enzymes (3). Other EFFECT OF PHENYLTHIOCYANATE ON IAOX functional groups of protein amino acids HYDRO-LYASE ACTIVITY~ such as 01- and e-amino groups, imidazole, IAN formed Y0 Inhibition guanidino or hydroxyl groups did not react Concentration of PTC (mpmoles) with PTC under similar conditions. 0 (Control) 345 The inhibition of IAOX hydro-lyase by 10-h 0 100 PTC (3) was very pronounced (Table II). 0 10-G 100 Even at lo-’ 31 about 90 % inhibition was 87 lo-’ 46 observed. Since the reaction of PTC with IO-8 12 305 sulfhydryl groups was not instantaneous a Enzyme I in 2 ml total volume of 0.1 M phosas with PHMB, the affinity of PTC with phate buffer, pH 7.0, was preincubated for 10 min IAOX hydro-lyase must be relatively high. with 10-J aliquots of different concentrations of In order to test whether inhibition by PTC PTC in alcohol to give the final desired concentracould be reversed by any of the activators of tion. Controls contained only 10 ~1 of alcohol. IAOX hydro-lyase, a l-ml sample of enThe reaction was started by the addition of 1 ml of IAOX solution (1 pmole), and incubation WOO zyme I was treated with PTC (1O-3 M final concentration) for 10 min at room tempercarried out for 30 min at 30”. to remove excess PHMB and IAOX. The eluted enzyme was totally inactive showing that IAOX did not protect the enzyme under these conditions. However, this inactive enzyme was reactivated to 75% of its original activity by the addition of 1O-3 M glutathione. DHA could not reverse the inhibition caused by PH&!IB.
170
SHUKLA
AND
ature and then gel filtered through Sephadex G-25 column to remove excess PTC. This enzyme was completely inactive and was not reactivated by GSH, DHA, AA, or PALP (all at 1OW M). C. Removal of BAL Inhibztion by DHA and Other Compounds: The inhibition of IAOX hydro-lyase by BAL could not be reversed by DHA (1). The inability to reverse inhibition by BAL was possibly due to the very potent inhibitory action of BAL even at low concentrations. Therefore, after treatment of the enzyme with BAL, excess reagent was removed by gel filtration, and the enzyme, so treated, was tested for reactivation with DHA and other compounds (Table III). The results indicated that (a) BAL-treated enzyme was inactive even after gel filtration, (b) enzyme activity was restored almost completely by DHA but not by GSH,
MAHADEVAN TABLE
IT
ACTIVATION OF IAOX HYDRO-LYASE BY FOLLOWING SATURATION OF THE ENZYME DHA AND ITS REMOVAL BY SEPHADEX GEL FILTRATION&
Substanceadded None DHA
(control) (lo+) Original
DHA WITH
G-25
IAN formed (mcrmoles) 143 241
enzyme*
80
a Two milliliters of enzyme I were treated with DHA (lo+ M) for 1 hr at 0”. Excess DHA was then removed by gel filtration through Sephadex G-25. Aliquots of the eluted enzyme were tested for activity. Reaction was started by the addition of 1 pmole of IAOX. Incubation was for 30 min at 30”. * Activity of an equivalent amount of enzyme I not treated with DHA is given.
(c) PALP or frozen storage restored much of the lost enzyme activity, and (d) Fe2+ or Fe3+ ions were poor activators. TABLE III In order to test whether the activation of REVERSAL OF BAL INHIBITION BY ADDITIVES the native enzyme by DHA was due to its FOLLOWING REMOVAL OF EXCESS BAL BY action on the enzyme protein or due to an SEPHADEX G-25 GEL FILTRATION” activation of the catalytic process per se, IAN formed (mpmoles) the enzyme was preincubated with DHA Additions to reaction mixture Experiment I Experiment JJ and then filtered through Sephadex G-25 to remove the excess (unbound) DHA. None 0 0 The eluate following gel filtration was tested DHA (1OW M) 67 247 for activity in the presence or absence of GSH (1O-3 M) 0 DHA. The results are presented in Table Ferrous sulfate (1OW M) 40 IV. Pretreatment with DHA did enhance Ferric citrate (10e3 M) 73 the enzyme activity over the control. PALP (1.7 X 1O-4 M) 146 However, addition of DHA to the reaction None (frozen and thawed)* 166 mixture further enhanced the enzyme activity. Untreated enzyme” 110 240 a One or two milliliters of enzyme I or II was incubated with BAL (1W3 M final concentration) for 20 min. The BAL-treated enzyme was then passed through the Sephadex G-25 column. Aliquots of this eluate were incubated with the various additives for 10 min. The reaction was started by adding 1 @mole IAOX. Incubation was for 30 min at 30”. (a) for Experiment I, enzyme I was used. (b) For Experiment II, enzyme II concentrated by lyophilization was used. * BAL enzyme was stored for frozen for 4 days and subsequently thawed and assayed for IAOX hydro-lyase activity (air-oxidized enzyme). c An equivalent aliquot of the original enzyme before treatment with BAL was assayed for activity.
D. Pyridoxal
Phosphate and the Activity IAOX Hydro-lyase:
of
E$ect of sodium borohydride reduction: The activation of IAOX hydro-lyase by PALP, but not by other pyridoxine derivatives (4), and the effect of sodium borohydride reduction on enzyme I have been reported earlier (1). The behavior of the purified enzyme II to sodium borohydride reduction was tested, and the results are presented in Table V. In experiment I both the untreated and reduced enzymes were equally active, and both were activated 2-3-fold by PALP, DHA, or ascorbic acid. The ac-
INDOLEACETALDOXIME TABLE
171
HYDRO-LYASE TABLE
V
EFFECT OF SODIUM BOROHYDRIDE THE ACTIVITY OF DEAE-CELLULOSE IAOX HYDRO-LYASEa
REDUCTION PURIFIED
ON
REVERSAL OF KCN LYASE ACTIVITY
VI
INHIBITION OF IAOX HYDROBY PYRIDOXAL PHOSPHATE”
Substances added
apt. “0.
I
II
Substances added
None Pyridoxal phosphate (3.3 x lk4 M) DHA (1O-3 M) Ascorbate (IO+ M) None Pyridoxal phosphate (3.3 x IO-4 M) DHA (1OW M) Ferrous sulfate (lo-3 M)
Untreated enzyme (Itl~lllOl~S IAS)
Sodiu? bor$b;k;de enzyme (mpmoles
IAN)
134
134
420 358 289
360 279 230
30
46
287 330
270 305
34
27
* Experiment I: Two milliliters of lyophilized enzyme II containing PALP (lo+ M) was brought to pH 6 with dilute phosphoric acid in a pHstat. It was divided into two l.O-ml lots. One lot was treated with 1.5 mg NaBH4 in 0.1 ml of 1OW N NaOH at 0”. The yellow color of the enzyme solution was completely discharged by NaBH4 reduction. Both lots were finally subjected to Sephadex G-25 gel filtration to remove small molecular weight compounds. The pH of the eluted enzyme was readjusted to 7.0. Aliquots of both untreated and NaBHa-reduced enzyme were incubated with DHA, PALP, or ascorbic acid for 10 min. IAOX (1 rmole) was added, and the incubation was continued for 30 min at 30”. Experiment II: Two milliliters of lyophilized enzyme II containing 1.2 X 1OW M PALP was filtered through Sephadex G-25 to remove excess PALP. The eluate (25 ml in 0.01 M phosphate buffer, pH 7.0) was concentrated to 2 ml by lyophilization. The concentrated enzyme was divided into two l.O-ml lots, and each lot was treated as follows: To 1 ml of enzyme was added 0.5 ml of pH 6.0 phosphate buffer (0.5 M). Three milligrams of NaBH4 in 0.1 ml of lo+ N NaOH was added to reduce the enzyme at 0”. To the reduced enzyme 10 mg of DHA was added. The mixture was gelfiltered to remove excess DHA and other small molecules. To the other 1 ml of enzyme was added 0.5 ml of pH 6.0 phosphate buffer (0.5 M) and 10 mg of DHA. This mixture was directly gel-filtered through Sephadex G-25. Aliquots from both NaBHrreduced and untreated enzyme were tested for activity after preincubation with DHA, PALP, or ferrous sulfate for 10 min. Reaction was started by adding 1 pmole of IAOX. Incubation was for 30 min at 30”.
None KCN KCN lo-4
KCN
(1.7 X
276 87 362
(W3 M)
96
(1O-3 M) (lo-” M) + PALP M)
(1OW M) + DHA
a Enzyme was preincubated with KCN for 10 min. PALP or DHA was then added and kept for 10 min. The reaction was started by the addition of 1 pmole of IAOX, and incubation was continned for 30 min at, 30”. TABLE
VII
EFFECT OF ADDITIVES ON IAOX HYDRO-LYASE ACTIVITY FOLLOWING KCN TREATMENT AND REMOVAL OF UNREACTED KCN BY GEL FILTRATION’” Substance added
IAN formed (m~moles)
None PALP (1.7 X 1O-4 M) DHA (1W M)
57 160 155
Original
113
enzymeb
0 Enzyme I, 1.5 ml, was treated with an inhibitory concentration of KCN, i.e., ~O+M for 30 min at 0” and subsequently filtered through Sephadex G-25 to remove unreacted KCN. Suitable aliquots of the enzyme were treated for 10 min with the above additives and finally incubated with IAOX (1 pmole) for 30 min at 30”. * Activity of an equivalent amount of enzyme not treated with KCN.
tivations of the untreated enzymes were somewhat higher (15-25 %). In experiment II an enzyme sample after reduction with sodium borohydride was treated with an excess of DHA to reoxidize any group on the enzyme oxidizable with DHA and was subsequently filtered through Sephadex G-25. Here the activities of both control and reduced enzyme were low. Both the reduced and untreated enzymes were strikingly activated (8-10 times) by either PALP or DHA. Ferrous sulfate did not activate the inhibited enzyme. Reversal of cyanide inhibition by PALP: Activation of enzyme I by low concentra-
172
SHUKLA
AND
tions and inhibition by higher concentrations of KCN have been described (1). As cyanide ions are known to interact with bound PALP and to inactivate PALPcontaining enzymes (7), the reversal of KCN inhibition by PALP was tested (Table VI). It was found that while PALP completely reversed the inhibition caused by KCN, DHA was ineffective. Since excess KCN was still present in the incubation mixture, another experiment was carried out wherein the enzyme was treated with KCN, and the unreacted KCN removed by passing through Sephadex G-25. As seen from Table VII, the gel-filtered enzyme, which was half as active as the control, was reactivated by PALP as well as by DHA. TABLE EFFECTS OF FOLIC THE
ACTIVITY
Additives
VIII
ACID, DHFA, AND THFA OF IAOX HYDRO-LYASE”
Concentration (Ml
ON
MAHADEVAN
E. Efects of Folic Acid (FA), Dihydrojolic Acid (DHFA), and Tetrahydrojolic Acid (THFA) on IAOX Hydro-lyase: Since several reducing agents were found to inhibit IAOX hydro-lyase (l), the effects of folic acid and its reduced forms were tested for their action on the enzyme. These results are presented in Table VIII. Whereas folic acid itself had no effect on enzyme activity, THFA was found to inhibit totally at 1O-4 M. However, dihydrofolic acid at lop3 M greatly activated the enzyme. Moreover, the activations by DHA (or AA) and DHFA were additive. F. E$ect of Freezing and Thawing on IAOX Hydro-lyase: A method for the concentration of dilute enzyme II using sucrose has been described under Methods. It was found that frozen storage of such a concentrated enzyme had a greater activity upon thawing. This ob-
IAN formed ‘% Inhibition C-J or % (mpmoles) activation (+)
TABLE EFFECT
None Folic acid
None DHFA
DHFA + DHA DHA DHFA + AA AA None THFA
10-a 10-h 10-b
IO-3 10-d IO-6 10-d 10-d 10-d 10-4
103 103 109 115 132 431 184 148
OF FREEZING
IX
AND THAWING
ON IAOX
HYDRO-LYASE~
-tG +10 +225 +40 +10
213
+60
167
+25
190
$44
10-d 10-d
150
+10
10-s 10-4 lo-5
143 0 17 46
-100 -90 -70
a Different concentrations of folic acid, DHFA, and THFA were incubated with the enzyme for 10 min. Subsequently, ascorbic acid or DHA was added and incubated for another 10 min. Finally, IAOX (1 pmole) was added and incubated for 30 min at 30°C.
Experiment
y0 Of initial activity after freezingb and thawing
I
100 175 270 (255c)
II III
a Experiment I: One milliliter of enzyme I (45-7Oyn ammonium sulfate fraction) was diluted to 30 ml with 0.1 M phosphate buffer containing PALP (5 X 1W M) and DHA (10-S M) It was then concentrated using sucrose to 4.0 ml volume. Experiment II: Forty-five milliliters of enzyme II (DEAE-cellulose purified) was concentrated to 3.0 ml using sucrose. Experiment III: Two milliliters of enzyme I was reduced with sodium borohydride and chromatographed on DEAE-cellulose. The eluate (45 ml) was concentrated using sucrose to 4.5 ml volume. Suitable aliquots of each enzyme preparation were used for enzyme assay. Incubation was for 30 min at 3” in the presence of DHA (10W M), PALP (2 X 1OW M), and IAOX (1 rmole) b Stored frozen for 24 hr at -10” before thawing. c Aliquots of concentrated enzyme assayed after second freezing and thawing.
INDOLEACETALDOXTME
servation has been confirmed by experiments given in Table IX. Both untreated and sodium borohydride-reduced enzyme samples purified by DEAE-cellulose chromatography showed increases over initial activities upon freezing and thawing. However, a repetition of freezing and thawing did not enhance the activity any further. A similar increment in activity was not observed with the less purified enzyme I. DISCUSSION
Several intriguing properties of IAOX hydra-lyase reported earlier (1) were investigated further using a more purified enzyme II preparation, and its activation by AA, DHA, and PALP as well as inhibitions by thiol reagents and thiol compounds were confirmed. Ferrous, but not ferric, ions activated the enzyme II. The pronounced inhibition of enzyme activity by thiol reagents such as PHMB, iYEi\i, PTC, and arsenite, the reversal of PH,1IB inhibition by GSH, and the partial protection of the enzyme by the competitive inhibitor, PAOX, from the action of NE&I (Table I) strongly suggest the involvement of an -- SH function in the catalytic activity of IAOX hydro-lyase. Such a thiol funct,ion could have the role of a base in an acidbase catalyzed dehydration mechanism, analogous to the enzymatic dehydration of malate (8) or citrate (9). However, neither the chemical dehydration of an aldoxime nor the enzymatic dehydration of IAOX described here is a reversible reaction like other dehydrations across carbon-carbon bonds. Indeed, the chemical hydration of a nitrile yields an amide and not an aldoxime (10). Hence a simple acid-base catalyzed mechanism for oxime dehydration appea,rs unlikely. An alternative role for the participation of a thiol function in this reaction can be proposed in the formation of an enzymebound thiohydroxamate intermediate as given in Fig. 2a. Such an intermediate can be visualized in analogy with the naturally occurring glucosinolate compounds (Fig. 2b). Thiohydroxamic acids (Fig. 2c) are known to decompose readily to the corresponding
HYDRO-LYASE
173
s s 7 Enz.-S-Fi G1”-S-$ HS-E N,OH K+ -O$O/N N, OH (a)
(b) FIG.~
(cl
nitriles (11). Nitriles are also formed from glucosinolate compounds by the action of myrosinase under certain conditions (12). In view of these observations, the enzymebound thiohydroxamate intermediate as proposed in Fig. 2a could decompose to give the nitrile. The requirement for an oxidized function on the enzyme for its activity was suggested earlier, based on the inhibition of the enzyme by several reducing agents, such as thiol compounds, sodium dithionite, and sodium borohydride, and its reversibility by an oxidant such as DHA (1). BAL inhibition was reversed by DHA following removal of unreacted BAL by gel filtration (Table III). Such a BAL-inhibited enzyme was also reactivated by frozen storage (Table III), a condition during which air oxidation of BAL-reduced function could have taken place (13). The inactivation of the enzyme by several reducing agents may be explained by the involvement of an oxidized function either in the catalytic process per se or in maintaining the structural integrity of the enzyme molecule. The nonspecificity of the reducing agents in causing inhibition makes the latter explanation more attractive in the case of IAOX hydro-lyase. It is now generally accepted that metabolically regulated enzymes have subunit structure and that their catalytic function is regulated by association-dissociation processes. Dissociation of such enzymes is facilitated by several means such as dilution, change in ionic concentration, or chemical agents including reductants, as has been shown with muscle phosphorylase (14). The following properties of IAOX hydro-lyase suggest that it may be a metabolically regulated enzyme composed of subunits: (a) the expression of its activit.y in the fungus is dependent on culture conditions such as an exposure to low temperature; (b) it is activated by com-
174
SHUKLA
AND
pounds such as DHA, AA, and DHFA which are not chemically related to the substrate; (c) it has a specific requirement for pyridoxal phosphate (4), and sodium borohydride reduction does not inactivate it (1) suggesting a structural role for the PALP; (d) it is unstable in dilute solution of low ionic concentration as obtained during DEAE-cellulose chromatography and is stabilized by concentration with sucrose; and (e) the concentrated enzyme is activated by freezing (Table IX). The activation of sucrose-concentrated enzyme by freezing and thawing (Table IX) is unusual since several enzymes are inactivated by freezing (13, 15). Aggregation of subunit polypeptide chains as a consequence of freezing to form hybrid structures (16) has been suggested by Chilson et al. (13) to explain the inactivation of lactic dehydrogenase. Such an inactivation was decreased, however, or abolished in the presence of sucrose (13). If aggregation of subunits dissociated by dilution and exposure to low ionic concentration during DEAEcellulose chromatography was the reason for activation of IAOX hydro-lyase on freezing, it is probable that this enzyme has only one type of subunits. ACKNOWLEDGMENTS Thanks are due to Dr. S. A. Kumar for helpful discussions and to Professor P. S. Sarma for facilities accorded. REFERENCES 1. SHUKLA, P. S., AND MAHADEVAN, S., Arch. B&hem. Biophys. 126, 873 (1968).
MAHADEVAN 2. BLAKLEY, R. L., Nature (London) 188, 231 (1960). 3. MAHADEVAN, S., SHUKLA, P. S., KALYANARAMAN, V. S., AND KUMAR, S. A., FEBS Letters 2, 149 (1969). 4. KUMAR, S. A., AND MAHADEVAN, S., Arch. Biochem. Biophys. 103, 516 (1963). 5. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 6. FLUHARTY, A. L., AND SANADI, D. R., J. Biol. Chem. 236, 2272 (1961). 7. DIXON, M., AND WEBB, E. C. (Eds.), “Enzymes, ” p. 376. Academic Press, New York (1958). 8. MALMSTROM, Bo G., in “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), Vol. 5, p. 455. Academic Press, New York (1961). 9. DICKMAN, S. It., in “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), Vol. 5, p. 495. Academic Press, New York (1961). 10. WESTHEIMER, F. H. in “Mechanism of Enzyme Action” (W. D. McElroy, and B. Glass, eds.), p. 342. Johns Hopkins Press, Baltimore (1954). 11. ETTLINGER, M. J., AND LUNDEEN, A. J., J. Am. Chem. Sot. 79, 1764 (1957). 12. VIRTANEN, A. I., Arch. Biochem. Biophys. Suppl. 1, 200 (1962). 13. CHILSON, 0. P., COSTELLO, L. A., AND KAPLAN, N. O., Fed. Proc. 24, Suppl. 15, S-55 (1965). 14. WANG, J. H., SHONKA, M. L., AND GRAVES, D. J., Biochem. 4, 2296 (1965). 15. SHALTIEL, S., HEDRICK, J., AND FISCHER, E. H., Biochem. 6, 2108 (1966). 16. TAPPEL, A. L., in “Cryobiology” (H. T. Meryman, ed.), p. 163. Academic Press, New York (1966). 17. LEVITT, J., J. Theoret. Biol. 3, 355 (1962).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
166-174 (1976)
13,
lndoleacetaldoxime
Hydro-Lyase
III. Further Studies on the Nature I’. S. SHUKLA Department
and Mode of Action of the Enzyme
AND
S. MAHADEVAN
of Biochemistry,
Indian
Received
21,1969; accepted
August
(4.2.1.29)
Institute
of Science, Bangalore-12, December
India
15,1969
Further purification of indoleacetaldoxime (IAOX) hydro-lyase from Gibberella fujikuroi by DEAE-cellulose chromatography is described. The purified enzyme was activated by dehydroascorbic acid (DHA), ascorbic acid (AA), and pyridoxal phosphate (PALP) and was inhibited by thiol compounds and thiol reagents including phenylthiocyanate. Ferrous ions but not ferric ions activated the purified enzyme. The enzyme was activated by dihydrofolic acid but inhibited by tetrahydrofolic acid. Phenylacetaldoxime, a competitive inhibitor, afforded partial protection of the enzyme from the action of N-ethylmaleimide suggesting the involvement of a thiol function at the active site or substrate-binding site. The inhibition of the enzyme by 2,3-dimercaptopropanol was reversed by DHA, PALP, or frozen storage. KCN inhibition of the enzyme was reversed by PALP. NaBHd reduction of the purified enzyme in the presence of PALP gave an active enzyme which was further activated by PALP or DHA but not by ferrous ions. These results suggested a “structural” role for PALP in the activity of IAOX hydro-lyase. Dilute solutions of the purified enzyme, obtained during DEAE-cellulose chromatography and concentrated using sucrose, showed enhanced activity upon frozen storage and thawing. The increase in activity of the enzyme during certain culture conditions, the activation and inhibition of the enzyme by several unrelated compounds, and the effect of freezing indicate that IAOX hydro-lyase is probably a metabolically regulated enzyme with a structure composed of subunits.
We have recently reported the partial purification of indoleacetaldoxime hydrolyase (EC4.2.1.29) from Gibberellafujikuroi, which catalyzes the conversion of indoleacetaldoxime (IAOX)’ to indoleacetonitrile (IAN) (1). The enzyme was found to be activated by dehydroascorbic acid or ascorbic acid, pyridoxal phosphate, and ferric ions. The sulfhydryl nature of the enzyme was indicated by its inhibition, heavy metal 1 Abbreviations used: IAOX, indoleacetaldoxime; PAOX, phenylacetaldoxime; IAN, indoleacetonitrile; DHA, dehydroascorbic acid; AA, ascorbic acid; PALP, pyridoxal phosphate; FA, folic acid; DHFA, dihydrofolic acid; THFA, tetrahydrofolic acid; PHMB, p-hydroxymercuribenzoate; NEM, N-ethylmaleimide; PTC, phenylthiocyanate; BAL, 2,3-dimercaptopropanol; BME, p-mercaptoethanol; GSH, reduced glutathione.
ions, and sulfhydryl reagents. Surprisingly, the enzyme was also inhibited by several thiol compounds such as 2,3-dimercaptopropanol (BAL), /3-mercaptoethanol (BME), cysteine, etc., suggesting a role for an oxidized function in enzyme activity. In this paper further studies on the properties of the enzyme are reported in an effort to elucidate the nature of the enzyme and its mode of action. MATERIALS
AND
METHODS
Chemicals Indoleacetaldoxime (IAOX), phenylacetaldoxime (PAOX), n-dehydroascorbic acid (DHA), recrystallized ammonium sulfate, and peroxidefree ether were prepared as described earlier (1). Dihydrofolic acid (DHFA) was prepared by dithionite reduction of folic acid in the presence of 166
INDOLEACETALDOXIME ascorbic acid (2). Phenylthiocyanate sytlthesized as reported earlier (3). folic acid (THFA) was a gift from Huennekens. All other chemicals were products of high purity.
(PTC) was TetrahydroI)r. F. M. commercial
Determinations IAN was estimated after conversion to indoleacetic acid (IAA) by the procedure outlined earlier (4). Protein was estimated by the method of Lowry et al. (5).
Gel Filtration
of the Enzyme on Xephadex G-25 Column:
Removal of small molecular weight compounds from the enzyme in several experiments reported in this paper was achieved by gel filtration through a Sephadex G-25 column. Coarse grade Sephadex G-25 was soaked in water and deaerated before being packed into a column of 2.5 cm diamet,er to a height of 35 cm. Void volume as determined by using blue dextran 2000 (Pharmacia) was found to be 60 ml. Ammonium sulfate was eluted after 95 ml. For desalting enzyme solutions, 1 or 2 ml volume of the enzyme solution was placed on the column and 15 or 25 ml of the eluate was collected aft,er t,he void volume. This eluate contained over 90y0 of the total protein and enzyme activity.
Culture of Gibberella fujikuroi: The requirement for a cold exposure (g-10”) for several days of the Gibberella fujikuroi (C.B.S., Baarn) mats for enhanced IAOX hydro-lyase act,ivity has already been reported (1). A screening of the following G. fujikuroi cultures, C.B.S. (Baarn), I.A.R.I., N.R.R.L.2633, N.R.R.L.2278, I.M.I. 45545, I.M.I. 112801, Sawada 6604 and Sawada 6349, indicated that Sawada 6604 showed maximum enzyme activity. This strain has been used in all experiment,s reported in this paper. Cultures were grown routinely between 19 and 22’ for 7 days on a potato-sucrose-yeast extract medium (4) and subsequently transferred to a cold chamber (g-10”) for 3-4 days. Mats from such cultures consistently showed high enzyme activity. The enzyme used for further purification by DEAE-cellulose chromatography was the 45p70”i0 ammonium sulfate fraction described earlier (1). This ammonium sulfate fract,ion will be referred to as enzyme I.
PuriJication of the Enzyme by DEAECellulose Column Chromatography: l>EAE-cellulose (Schleicher and Schull, 0.84 meq/g) was regenerated by soaking in 2 M K2HPOa
HYDRO-LYBSE
167
overnight and washed exhaustively with distilled water until free of phosphate. The washed DEAEcellulose was packed to a height of 10 cm in a small jacketed column of a l-cm diameter. The packed column was equilibrated with 1 X 1O-4 111phosphate buffer, pH 7.0, containing ascorbic acid phosphate (l@* M), EDTA (1e5 M), and pyridoxal (lOme M); the pH was adjusted to 7.0. Ice-cold water was circulated around the column. 911 operations were carried out in a cold room (4-8”). One to two milliliters of enzyme I containing 30-60 mg of protein was desalted by passing through the Sephadex G-25 column as described above. Phosphate buffer (1 X IF4 M, pH 7.0) containing PALP (10-O M), EDTA (10-j .II), and ascorbic acid (10m4 M) was used for eluting the protein during gel filtration, as these additives were found to stabilize the enzyme during gel filtration and subsequent fractionation on l>EAEcellulose. The Sephadex eluate was loaded on the l)EAE-cellulose column under slight positive pressure using an inflated rubber bladder. The enzyme was eluted wit,h phosphate buffer, pH 7.0, using a linear gradient between 1 X 10m4M (250 ml) and 2.5 X lo* M (250 ml) phosphate buffer, pH 7.0. The initial buffer contained the above additives . Three-milliliter fractions were collected in an automatic fraction collector. IAOX hydro-lyase activity and the protein content in the fractions were estimated. A typical elution pattern is given in Fig. 1. The enzyme act,ivity was eluted as a sharp peak between tubes 30 and 45, whereas the protein was distributed in all tubes. When these fractions were pooled (enzyme II), a twofold increase in specific activity over enzyme I was obtained. The purification achieved was low, due mainly to a loss of 65y0 in total activity. However, four-fifths of the extraneous proteins present in enzyme I were removed by DEAE-cellulose chromatography. Isnzyme II was either used immediately or was concentrated and stored in the following manner. Excess buffer salts in the enzyme solution could not be removed by dialysis owing to the instability of the enzyme during dialysis. The pooled samples were lyophilized, therefore, to a small volume (2 to 4 ml), and this solution was chilled to 0” in an ice bath until most of the buffer salts had crystallized; the latter were removed by centrifugation. It was found, however, that this method of concentration resulted in an appreciable loss (50@80y0) of enzyme activity. In later experiments, the enzyme II solution containing EDTA (1w4 M) and PALP (10-h M) was concentrated by dehydration using sucrose. For dehydration, the dialysis tubing was pretreated as follows: Visking dialysis tubing was boiled for
168
SHUKLA
AND
MAHADEVAN
1920 Y 5
::
1660
- 700 i - 600
-500
I$
-400
5 a
2
z i2 -300
6 d a 2
-200
-100
I
0
10
20
a
I
30
50 TUBE
FIG. 1. DEAE-cellulose chromatography hydro-lyase activity; Broken line, Protein
RESULTS
on the
The activation of enzyme I by DHA, AA, and PALP and the inhibition by thiol reagents and thiol compounds has been reported (1). These findings have been con-
60
I
60
90
I
Continuous
line,
100
NUMBER
of IAOX content.
20 min in 0.1 N NaOH, washed with distilled water, boiled again for 20 min in distilled water, soaked overnight in lOma M EDTA solution, pH 7.0, and finally washed in water. Sucrose (commercial grade) was stirred in 90% ethanol containing EDTA (200 mg/l), filtered on a Buchner funnel, and air-dried before use. The enzyme solution was placed in the treated dialysis tubing and buried in a tray containing chilled sucrose. By this procedure enzyme II was concentrated to one-tenth its volume in about 8 hr at 4-8”. The concentrated enzyme retained over 40y0 activity and could be stored frozen for several days.
A. Effects of Activators and Inhibitors PuriJied Enzyme II:
I
70
I
40
hydro-lyase.
IAOX
firmed with the purified enzyme II preparation
described
here.
Ferric
ions
(ferric
citrate), which were found to activate several preparations of enzyme I (l), had surprisingly no effect on enzyme II, whereas ferrous ions (ferrous sulfate) at 10e3 M activated enzyme II two- to threefold over controls in several experiments. B. Involvement of Sulfhydryl Group in the Activity of IAOX Hydro-lyase: It has been shown that PHMB at 10e5 M totally inhibited the enzyme, and the inhibition is partially reversed (20%) by the addition of 10B3 M glutathione (GSH) (1). In order to examine whether the enzyme could be protected against PHMB-inhibition by its substrate, IAOX, the enzyme was treated with PHMB in the presence of IAOX, and passed through Sephadex G-25
INDOLEACETALDOXIME
HYDRO-LYASE
169
Protection of IAOX hydro-lyase by PAOX from NEN inhibition. Since the affinity of PHMB to sulfhydryl groups is very high, it is not always possible to demonstrate the protection of an active -SH group by the substrate from the action of PHMB. On the other hand, NE11 reacts much more slowly with -SH groups, forming stable covalent bonds. Under these conditions it may be TABLE I possible to demonstrate the protection of an active -SH group from NEM action by the PROTECTION OF IAOX HYDRO-LYASE FROM NEM INHIBITION BY PAOXa substrate or its analog. Such an experiment was carried out, and the results are preIAN formed (qmoles) sented in Table I. The enzyme was treated Additive during incubation with NEM Activity of enzyme with NEM both in the presence and the without NEM PAOX NOW treatment (control) absence of PAOX, a competitive inhibitor of the enzyme (Ki = 2.2 X lo-* 11) (1). 0 58 460 The enzyme sample treated with NE_\1 alone a Two lots of 1 ml each of enzyme I in 0.1 M was totally inactivated, whereas the enzyme phosphate buffer, pH 7.0, were taken, and to one treated with ?JEM in the presence of PAOX lot 0.1 rmole of PAOX was added. After 2 min, retained some enzyme activity. This exNEM was added to both lots to give a final con periment shows that an -SH group is centration of 1OV M. The tubes were placed at present at the active or substrate-binding room temperature (25”) for 30 min for NEM to site of the enzyme. react with the enzyme. The contents of each tube Effect of sodium arsenite on IAOX hyclrowere filtered separately through a column of Sephlyase: Sodium arsenite is shown to inhibit adex G-25 using 0.01 M phosphate buffer, pH 7.0, sulfhydryl enzymes, particularly those conas the eluant to remove unreacted NEM and PAOX. Fifteen milliliters of the eluate following taining vicinal dithiols (6). Eighty-five and the void volume was collected in each case. Alisixty-five per cent inhibitions were obtained quots of the eluates were tested for activity with by sodium arsenite at lop3 and lo-* WI, IAOX (1 pmole). Incubation was for 30 min at 30”. respectively. An equivalent amount of enzyme not treated with Effect of phenylthiocyanate (PTC) on NEM was also tested for determining the original IAOX hydra-lyase activity: PTC has been activity of the enzyme. shown to interact specifically with -SH groups of simple thiol compounds and also TABLE II to inhibit several -SH enzymes (3). Other EFFECT OF PHENYLTHIOCYANATE ON IAOX functional groups of protein amino acids HYDRO-LYASE ACTIVITY~ such as 01- and e-amino groups, imidazole, IAN formed Y0 Inhibition guanidino or hydroxyl groups did not react Concentration of PTC (mpmoles) with PTC under similar conditions. 0 (Control) 345 The inhibition of IAOX hydro-lyase by 10-h 0 100 PTC (3) was very pronounced (Table II). 0 10-G 100 Even at lo-’ 31 about 90 % inhibition was 87 lo-’ 46 observed. Since the reaction of PTC with IO-8 12 305 sulfhydryl groups was not instantaneous a Enzyme I in 2 ml total volume of 0.1 M phosas with PHMB, the affinity of PTC with phate buffer, pH 7.0, was preincubated for 10 min IAOX hydro-lyase must be relatively high. with 10-J aliquots of different concentrations of In order to test whether inhibition by PTC PTC in alcohol to give the final desired concentracould be reversed by any of the activators of tion. Controls contained only 10 ~1 of alcohol. IAOX hydro-lyase, a l-ml sample of enThe reaction was started by the addition of 1 ml of IAOX solution (1 pmole), and incubation WOO zyme I was treated with PTC (1O-3 M final concentration) for 10 min at room tempercarried out for 30 min at 30”. to remove excess PHMB and IAOX. The eluted enzyme was totally inactive showing that IAOX did not protect the enzyme under these conditions. However, this inactive enzyme was reactivated to 75% of its original activity by the addition of 1O-3 M glutathione. DHA could not reverse the inhibition caused by PH&!IB.
170
SHUKLA
AND
ature and then gel filtered through Sephadex G-25 column to remove excess PTC. This enzyme was completely inactive and was not reactivated by GSH, DHA, AA, or PALP (all at 1OW M). C. Removal of BAL Inhibztion by DHA and Other Compounds: The inhibition of IAOX hydro-lyase by BAL could not be reversed by DHA (1). The inability to reverse inhibition by BAL was possibly due to the very potent inhibitory action of BAL even at low concentrations. Therefore, after treatment of the enzyme with BAL, excess reagent was removed by gel filtration, and the enzyme, so treated, was tested for reactivation with DHA and other compounds (Table III). The results indicated that (a) BAL-treated enzyme was inactive even after gel filtration, (b) enzyme activity was restored almost completely by DHA but not by GSH,
MAHADEVAN TABLE
IT
ACTIVATION OF IAOX HYDRO-LYASE BY FOLLOWING SATURATION OF THE ENZYME DHA AND ITS REMOVAL BY SEPHADEX GEL FILTRATION&
Substanceadded None DHA
(control) (lo+) Original
DHA WITH
G-25
IAN formed (mcrmoles) 143 241
enzyme*
80
a Two milliliters of enzyme I were treated with DHA (lo+ M) for 1 hr at 0”. Excess DHA was then removed by gel filtration through Sephadex G-25. Aliquots of the eluted enzyme were tested for activity. Reaction was started by the addition of 1 pmole of IAOX. Incubation was for 30 min at 30”. * Activity of an equivalent amount of enzyme I not treated with DHA is given.
(c) PALP or frozen storage restored much of the lost enzyme activity, and (d) Fe2+ or Fe3+ ions were poor activators. TABLE III In order to test whether the activation of REVERSAL OF BAL INHIBITION BY ADDITIVES the native enzyme by DHA was due to its FOLLOWING REMOVAL OF EXCESS BAL BY action on the enzyme protein or due to an SEPHADEX G-25 GEL FILTRATION” activation of the catalytic process per se, IAN formed (mpmoles) the enzyme was preincubated with DHA Additions to reaction mixture Experiment I Experiment JJ and then filtered through Sephadex G-25 to remove the excess (unbound) DHA. None 0 0 The eluate following gel filtration was tested DHA (1OW M) 67 247 for activity in the presence or absence of GSH (1O-3 M) 0 DHA. The results are presented in Table Ferrous sulfate (1OW M) 40 IV. Pretreatment with DHA did enhance Ferric citrate (10e3 M) 73 the enzyme activity over the control. PALP (1.7 X 1O-4 M) 146 However, addition of DHA to the reaction None (frozen and thawed)* 166 mixture further enhanced the enzyme activity. Untreated enzyme” 110 240 a One or two milliliters of enzyme I or II was incubated with BAL (1W3 M final concentration) for 20 min. The BAL-treated enzyme was then passed through the Sephadex G-25 column. Aliquots of this eluate were incubated with the various additives for 10 min. The reaction was started by adding 1 @mole IAOX. Incubation was for 30 min at 30”. (a) for Experiment I, enzyme I was used. (b) For Experiment II, enzyme II concentrated by lyophilization was used. * BAL enzyme was stored for frozen for 4 days and subsequently thawed and assayed for IAOX hydro-lyase activity (air-oxidized enzyme). c An equivalent aliquot of the original enzyme before treatment with BAL was assayed for activity.
D. Pyridoxal
Phosphate and the Activity IAOX Hydro-lyase:
of
E$ect of sodium borohydride reduction: The activation of IAOX hydro-lyase by PALP, but not by other pyridoxine derivatives (4), and the effect of sodium borohydride reduction on enzyme I have been reported earlier (1). The behavior of the purified enzyme II to sodium borohydride reduction was tested, and the results are presented in Table V. In experiment I both the untreated and reduced enzymes were equally active, and both were activated 2-3-fold by PALP, DHA, or ascorbic acid. The ac-
INDOLEACETALDOXIME TABLE
171
HYDRO-LYASE TABLE
V
EFFECT OF SODIUM BOROHYDRIDE THE ACTIVITY OF DEAE-CELLULOSE IAOX HYDRO-LYASEa
REDUCTION PURIFIED
ON
REVERSAL OF KCN LYASE ACTIVITY
VI
INHIBITION OF IAOX HYDROBY PYRIDOXAL PHOSPHATE”
Substances added
apt. “0.
I
II
Substances added
None Pyridoxal phosphate (3.3 x lk4 M) DHA (1O-3 M) Ascorbate (IO+ M) None Pyridoxal phosphate (3.3 x IO-4 M) DHA (1OW M) Ferrous sulfate (lo-3 M)
Untreated enzyme (Itl~lllOl~S IAS)
Sodiu? bor$b;k;de enzyme (mpmoles
IAN)
134
134
420 358 289
360 279 230
30
46
287 330
270 305
34
27
* Experiment I: Two milliliters of lyophilized enzyme II containing PALP (lo+ M) was brought to pH 6 with dilute phosphoric acid in a pHstat. It was divided into two l.O-ml lots. One lot was treated with 1.5 mg NaBH4 in 0.1 ml of 1OW N NaOH at 0”. The yellow color of the enzyme solution was completely discharged by NaBH4 reduction. Both lots were finally subjected to Sephadex G-25 gel filtration to remove small molecular weight compounds. The pH of the eluted enzyme was readjusted to 7.0. Aliquots of both untreated and NaBHa-reduced enzyme were incubated with DHA, PALP, or ascorbic acid for 10 min. IAOX (1 rmole) was added, and the incubation was continued for 30 min at 30”. Experiment II: Two milliliters of lyophilized enzyme II containing 1.2 X 1OW M PALP was filtered through Sephadex G-25 to remove excess PALP. The eluate (25 ml in 0.01 M phosphate buffer, pH 7.0) was concentrated to 2 ml by lyophilization. The concentrated enzyme was divided into two l.O-ml lots, and each lot was treated as follows: To 1 ml of enzyme was added 0.5 ml of pH 6.0 phosphate buffer (0.5 M). Three milligrams of NaBH4 in 0.1 ml of lo+ N NaOH was added to reduce the enzyme at 0”. To the reduced enzyme 10 mg of DHA was added. The mixture was gelfiltered to remove excess DHA and other small molecules. To the other 1 ml of enzyme was added 0.5 ml of pH 6.0 phosphate buffer (0.5 M) and 10 mg of DHA. This mixture was directly gel-filtered through Sephadex G-25. Aliquots from both NaBHrreduced and untreated enzyme were tested for activity after preincubation with DHA, PALP, or ferrous sulfate for 10 min. Reaction was started by adding 1 pmole of IAOX. Incubation was for 30 min at 30”.
None KCN KCN lo-4
KCN
(1.7 X
276 87 362
(W3 M)
96
(1O-3 M) (lo-” M) + PALP M)
(1OW M) + DHA
a Enzyme was preincubated with KCN for 10 min. PALP or DHA was then added and kept for 10 min. The reaction was started by the addition of 1 pmole of IAOX, and incubation was continned for 30 min at, 30”. TABLE
VII
EFFECT OF ADDITIVES ON IAOX HYDRO-LYASE ACTIVITY FOLLOWING KCN TREATMENT AND REMOVAL OF UNREACTED KCN BY GEL FILTRATION’” Substance added
IAN formed (m~moles)
None PALP (1.7 X 1O-4 M) DHA (1W M)
57 160 155
Original
113
enzymeb
0 Enzyme I, 1.5 ml, was treated with an inhibitory concentration of KCN, i.e., ~O+M for 30 min at 0” and subsequently filtered through Sephadex G-25 to remove unreacted KCN. Suitable aliquots of the enzyme were treated for 10 min with the above additives and finally incubated with IAOX (1 pmole) for 30 min at 30”. * Activity of an equivalent amount of enzyme not treated with KCN.
tivations of the untreated enzymes were somewhat higher (15-25 %). In experiment II an enzyme sample after reduction with sodium borohydride was treated with an excess of DHA to reoxidize any group on the enzyme oxidizable with DHA and was subsequently filtered through Sephadex G-25. Here the activities of both control and reduced enzyme were low. Both the reduced and untreated enzymes were strikingly activated (8-10 times) by either PALP or DHA. Ferrous sulfate did not activate the inhibited enzyme. Reversal of cyanide inhibition by PALP: Activation of enzyme I by low concentra-
172
SHUKLA
AND
tions and inhibition by higher concentrations of KCN have been described (1). As cyanide ions are known to interact with bound PALP and to inactivate PALPcontaining enzymes (7), the reversal of KCN inhibition by PALP was tested (Table VI). It was found that while PALP completely reversed the inhibition caused by KCN, DHA was ineffective. Since excess KCN was still present in the incubation mixture, another experiment was carried out wherein the enzyme was treated with KCN, and the unreacted KCN removed by passing through Sephadex G-25. As seen from Table VII, the gel-filtered enzyme, which was half as active as the control, was reactivated by PALP as well as by DHA. TABLE EFFECTS OF FOLIC THE
ACTIVITY
Additives
VIII
ACID, DHFA, AND THFA OF IAOX HYDRO-LYASE”
Concentration (Ml
ON
MAHADEVAN
E. Efects of Folic Acid (FA), Dihydrojolic Acid (DHFA), and Tetrahydrojolic Acid (THFA) on IAOX Hydro-lyase: Since several reducing agents were found to inhibit IAOX hydro-lyase (l), the effects of folic acid and its reduced forms were tested for their action on the enzyme. These results are presented in Table VIII. Whereas folic acid itself had no effect on enzyme activity, THFA was found to inhibit totally at 1O-4 M. However, dihydrofolic acid at lop3 M greatly activated the enzyme. Moreover, the activations by DHA (or AA) and DHFA were additive. F. E$ect of Freezing and Thawing on IAOX Hydro-lyase: A method for the concentration of dilute enzyme II using sucrose has been described under Methods. It was found that frozen storage of such a concentrated enzyme had a greater activity upon thawing. This ob-
IAN formed ‘% Inhibition C-J or % (mpmoles) activation (+)
TABLE EFFECT
None Folic acid
None DHFA
DHFA + DHA DHA DHFA + AA AA None THFA
10-a 10-h 10-b
IO-3 10-d IO-6 10-d 10-d 10-d 10-4
103 103 109 115 132 431 184 148
OF FREEZING
IX
AND THAWING
ON IAOX
HYDRO-LYASE~
-tG +10 +225 +40 +10
213
+60
167
+25
190
$44
10-d 10-d
150
+10
10-s 10-4 lo-5
143 0 17 46
-100 -90 -70
a Different concentrations of folic acid, DHFA, and THFA were incubated with the enzyme for 10 min. Subsequently, ascorbic acid or DHA was added and incubated for another 10 min. Finally, IAOX (1 pmole) was added and incubated for 30 min at 30°C.
Experiment
y0 Of initial activity after freezingb and thawing
I
100 175 270 (255c)
II III
a Experiment I: One milliliter of enzyme I (45-7Oyn ammonium sulfate fraction) was diluted to 30 ml with 0.1 M phosphate buffer containing PALP (5 X 1W M) and DHA (10-S M) It was then concentrated using sucrose to 4.0 ml volume. Experiment II: Forty-five milliliters of enzyme II (DEAE-cellulose purified) was concentrated to 3.0 ml using sucrose. Experiment III: Two milliliters of enzyme I was reduced with sodium borohydride and chromatographed on DEAE-cellulose. The eluate (45 ml) was concentrated using sucrose to 4.5 ml volume. Suitable aliquots of each enzyme preparation were used for enzyme assay. Incubation was for 30 min at 3” in the presence of DHA (10W M), PALP (2 X 1OW M), and IAOX (1 rmole) b Stored frozen for 24 hr at -10” before thawing. c Aliquots of concentrated enzyme assayed after second freezing and thawing.
INDOLEACETALDOXTME
servation has been confirmed by experiments given in Table IX. Both untreated and sodium borohydride-reduced enzyme samples purified by DEAE-cellulose chromatography showed increases over initial activities upon freezing and thawing. However, a repetition of freezing and thawing did not enhance the activity any further. A similar increment in activity was not observed with the less purified enzyme I. DISCUSSION
Several intriguing properties of IAOX hydra-lyase reported earlier (1) were investigated further using a more purified enzyme II preparation, and its activation by AA, DHA, and PALP as well as inhibitions by thiol reagents and thiol compounds were confirmed. Ferrous, but not ferric, ions activated the enzyme II. The pronounced inhibition of enzyme activity by thiol reagents such as PHMB, iYEi\i, PTC, and arsenite, the reversal of PH,1IB inhibition by GSH, and the partial protection of the enzyme by the competitive inhibitor, PAOX, from the action of NE&I (Table I) strongly suggest the involvement of an -- SH function in the catalytic activity of IAOX hydro-lyase. Such a thiol funct,ion could have the role of a base in an acidbase catalyzed dehydration mechanism, analogous to the enzymatic dehydration of malate (8) or citrate (9). However, neither the chemical dehydration of an aldoxime nor the enzymatic dehydration of IAOX described here is a reversible reaction like other dehydrations across carbon-carbon bonds. Indeed, the chemical hydration of a nitrile yields an amide and not an aldoxime (10). Hence a simple acid-base catalyzed mechanism for oxime dehydration appea,rs unlikely. An alternative role for the participation of a thiol function in this reaction can be proposed in the formation of an enzymebound thiohydroxamate intermediate as given in Fig. 2a. Such an intermediate can be visualized in analogy with the naturally occurring glucosinolate compounds (Fig. 2b). Thiohydroxamic acids (Fig. 2c) are known to decompose readily to the corresponding
HYDRO-LYASE
173
s s 7 Enz.-S-Fi G1”-S-$ HS-E N,OH K+ -O$O/N N, OH (a)
(b) FIG.~
(cl
nitriles (11). Nitriles are also formed from glucosinolate compounds by the action of myrosinase under certain conditions (12). In view of these observations, the enzymebound thiohydroxamate intermediate as proposed in Fig. 2a could decompose to give the nitrile. The requirement for an oxidized function on the enzyme for its activity was suggested earlier, based on the inhibition of the enzyme by several reducing agents, such as thiol compounds, sodium dithionite, and sodium borohydride, and its reversibility by an oxidant such as DHA (1). BAL inhibition was reversed by DHA following removal of unreacted BAL by gel filtration (Table III). Such a BAL-inhibited enzyme was also reactivated by frozen storage (Table III), a condition during which air oxidation of BAL-reduced function could have taken place (13). The inactivation of the enzyme by several reducing agents may be explained by the involvement of an oxidized function either in the catalytic process per se or in maintaining the structural integrity of the enzyme molecule. The nonspecificity of the reducing agents in causing inhibition makes the latter explanation more attractive in the case of IAOX hydro-lyase. It is now generally accepted that metabolically regulated enzymes have subunit structure and that their catalytic function is regulated by association-dissociation processes. Dissociation of such enzymes is facilitated by several means such as dilution, change in ionic concentration, or chemical agents including reductants, as has been shown with muscle phosphorylase (14). The following properties of IAOX hydro-lyase suggest that it may be a metabolically regulated enzyme composed of subunits: (a) the expression of its activit.y in the fungus is dependent on culture conditions such as an exposure to low temperature; (b) it is activated by com-
174
SHUKLA
AND
pounds such as DHA, AA, and DHFA which are not chemically related to the substrate; (c) it has a specific requirement for pyridoxal phosphate (4), and sodium borohydride reduction does not inactivate it (1) suggesting a structural role for the PALP; (d) it is unstable in dilute solution of low ionic concentration as obtained during DEAE-cellulose chromatography and is stabilized by concentration with sucrose; and (e) the concentrated enzyme is activated by freezing (Table IX). The activation of sucrose-concentrated enzyme by freezing and thawing (Table IX) is unusual since several enzymes are inactivated by freezing (13, 15). Aggregation of subunit polypeptide chains as a consequence of freezing to form hybrid structures (16) has been suggested by Chilson et al. (13) to explain the inactivation of lactic dehydrogenase. Such an inactivation was decreased, however, or abolished in the presence of sucrose (13). If aggregation of subunits dissociated by dilution and exposure to low ionic concentration during DEAEcellulose chromatography was the reason for activation of IAOX hydro-lyase on freezing, it is probable that this enzyme has only one type of subunits. ACKNOWLEDGMENTS Thanks are due to Dr. S. A. Kumar for helpful discussions and to Professor P. S. Sarma for facilities accorded. REFERENCES 1. SHUKLA, P. S., AND MAHADEVAN, S., Arch. B&hem. Biophys. 126, 873 (1968).
MAHADEVAN 2. BLAKLEY, R. L., Nature (London) 188, 231 (1960). 3. MAHADEVAN, S., SHUKLA, P. S., KALYANARAMAN, V. S., AND KUMAR, S. A., FEBS Letters 2, 149 (1969). 4. KUMAR, S. A., AND MAHADEVAN, S., Arch. Biochem. Biophys. 103, 516 (1963). 5. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 6. FLUHARTY, A. L., AND SANADI, D. R., J. Biol. Chem. 236, 2272 (1961). 7. DIXON, M., AND WEBB, E. C. (Eds.), “Enzymes, ” p. 376. Academic Press, New York (1958). 8. MALMSTROM, Bo G., in “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), Vol. 5, p. 455. Academic Press, New York (1961). 9. DICKMAN, S. It., in “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), Vol. 5, p. 495. Academic Press, New York (1961). 10. WESTHEIMER, F. H. in “Mechanism of Enzyme Action” (W. D. McElroy, and B. Glass, eds.), p. 342. Johns Hopkins Press, Baltimore (1954). 11. ETTLINGER, M. J., AND LUNDEEN, A. J., J. Am. Chem. Sot. 79, 1764 (1957). 12. VIRTANEN, A. I., Arch. Biochem. Biophys. Suppl. 1, 200 (1962). 13. CHILSON, 0. P., COSTELLO, L. A., AND KAPLAN, N. O., Fed. Proc. 24, Suppl. 15, S-55 (1965). 14. WANG, J. H., SHONKA, M. L., AND GRAVES, D. J., Biochem. 4, 2296 (1965). 15. SHALTIEL, S., HEDRICK, J., AND FISCHER, E. H., Biochem. 6, 2108 (1966). 16. TAPPEL, A. L., in “Cryobiology” (H. T. Meryman, ed.), p. 163. Academic Press, New York (1966). 17. LEVITT, J., J. Theoret. Biol. 3, 355 (1962).