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Isocitrate lyase from Pseudomonas indigofera III. Sulfhydryl groups and enzyme activity
BIOCHIMICA ET BIOPHYSICA ACTA BBA
65273
ISOCITRATE LYASE FROM PSEUDOMONAS INDIGOFERA III. SULFHYDRYL GROUPS AND ENZYME ACTIVITY
ISAMU SHIIO*
AND BRUCE A. McFADDEN"* Department of Chemistry, Washington State University, Pullman, Wash. (U.S.A.) (Received February 2nd, 1965)
SUMMARY
1. The number of sulfhydryl groups per enzyme molecule was examined for various preparations ofisocitrate lyase (threo-Ds-isocitrateglyoxalate-Iyase, EC 4.1.3.1) from Pseudomonas indigo/era by spectrophotometry using p-hydroxymercuribenzoate. It was: 9 for "active" enzyme, i,e., enzyme for which EDTA could replace GSH in the assay; 4 for "inactive" enzyme prepared by treatment with GSSG, i.e., enzyme for which EDTA could not replace HSG; and 4-5 for either active or inactive enzyme pretreated with N-ethylmaleimide. Addition of 7-8 moles N-ethylmaleimide per mole active enzyme resulted in maximum inhibition. 2. Isocitrate plus Mg2+ rather specifically protected enzyme against inactivation by GSSG, when several combinations of substrates and cofactors (isocitrate, succinate, glyoxylate, Mg2+, and EDTA) were examined. Of several other organic compounds tested only malonate afforded some protection in the presence of Mg2+. Mn2+ and Co2+ replaced Mg2+ to some degree in protection that was dependent upon the presence of isocitrate. 3. A possible method for selective labeling of enzymically essential SH by N-[14C]ethylmaleimide was examined and appears promising.
INTRODUCTION
In the previous paper- dealing with crystalline isocitrate lyase (threo-Ds-isocitrate glyoxalate-Iyase, EC 4.1.3.1) from Pseudomonas indigo/era, data were reported which were consistent with the generation of enzymically essential sulfhydryl group(s) during the activation of "inactive" or aged enzyme by GSH. In different preparations the extent of activation, i.e., the extent to which EDTA could replace GSH in the assay, was paralleled by increasing sensitivity to inhibition by NEM. It was also shown Abbreviations: NEM, N-ethylmaleimicle; PMB, p-hydroxymcrcuribenzoate, * Present address: Central Research Laboratory, Ajinomoto Co., Inc., Kawasaki (Japan). ** The author to whom inquiries should be addressed.
Biochim. Biophys. Acta. 105 (1965) 496-505
ISOCITRATE LYASE. III
497
that "active" enzyme was inhibited by PMB and could be reversibly inactivated by GSSG. The present communication further defines the active and inactive st at es and provides evidence suggesting that one (or some) of the SH groups generated during activation is in the vicinity of the catalytic site(s) of isocitrate lyase. MATERIALS AND METHODS Materials The following were suppliers of materials: Matheson, Coleman and Bell, disodium EDTA; Pharmacia Fine Chemicals, Inc., Sephadex G-50, coarse; Sigma Chemical Co., NEM, PMB (sodium salt) and 'Iris (Sigma 7-9) ; Schwarz BioResearch, Inc ., monosodium GSH, GSSG and [14CJNEM. Preparation and assay of isocitrate lyase Crystalline preparations were used in all experiments and were prepared from P . indigo/e ra Ml as described earliers, Essentially the same methods as described by SHIIO, SHIIO AND McFADDEN 1 , 2 were used for the preparation of active and inactive isocitrate lyase, for Sephadex G-50 treatment, and for determination of enzyme activity and protein concentration. Determination of SH grottps with PMB The determinations were performed by the spectrophotometric method of BOYERs. For example, I mM PMB in 0.05 M phosphate (pH 7.0) was added to an isocitrate lyase solution (1.5-1 .6 mg protein per ml) in the sam e buffer and to a buffer blank. Then the difference in absorbancy at 250 mfL was measured (path length, 1.0
0 .05
1.4 :l-
E
o
on
0. 2
'"
'"
1.2
( 0.05 1.0
-------"
O.B L...._-'--_-'--_--'-_-'-_--'-_---'_ _L . - - _. L - - - - l o 10 20 30 40
Time (min) wilh PMB
Fig.!. Time-cou rse of spectrophotometric determination of SH groups in active isocit rate lyase wit h PMB. 0.05 m! or 0 . 20 mI of 0.g8 mM PMB in 0 .05 M phosphate buffer (pH 7.0) wa s ad ded (as indicated by arrows) to a solution of active isocitrate lyase in the same buffer (initially 2.5 ml with r.64 mg enzyme per rnl) and to a buffer blank. Absorbancy at 250 IDfL. Am. was then measured at intervals. The plot is uncorrected for changes in absorbancy due to dilution.
1. SHIlO, B. A. MCFADDEN
em) from which the molar extinction increment for the mercaptide formed was calculated. Addition of the PMB solution was continued and absorbancy changes followed spectrophotometrically until readings at intervals became constant. The number of PlIiIB-reactive SH groups per molecule was then estimated from the molar extinction increment at 250 m,.,. for the mercaptide formed, the total increment in absorbancy and the final molarity ofisocitrate lyase", Active and inactive enzyme preparations were made by incubation for 10 min at room temperature of enzyme 'with 0.02 111: GSH and O.OI M GSSG, respectively, in 0.05 M Tris (pH 7.7). Each preparation was then transferred into 0.05 M phosphate (pH 7.0) by Scphac1ex G-50 treatment. RESULTS
SH groups in active, inactive and NEM-treated isocitraie lyase Fig. I represents raw data obtained using PMB to determine the sulfhydryl content of isocitrate lyase and is presented to illustrate that a satisfactory end point was achieved. As shown in Table I, 9 of 2I half-cystines- in active isocitrate lyase were reactive with PMB while only 4 of them were PMB-reaetive in the inactive preparaTABLE I DETERMINATION OF TITRATION
SH
GROUPS IN VA"RlOUS PREPARATIONS OF ISOCITRATE LYASE BY
PMB
The general procedure was as given foi Fig.!. Molar extinction increments for the mercaptide formed in active and inactive enzyme were 7.13' 108 and 8.40' 10", respectively. For both active and inactive enzyme preparations treated with NEM, 8'40' 10 8 was used for the calculation. If 7.13' 10 8 is used for NEM-treated active enzyme, the calculation results in 5.0 SH groups per enzyme molecule.
En:yme preparation
SH groups per molecule enzyme
Active enzyme Inactive enzyme Active enzyme after NEM-treatment* Inactive enzyme after NEM-treatment·
92 4. 1 4·3 5·1
* The enzyme solution was preineubated with NEM (final concn., 0.5 mM) for 20 min at room temperature before additio'iJ. Df PMB. Absorbaney of NEM, whieh was provided in large excess to enzyme, was corrected for by addition of NEM to the buffer blank.
tion, establishing that an increase in PMB-reactivc sulfhydryl corresponded to the acquisition of enzyme activity. It is also of interest that there was essentially no difference in the number of PMB-reactive sulfhydryl groups between inactive enzyme, NEM-treated active enzyme and NEM-treated inactive enzyme. There was no significant difference in the sedimentation constant between active and inactive enzymes (at approx. l.5 rug/ml).
Reaction with NEM When a high concentration of active isocitrate lyase was used with various high dilutions of NEM, it was possible to relate the degree of inhibition with NEM added Biochim, Biophys. A eta, 105 (1965) 496-5°5
ISOCITRATE LYASE.
III
499
100
x 80
t!-
60
<::
o
:.a .s:
<::
40
20
2
4
6
8
10
12
14
16 /
100
NEM moles/mole enzyme
Fig. 2. Relationship between molar ratio of NEM per enzyme and inhibition of isocitrate lyase. Active isocitrate lyase (final concn., 4.65' 10- 6 M) was incubated for 20 min with NEM in 0.05 M phosphate (pH 7.0) at room temperature and then the activity was assayed by the GSH method.
per enzyme molecule (Fig. 2). The initial addition of I mole of NEM had a negligible effect, while the next 6 or 7 per mole of enzyme were much more effective and resulted in maximum inhibition. Fig. 3 demonstrates that the reaction with NEM was fairly fast and was complete within 10 min at room temperature (27°). At 0°, the maximum degree of inhibition itself was very low (about 40%). Evidence for binding of NEM with sulfhydryl groups of isocitrate lyase was obtained from the identity with authentic NEM~ysteine of a product in acid hydrolysates of [14C]NEM-isocitrate lyase which had been isolated by Sephadex G-50 treatment after reaction with [14CJNEM. The hydrolysis of [14C]NEM-isocitrate lyase (about 20 000 counts/min of radioactivity) in 6 N Hel was carried out at r05° in a sealed, evacuated tube for r4 h. The hydrolysate was submitted to two-dimensional paper-chromatographic analysis with n-butanol-acetic acid-water (4:r :1, v/v) and phenol-water (4: I. W Iv) as solvents. Two radioactive spots were detected radioautographically using Kodak No-Screen Medical X-ray film (10 days exposure) and each was then rechromatographed after elution. One radioactive spot detected by radioautography coincided with the ninhydrin-developed spot on paper chromatograms due to NEM--cysteine itself. NEM-cysteine was prepared by incubation of 0.6 M cysteine (pH 6) with 2 vol. of 0.3 M NEM for 30 min at room temperature. The recovery of NEM--cysteine, after hydrolysis of the altered protein with 6 N Biochim, Biopltys. Acta, 105 (1965) 496-505
500
1. SHIlO, B.
A. MCFADDEN
100
ao ~
~0
/
o·
Q)
E e-, N
c::
UJ
.,
-
40
..,....-
.~
27·
c
q; a:
20
o
5
:30
10
Time (min) with NEM
Fig. 3. Time-course of NEM reaction with isocitrate lyase. Aged preparation of isocitrate lyase was incubated with 6 mM GSH for 10 min at room temperature in 0.05 M phosphate (pH 7.0). Then NEM (final concn., 0.01 M) was added to the incubation mixture at the temperature shown and incubation conducted. The reaction was stopped by the addition of excess GSH and the activity assayed by the GSH method. Failure to observe a higher degree of inhibition at room temperature was probably due to incomplete activation of the isocitrate lyase preparation" used in this experiment.
Hel for 14 h at IOSo is of some interest because it confirms the expectation that it would be a major product after hydrolysis under these conditions-A The other spot may have been due to S-succinyl-L-cysteine4 , 5. Although diastereomers of NEMcysteine are known to forms, our finding of a single spot after paper chromatography of the standard is in accord with the experience of LEE AND SAMUELS 5 , 6. TABLE II EFFECT OF SUBSTRATE AND COFACTORS ON ENZYME INACTIVATION BY
GSSG
Final reaction mixtures contained 8 ,umoles of sodium DL-isocitrate, 6 ftmoles of MgC12, 2 ftmoles of EDTA, 20,umoles of GSSG (or 0.02 ,umole of PMB) and ISO ,umoles of Tris (pH 7.7) in a final volume of 2.0 ml. After 5 min of preincubation with the designated components at 30., GSSG (or PMB) was added. After 10 min of further incubation the reaction was initiated by addition of the remaining components. In each case a control lacking only GSSG and PMB was conducted exactly as its experimental counterpart.
Preinc-ubation with
nt-Isooitrate nr.-Isocitrate
+ MgH
+ EDTA
DL-Isocitrate MgH EDTA MgH EDTA None
+
Enzyme activity as % of corresponding control + GSSG
+PMB
21
2
3 3
2
0
0
1
1
1
3
2
I
I
Biochim, Biophys. Acta, 105 (1965) 496-.,505
ISOCITRATE LYASE.
III
SOI
TABLE III EFFECT OF ISOCITRATE AND MgH LEVELS UPON PROTECTION AGAINST GSSG-INACTIVATION
Experimental procedures were as given for Table II. At zero time when EDTA was added to initiate the reaction sufficient nr.-lsocitrate or MgCl, was also added where necessary to bring the concentration to 4 and 3 mM, respectively.
Preincubation with I socitrate Mg'+
Enzyme activity in presence of esse as % of control
(lImo/as/tube) 8 8 8 8 8
8 8 S 16
32 16 8 4 2 0.8 0.08 0
16 16 20
18 16 15 5·7
8
8
2.8 20 20
4
8
14
2
8
10
I
8 8
7
0
8
I
Protection by substrate and cofactors against GSSG-inactivation Since catalysis of the forward reaction by active isocitrate lyase requires Mg2+, EDTA and substrate, isocitrate, the effect upon GSSG-inactivation of preincubation of active enzyme with these components both singly and in combination was examined. In these experiments active enzyme was preincubated for S min with the designated components, GSSG was added and the reaction was started IO min later by addition of all of the remaining reaction components. Results are shown in Table II. Only one (isocitrate + Mg2+) of six possible combinations was effective in protecting against GSSG-inactivation and none was significantly effective in the case of PMB inhibition. Although the data are not shown, results with NEM were similar to those with PMB. The effect of concentration of isocitrate and Mg2+ on protection against inactivation by GSSG is shown in Table III. As shown in Table IV, certain other divalent metallic ions such as Mn2+, Co2+, Zn 2+ and Cd2+ when tested with isocitrate afforded some protection against inactivation when preincubated with enzyme. However, two of these four, Zn2+ and Cd 2 +, showed a similar effect in the absence of substrate suggesting that protection by these two species was non-specific. The fact that the effects of Mn2+ and Co2+ absolutely required the presence of substrate as did that of Mg2+ (Table II) is of interest because either Mn2+ or Co2+ can partially replace the Mg2+ requirement of th.e enzymic reaction'. Table V illustrates the effect in the presence of Mg2+ of salts of various di- and tricarboxylic acids in protecting isocitrate lyase against GSSG-inactivation. Only malonate, of the compounds tested, provided protection in addition to isocitrate. It is of interest in this connection that malonate has been shown in our laboratory to be a Biochim, Biophys. Acta, ID5 (1965) 496-5°5
502
1. SHIIO, B. A. MCFADDEN
TABLE
IV
SPECIFICITY OF Mga+ IN PROTECTING AGAINST INACTlVATION OF ISOCITRATE LYASE BY GSSG Experimental procedures were essentially the same as given for Table II except that the reaction mixtures contained 1.4 flmoles of EDTA. I ,llmole of MgH, and I limole of the metallic ion with which enz yrne had been preincubatcd. EDT A and MgH were added at zero time.
Preincubation with
Enzyme activity in presence of esse as % of conn ot
+ + + + + +
MgCI a DL-isocitrate MnCl a DL-isocitrate CoCl a + DL-isocitrate NiCI. DL-isocitrate Fe(SO.la(NH.).SO. + DL-isocitrate ZnCl 2 nn-isocitrate CdCIa + nr.-Isocrtrate CaCl. Dr.-isocitrate None DL-isocitrate MgCl 2 MuCI.
ceci,
17
28 8.8 2
o 37 37 o
2·7 I
o
o
ZnCI. CdCI.
9 20
competitive inhibitor of the forward reaction (unpublished observation). The apparent, high degree of protective specificity by isocitrate plus Mg 2+ was of interest and these studies were extended to a more detailed investigation of effects of the substrates for the back reaction. In these experiments it was necessary to remove glyoxylate by Sephadex G-50 treatment before the assay. As can be seen in Table VI neither glyoxylate plus Mg 2+ nor glyoxylate plus succinate and Mg2+ afforded significant protection against inactivation by GSSG. For the ratios of enzyme activity deterTABLE V SPECIFICU:Y OF ISOCITRATE IN PROTECTING AGAINST INACTIVATION BY GSSG Experimental procedures were essentially the same as in Table II. After preincubation with 21imoles of MgCI. plus 4 pmoles of the designated organic compound (or 8 flmoles of nr-compounds) and then with 2o/iffioies of GSSG, the enzyme reaction was initiated by addition of 0.5 pmole of EDTA plus 8,imoles of nr.-isocitrate.
Preincubation with MgH and
Enzyme activity in presence of esse (is % of control
nr-Esocitrate
28 0·4
cis-Aeonitate Citrate Fumarate ut-Mala.te Malonate Succinate None
0·5 0·5
0·7 I3 0·3 0·3
Bioch im, Biophys, Acta, 105 (Ig65) 496-505
IS0CITRATE LYASE.
III
503
TABLE VI COMPARATIVE EFFECTS OF SUBSTRATES UPON PROTECTION AGAINST GSSG-INACTIVATION
A 5-min incubation of active enzyme in 0.8 ml 0.06 M 'Iris (pH 7.7) containing 2 flmoles of MgCl 2 plus 4 flmoles of the specified compound(s) was followed by a ro-min incubation period with 10 pmoles of GSSG. The resulting reaction mixtures (1.0 ml) were then passed through Sephadex G-50 with 0.o5lVI 'Iris (pH 7.7). Enzyme activity was assayed in an aliquot of the effluent. In tests for NEM-inhibition, enzyme in the Sephadex G-so effluent was preincubated with 2 ftmoles of NEM in 1.2 ml of 0.1 M 'Iris (pH 7.7) for 20 min at room temperature before GSH-assay.
Preincubatiow with MgC1 2 and
GSSG
Enzyme activity ( EDTA-assay/GSH-assay) x IDD
% inhibition byNEM
None DL-Isocitrate Glyoxylate Glyoxylate + succinate None
+ + + +
1.6 41.8 2·9 4·7 96
10 5 1.5 17 18 92
mined by the two different assay techniques, activity with GSH was referred to as 100 because GSSG-inactivated enzyme is known to be reactivated by GSH in the assay medium'. A possible approach to selective labeling of sulfhydryl at the catalytic site(s) Because substrate and cofactor provided specific protection of enzyme against GSSG-inactivation, a process involving the loss of PMB-reactive groups, it seemed likely that one (or more) SH group was in the region of a substrate-binding site. The possibility of selectively labeling this SH was intriguing and was examined by measuring the incorporation of [14CJNEM into: active enzyme, and the same preparations which had been treated with GSSG in the presence and absence of isocitrate Mg2+. The extent of inhibition was also measured for each of these preparations. In these experiments active enzyme was prepared from stock enzyme which had been inactivated with GSSG and then incubated with NEM at pH 7.0 prior to reactivation. In this way, the possibility of subsequent binding of [l4C]NEM at non-sulfhydryl sites was minimized. The results in Table VII show that the efficacy of NEM as an inhibitor (liN) increases about two-fold after GSSG-treatment in the presence of isocitrate and Mg2+. Data in Table VII also suggest considerable radioactivity in the GSSG-inactivated enzyme (Preparation C) after treatment with [14CJNEM and subsequent removal of unreacted [14C]NEM. This is surprising because the data in Table I reveal essentially no difference in reactivity to PMB between inactive enzyme and NEM-treated inactive enzyme; in general, PMB-reactive sulfhydryl groups seem to include NEMreactive ones". It is possible that this radioactivity is from radioactive contaminants in the reagent or decomposition and polymerization products of [14C]NEM itselfs.", since, in a separate experiment using another bottle of the radioactive reagent, [14CJNEM binding by inactive aged preparation (5.3 % active, measured by NEMinhibition method) was only 8.8 % of that by the activated preparation (75 % active). If the contaminants were non-specifically bound to enzyme or were of high molecular
+
Biocbim. Biophys. Acta, !OS (19 65) 496-505
1. SHIIO, B. A. MCFADDEN
TABLE VII SELECTIVE LABELING
OF
ESSENTIAL SULFHYDRYL GROUP(S)
BY
[14C)NEM
Stock enzyme preparation was inactivated in the standard way and was then treated for 20 min at room temperature with I mM NEM. It was then activated with 0.013 M GSH in 0.1 M Tris (pH 7.7) for 10 min at room temperature, and then passed through Sephadex G-50 to remove reagents (Preparation A). An aliquot of Preparation A was incubated for 5 min with 4 mM DL-isocitrate plus I mM MgCl 2 and then for 10 min at 300 with 0.01 M GSSG in 0.1 M Tris (pH 7.7) (Preparation B). Another aliquot of Preparation A was incubated only with GSSG under the conditions described (Preparation C). After Sephadex G-50 treatment of E and C, enzyme preparations A, Band C were incubated with 0.5 mM [14C]NEM (o.5 floCfflomole) in 0.05 M phosphate (pH 7.0) for 20 min at room temperature. and then again passed through a Sephadex G-50 column. The column eluants were assayed for enzyme activity and protein. An aliquot of the protein fraction of the eluant was placed on a paper disc. dried and washed consecutively at 300 with 10% trichloroacetic acid (w/v) twice, diethyl ether-ethanol (I: I, v/v) twice and finally with diethyl ether. The disc was then dried and the radioactivity assayed in a toluene scintillator solution with a Packard Tri-Carb Spectrometer. Washing of the disc as described removed very little radioactivity.
Enzyme preparation
Per cent inkibit'ion byNEM
[14C]NEM bound
I
LJI'
N
LIN'
l/N
89.4
(100)
83. 8
0.944
2.2
1.75
Active enzyme (A) 94.4 Preparation A treated with GSSG in presence of isocitrate Mg2+ (E) 32 Preparation A treated with GSSG to yield inactive enzyme (C) 5
+
27
18·4 16.2
Efficiency of bound N EM in inhibition LJl/LJN 1.07 12
0.3 1
• Difference from the value for Preparation C.
weight and insoluble in 10% trichloroacetic acid (see protocol in Table VII) they could account for the radioactivity in inactivated enzyme. If this radioactivity is subtracted from the other data to obtain the denominator in the efficiency term (LJIILJN) for preparation treated with GSSG in the presence of substrate Mg2+, the ratio becomes much higher than liN.
+
DISCUSSION
Information from the PMB titrations suggests that there is the net generation of five SH groups per molecule of isocitrate lyase during activation of the enzyme. A molecule of inactive enzyme has four SH groups which seem to be unreactive to NEM. About five SH groups in active enzyme are NEM-reactive and these are presumably identical with the thiol groups generated during activation. The thiols appearing include enzymically essential groups since reaction of active enzyme with GSSG or NEM results in inactivation, and GSH activates inactive enzyme (ref. 7. p. 264). In general accordance with the above data, maximum inhibition of active enzyme was obtained when seven to eight moles of NEM per mole enzyme were added, of which the first mole of NEM had little effect. The need for a slight excess of NEM is Biochim, Biophys. Acta. 105 (1965) 496-505
ISOCITRATE LYASE.
III
505
probably due to instability of NEM8,9 and/or to the rate of reversible loss of NEM~ reactive sulfhydryl groups of the enzyme- which may be comparable to the rate of reaction of enzyme with high dilutions of NEM. At these high dilutions of reagent, some excess may be required to approach a reaction velocity of enzymic thiol with NEM which is independent of the NEM/enzyme ratio and therefore to approach maximal inhibition. Formation of NEM-cysteine residues was confirmed. The data, then, raise the important question about the nature of the activation process. Dissociation of enzyme into separate components is unlikely since sedimentation coefficients were the same for active and inactive enzyme. Additional research will be required to define the activation process more thoroughly. One of the most significant findings of the present work is the specific protection exerted by isocitrate plus Mg2+ against enzyme inactivation by GSSG, suggesting that some (one) of the :five thiols generated by activation are components of the isocitratebinding site. It also suggests formation of a complex involving isocitrate, Mg2+ and enzymic SH, in the enzymic reaction, although Mg2+-facilit at ed binding of isocitrate may simply render the thiol(s) inaccessible to some reagents. Selective labeling of sulfhydryl at the substrate-binding site would be possible by incubating active isocitrate lyase with GSSG in the presence of isocitrate plus Mg2+ so that all SH groups except those (that) at the substrate-binding site(s) become unreactive to NEM, and then treating the enzyme with [l4CJNEM after removal of the reagents. Supporting this is the fact that the efficiency of NEM inhibition as a function of NEM bound increases about two-fold when compared with that for active enzyme, even without consideration of the large amount of radioactivity in enzyme inactivated in the absence of substrate. Were this radioactivity subtracted from that for substrateprotected enzyme, the efficiency would increase about r r-fold (see Table VII) suggesting the presence of only one SH group at the substrate binding site and one binding site per enzyme molecule. Since this increase is greater than the theoretical limiting value of a S-fold increase, the subtraction may not be wholly valid. It will be necessary to separate [l4C]NEM--eysteine from hydrolysates of treated enzyme preparations to precisely evaluate the quantity of NEM which is bound to enzymic thiol. ACKNOWLEDGEMENTS
This investigation was supported in part by Research Grant GM-ogo39 from the National Institutes of Health and a Research Career Development Award (No. I - K3-AI5268) from the Institute of Allergy and Infectious Diseases, Public Health Service. REFERENCES
1. SHUO, T. SHIIO AND B. A. McFADDEN, Biochim, Biophys. Acta, 96 (I965) I23. 1. SHUO, T. SHIIO AND B. A. McFADDEN, Biochim. Biophys. Acta, 96 (1965) II4. P. D. BOYER, J. Am. Chem, Soc., 76 (1954) 433I. D. G. SMYTH, O. O. BLUMENFELD AND vv. KONIGSBERG, Biochem, j., 91 (1964) 589. C. C. LEE AND E. R. SAMUELS, Can. J. Chem., 42 (I964) I64. C. C. LEE AND E. R. SAMUELS, Can. J. Chern: 40 (I962) I040. 7 H. FRAENKEL-CONRAT, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 4, Academic Press, New York, 1957, p. ~56. 8 D. G. SMYTH, A. NAGAMATSU AND J. S. FRUTON, l- Am. Chem. Soc., 82 (1960) 4600. 9 J. D. GREGORY, J. Am. Chern. Soc., 77 (1955) 3922.
I 2 3 4 5 6
Biocbim. Biophys. Acta, 105 (1905) 496-505
65273
ISOCITRATE LYASE FROM PSEUDOMONAS INDIGOFERA III. SULFHYDRYL GROUPS AND ENZYME ACTIVITY
ISAMU SHIIO*
AND BRUCE A. McFADDEN"* Department of Chemistry, Washington State University, Pullman, Wash. (U.S.A.) (Received February 2nd, 1965)
SUMMARY
1. The number of sulfhydryl groups per enzyme molecule was examined for various preparations ofisocitrate lyase (threo-Ds-isocitrateglyoxalate-Iyase, EC 4.1.3.1) from Pseudomonas indigo/era by spectrophotometry using p-hydroxymercuribenzoate. It was: 9 for "active" enzyme, i,e., enzyme for which EDTA could replace GSH in the assay; 4 for "inactive" enzyme prepared by treatment with GSSG, i.e., enzyme for which EDTA could not replace HSG; and 4-5 for either active or inactive enzyme pretreated with N-ethylmaleimide. Addition of 7-8 moles N-ethylmaleimide per mole active enzyme resulted in maximum inhibition. 2. Isocitrate plus Mg2+ rather specifically protected enzyme against inactivation by GSSG, when several combinations of substrates and cofactors (isocitrate, succinate, glyoxylate, Mg2+, and EDTA) were examined. Of several other organic compounds tested only malonate afforded some protection in the presence of Mg2+. Mn2+ and Co2+ replaced Mg2+ to some degree in protection that was dependent upon the presence of isocitrate. 3. A possible method for selective labeling of enzymically essential SH by N-[14C]ethylmaleimide was examined and appears promising.
INTRODUCTION
In the previous paper- dealing with crystalline isocitrate lyase (threo-Ds-isocitrate glyoxalate-Iyase, EC 4.1.3.1) from Pseudomonas indigo/era, data were reported which were consistent with the generation of enzymically essential sulfhydryl group(s) during the activation of "inactive" or aged enzyme by GSH. In different preparations the extent of activation, i.e., the extent to which EDTA could replace GSH in the assay, was paralleled by increasing sensitivity to inhibition by NEM. It was also shown Abbreviations: NEM, N-ethylmaleimicle; PMB, p-hydroxymcrcuribenzoate, * Present address: Central Research Laboratory, Ajinomoto Co., Inc., Kawasaki (Japan). ** The author to whom inquiries should be addressed.
Biochim. Biophys. Acta. 105 (1965) 496-505
ISOCITRATE LYASE. III
497
that "active" enzyme was inhibited by PMB and could be reversibly inactivated by GSSG. The present communication further defines the active and inactive st at es and provides evidence suggesting that one (or some) of the SH groups generated during activation is in the vicinity of the catalytic site(s) of isocitrate lyase. MATERIALS AND METHODS Materials The following were suppliers of materials: Matheson, Coleman and Bell, disodium EDTA; Pharmacia Fine Chemicals, Inc., Sephadex G-50, coarse; Sigma Chemical Co., NEM, PMB (sodium salt) and 'Iris (Sigma 7-9) ; Schwarz BioResearch, Inc ., monosodium GSH, GSSG and [14CJNEM. Preparation and assay of isocitrate lyase Crystalline preparations were used in all experiments and were prepared from P . indigo/e ra Ml as described earliers, Essentially the same methods as described by SHIIO, SHIIO AND McFADDEN 1 , 2 were used for the preparation of active and inactive isocitrate lyase, for Sephadex G-50 treatment, and for determination of enzyme activity and protein concentration. Determination of SH grottps with PMB The determinations were performed by the spectrophotometric method of BOYERs. For example, I mM PMB in 0.05 M phosphate (pH 7.0) was added to an isocitrate lyase solution (1.5-1 .6 mg protein per ml) in the sam e buffer and to a buffer blank. Then the difference in absorbancy at 250 mfL was measured (path length, 1.0
0 .05
1.4 :l-
E
o
on
0. 2
'"
'"
1.2
( 0.05 1.0
-------"
O.B L...._-'--_-'--_--'-_-'-_--'-_---'_ _L . - - _. L - - - - l o 10 20 30 40
Time (min) wilh PMB
Fig.!. Time-cou rse of spectrophotometric determination of SH groups in active isocit rate lyase wit h PMB. 0.05 m! or 0 . 20 mI of 0.g8 mM PMB in 0 .05 M phosphate buffer (pH 7.0) wa s ad ded (as indicated by arrows) to a solution of active isocitrate lyase in the same buffer (initially 2.5 ml with r.64 mg enzyme per rnl) and to a buffer blank. Absorbancy at 250 IDfL. Am. was then measured at intervals. The plot is uncorrected for changes in absorbancy due to dilution.
1. SHIlO, B. A. MCFADDEN
em) from which the molar extinction increment for the mercaptide formed was calculated. Addition of the PMB solution was continued and absorbancy changes followed spectrophotometrically until readings at intervals became constant. The number of PlIiIB-reactive SH groups per molecule was then estimated from the molar extinction increment at 250 m,.,. for the mercaptide formed, the total increment in absorbancy and the final molarity ofisocitrate lyase", Active and inactive enzyme preparations were made by incubation for 10 min at room temperature of enzyme 'with 0.02 111: GSH and O.OI M GSSG, respectively, in 0.05 M Tris (pH 7.7). Each preparation was then transferred into 0.05 M phosphate (pH 7.0) by Scphac1ex G-50 treatment. RESULTS
SH groups in active, inactive and NEM-treated isocitraie lyase Fig. I represents raw data obtained using PMB to determine the sulfhydryl content of isocitrate lyase and is presented to illustrate that a satisfactory end point was achieved. As shown in Table I, 9 of 2I half-cystines- in active isocitrate lyase were reactive with PMB while only 4 of them were PMB-reaetive in the inactive preparaTABLE I DETERMINATION OF TITRATION
SH
GROUPS IN VA"RlOUS PREPARATIONS OF ISOCITRATE LYASE BY
PMB
The general procedure was as given foi Fig.!. Molar extinction increments for the mercaptide formed in active and inactive enzyme were 7.13' 108 and 8.40' 10", respectively. For both active and inactive enzyme preparations treated with NEM, 8'40' 10 8 was used for the calculation. If 7.13' 10 8 is used for NEM-treated active enzyme, the calculation results in 5.0 SH groups per enzyme molecule.
En:yme preparation
SH groups per molecule enzyme
Active enzyme Inactive enzyme Active enzyme after NEM-treatment* Inactive enzyme after NEM-treatment·
92 4. 1 4·3 5·1
* The enzyme solution was preineubated with NEM (final concn., 0.5 mM) for 20 min at room temperature before additio'iJ. Df PMB. Absorbaney of NEM, whieh was provided in large excess to enzyme, was corrected for by addition of NEM to the buffer blank.
tion, establishing that an increase in PMB-reactivc sulfhydryl corresponded to the acquisition of enzyme activity. It is also of interest that there was essentially no difference in the number of PMB-reactive sulfhydryl groups between inactive enzyme, NEM-treated active enzyme and NEM-treated inactive enzyme. There was no significant difference in the sedimentation constant between active and inactive enzymes (at approx. l.5 rug/ml).
Reaction with NEM When a high concentration of active isocitrate lyase was used with various high dilutions of NEM, it was possible to relate the degree of inhibition with NEM added Biochim, Biophys. A eta, 105 (1965) 496-5°5
ISOCITRATE LYASE.
III
499
100
x 80
t!-
60
<::
o
:.a .s:
<::
40
20
2
4
6
8
10
12
14
16 /
100
NEM moles/mole enzyme
Fig. 2. Relationship between molar ratio of NEM per enzyme and inhibition of isocitrate lyase. Active isocitrate lyase (final concn., 4.65' 10- 6 M) was incubated for 20 min with NEM in 0.05 M phosphate (pH 7.0) at room temperature and then the activity was assayed by the GSH method.
per enzyme molecule (Fig. 2). The initial addition of I mole of NEM had a negligible effect, while the next 6 or 7 per mole of enzyme were much more effective and resulted in maximum inhibition. Fig. 3 demonstrates that the reaction with NEM was fairly fast and was complete within 10 min at room temperature (27°). At 0°, the maximum degree of inhibition itself was very low (about 40%). Evidence for binding of NEM with sulfhydryl groups of isocitrate lyase was obtained from the identity with authentic NEM~ysteine of a product in acid hydrolysates of [14C]NEM-isocitrate lyase which had been isolated by Sephadex G-50 treatment after reaction with [14CJNEM. The hydrolysis of [14C]NEM-isocitrate lyase (about 20 000 counts/min of radioactivity) in 6 N Hel was carried out at r05° in a sealed, evacuated tube for r4 h. The hydrolysate was submitted to two-dimensional paper-chromatographic analysis with n-butanol-acetic acid-water (4:r :1, v/v) and phenol-water (4: I. W Iv) as solvents. Two radioactive spots were detected radioautographically using Kodak No-Screen Medical X-ray film (10 days exposure) and each was then rechromatographed after elution. One radioactive spot detected by radioautography coincided with the ninhydrin-developed spot on paper chromatograms due to NEM--cysteine itself. NEM-cysteine was prepared by incubation of 0.6 M cysteine (pH 6) with 2 vol. of 0.3 M NEM for 30 min at room temperature. The recovery of NEM--cysteine, after hydrolysis of the altered protein with 6 N Biochim, Biopltys. Acta, 105 (1965) 496-505
500
1. SHIlO, B.
A. MCFADDEN
100
ao ~
~0
/
o·
Q)
E e-, N
c::
UJ
.,
-
40
..,....-
.~
27·
c
q; a:
20
o
5
:30
10
Time (min) with NEM
Fig. 3. Time-course of NEM reaction with isocitrate lyase. Aged preparation of isocitrate lyase was incubated with 6 mM GSH for 10 min at room temperature in 0.05 M phosphate (pH 7.0). Then NEM (final concn., 0.01 M) was added to the incubation mixture at the temperature shown and incubation conducted. The reaction was stopped by the addition of excess GSH and the activity assayed by the GSH method. Failure to observe a higher degree of inhibition at room temperature was probably due to incomplete activation of the isocitrate lyase preparation" used in this experiment.
Hel for 14 h at IOSo is of some interest because it confirms the expectation that it would be a major product after hydrolysis under these conditions-A The other spot may have been due to S-succinyl-L-cysteine4 , 5. Although diastereomers of NEMcysteine are known to forms, our finding of a single spot after paper chromatography of the standard is in accord with the experience of LEE AND SAMUELS 5 , 6. TABLE II EFFECT OF SUBSTRATE AND COFACTORS ON ENZYME INACTIVATION BY
GSSG
Final reaction mixtures contained 8 ,umoles of sodium DL-isocitrate, 6 ftmoles of MgC12, 2 ftmoles of EDTA, 20,umoles of GSSG (or 0.02 ,umole of PMB) and ISO ,umoles of Tris (pH 7.7) in a final volume of 2.0 ml. After 5 min of preincubation with the designated components at 30., GSSG (or PMB) was added. After 10 min of further incubation the reaction was initiated by addition of the remaining components. In each case a control lacking only GSSG and PMB was conducted exactly as its experimental counterpart.
Preinc-ubation with
nt-Isooitrate nr.-Isocitrate
+ MgH
+ EDTA
DL-Isocitrate MgH EDTA MgH EDTA None
+
Enzyme activity as % of corresponding control + GSSG
+PMB
21
2
3 3
2
0
0
1
1
1
3
2
I
I
Biochim, Biophys. Acta, 105 (1965) 496-.,505
ISOCITRATE LYASE.
III
SOI
TABLE III EFFECT OF ISOCITRATE AND MgH LEVELS UPON PROTECTION AGAINST GSSG-INACTIVATION
Experimental procedures were as given for Table II. At zero time when EDTA was added to initiate the reaction sufficient nr.-lsocitrate or MgCl, was also added where necessary to bring the concentration to 4 and 3 mM, respectively.
Preincubation with I socitrate Mg'+
Enzyme activity in presence of esse as % of control
(lImo/as/tube) 8 8 8 8 8
8 8 S 16
32 16 8 4 2 0.8 0.08 0
16 16 20
18 16 15 5·7
8
8
2.8 20 20
4
8
14
2
8
10
I
8 8
7
0
8
I
Protection by substrate and cofactors against GSSG-inactivation Since catalysis of the forward reaction by active isocitrate lyase requires Mg2+, EDTA and substrate, isocitrate, the effect upon GSSG-inactivation of preincubation of active enzyme with these components both singly and in combination was examined. In these experiments active enzyme was preincubated for S min with the designated components, GSSG was added and the reaction was started IO min later by addition of all of the remaining reaction components. Results are shown in Table II. Only one (isocitrate + Mg2+) of six possible combinations was effective in protecting against GSSG-inactivation and none was significantly effective in the case of PMB inhibition. Although the data are not shown, results with NEM were similar to those with PMB. The effect of concentration of isocitrate and Mg2+ on protection against inactivation by GSSG is shown in Table III. As shown in Table IV, certain other divalent metallic ions such as Mn2+, Co2+, Zn 2+ and Cd2+ when tested with isocitrate afforded some protection against inactivation when preincubated with enzyme. However, two of these four, Zn2+ and Cd 2 +, showed a similar effect in the absence of substrate suggesting that protection by these two species was non-specific. The fact that the effects of Mn2+ and Co2+ absolutely required the presence of substrate as did that of Mg2+ (Table II) is of interest because either Mn2+ or Co2+ can partially replace the Mg2+ requirement of th.e enzymic reaction'. Table V illustrates the effect in the presence of Mg2+ of salts of various di- and tricarboxylic acids in protecting isocitrate lyase against GSSG-inactivation. Only malonate, of the compounds tested, provided protection in addition to isocitrate. It is of interest in this connection that malonate has been shown in our laboratory to be a Biochim, Biophys. Acta, ID5 (1965) 496-5°5
502
1. SHIIO, B. A. MCFADDEN
TABLE
IV
SPECIFICITY OF Mga+ IN PROTECTING AGAINST INACTlVATION OF ISOCITRATE LYASE BY GSSG Experimental procedures were essentially the same as given for Table II except that the reaction mixtures contained 1.4 flmoles of EDTA. I ,llmole of MgH, and I limole of the metallic ion with which enz yrne had been preincubatcd. EDT A and MgH were added at zero time.
Preincubation with
Enzyme activity in presence of esse as % of conn ot
+ + + + + +
MgCI a DL-isocitrate MnCl a DL-isocitrate CoCl a + DL-isocitrate NiCI. DL-isocitrate Fe(SO.la(NH.).SO. + DL-isocitrate ZnCl 2 nn-isocitrate CdCIa + nr.-Isocrtrate CaCl. Dr.-isocitrate None DL-isocitrate MgCl 2 MuCI.
ceci,
17
28 8.8 2
o 37 37 o
2·7 I
o
o
ZnCI. CdCI.
9 20
competitive inhibitor of the forward reaction (unpublished observation). The apparent, high degree of protective specificity by isocitrate plus Mg 2+ was of interest and these studies were extended to a more detailed investigation of effects of the substrates for the back reaction. In these experiments it was necessary to remove glyoxylate by Sephadex G-50 treatment before the assay. As can be seen in Table VI neither glyoxylate plus Mg 2+ nor glyoxylate plus succinate and Mg2+ afforded significant protection against inactivation by GSSG. For the ratios of enzyme activity deterTABLE V SPECIFICU:Y OF ISOCITRATE IN PROTECTING AGAINST INACTIVATION BY GSSG Experimental procedures were essentially the same as in Table II. After preincubation with 21imoles of MgCI. plus 4 pmoles of the designated organic compound (or 8 flmoles of nr-compounds) and then with 2o/iffioies of GSSG, the enzyme reaction was initiated by addition of 0.5 pmole of EDTA plus 8,imoles of nr.-isocitrate.
Preincubation with MgH and
Enzyme activity in presence of esse (is % of control
nr-Esocitrate
28 0·4
cis-Aeonitate Citrate Fumarate ut-Mala.te Malonate Succinate None
0·5 0·5
0·7 I3 0·3 0·3
Bioch im, Biophys, Acta, 105 (Ig65) 496-505
IS0CITRATE LYASE.
III
503
TABLE VI COMPARATIVE EFFECTS OF SUBSTRATES UPON PROTECTION AGAINST GSSG-INACTIVATION
A 5-min incubation of active enzyme in 0.8 ml 0.06 M 'Iris (pH 7.7) containing 2 flmoles of MgCl 2 plus 4 flmoles of the specified compound(s) was followed by a ro-min incubation period with 10 pmoles of GSSG. The resulting reaction mixtures (1.0 ml) were then passed through Sephadex G-50 with 0.o5lVI 'Iris (pH 7.7). Enzyme activity was assayed in an aliquot of the effluent. In tests for NEM-inhibition, enzyme in the Sephadex G-so effluent was preincubated with 2 ftmoles of NEM in 1.2 ml of 0.1 M 'Iris (pH 7.7) for 20 min at room temperature before GSH-assay.
Preincubatiow with MgC1 2 and
GSSG
Enzyme activity ( EDTA-assay/GSH-assay) x IDD
% inhibition byNEM
None DL-Isocitrate Glyoxylate Glyoxylate + succinate None
+ + + +
1.6 41.8 2·9 4·7 96
10 5 1.5 17 18 92
mined by the two different assay techniques, activity with GSH was referred to as 100 because GSSG-inactivated enzyme is known to be reactivated by GSH in the assay medium'. A possible approach to selective labeling of sulfhydryl at the catalytic site(s) Because substrate and cofactor provided specific protection of enzyme against GSSG-inactivation, a process involving the loss of PMB-reactive groups, it seemed likely that one (or more) SH group was in the region of a substrate-binding site. The possibility of selectively labeling this SH was intriguing and was examined by measuring the incorporation of [14CJNEM into: active enzyme, and the same preparations which had been treated with GSSG in the presence and absence of isocitrate Mg2+. The extent of inhibition was also measured for each of these preparations. In these experiments active enzyme was prepared from stock enzyme which had been inactivated with GSSG and then incubated with NEM at pH 7.0 prior to reactivation. In this way, the possibility of subsequent binding of [l4C]NEM at non-sulfhydryl sites was minimized. The results in Table VII show that the efficacy of NEM as an inhibitor (liN) increases about two-fold after GSSG-treatment in the presence of isocitrate and Mg2+. Data in Table VII also suggest considerable radioactivity in the GSSG-inactivated enzyme (Preparation C) after treatment with [14CJNEM and subsequent removal of unreacted [14C]NEM. This is surprising because the data in Table I reveal essentially no difference in reactivity to PMB between inactive enzyme and NEM-treated inactive enzyme; in general, PMB-reactive sulfhydryl groups seem to include NEMreactive ones". It is possible that this radioactivity is from radioactive contaminants in the reagent or decomposition and polymerization products of [14C]NEM itselfs.", since, in a separate experiment using another bottle of the radioactive reagent, [14CJNEM binding by inactive aged preparation (5.3 % active, measured by NEMinhibition method) was only 8.8 % of that by the activated preparation (75 % active). If the contaminants were non-specifically bound to enzyme or were of high molecular
+
Biocbim. Biophys. Acta, !OS (19 65) 496-505
1. SHIIO, B. A. MCFADDEN
TABLE VII SELECTIVE LABELING
OF
ESSENTIAL SULFHYDRYL GROUP(S)
BY
[14C)NEM
Stock enzyme preparation was inactivated in the standard way and was then treated for 20 min at room temperature with I mM NEM. It was then activated with 0.013 M GSH in 0.1 M Tris (pH 7.7) for 10 min at room temperature, and then passed through Sephadex G-50 to remove reagents (Preparation A). An aliquot of Preparation A was incubated for 5 min with 4 mM DL-isocitrate plus I mM MgCl 2 and then for 10 min at 300 with 0.01 M GSSG in 0.1 M Tris (pH 7.7) (Preparation B). Another aliquot of Preparation A was incubated only with GSSG under the conditions described (Preparation C). After Sephadex G-50 treatment of E and C, enzyme preparations A, Band C were incubated with 0.5 mM [14C]NEM (o.5 floCfflomole) in 0.05 M phosphate (pH 7.0) for 20 min at room temperature. and then again passed through a Sephadex G-50 column. The column eluants were assayed for enzyme activity and protein. An aliquot of the protein fraction of the eluant was placed on a paper disc. dried and washed consecutively at 300 with 10% trichloroacetic acid (w/v) twice, diethyl ether-ethanol (I: I, v/v) twice and finally with diethyl ether. The disc was then dried and the radioactivity assayed in a toluene scintillator solution with a Packard Tri-Carb Spectrometer. Washing of the disc as described removed very little radioactivity.
Enzyme preparation
Per cent inkibit'ion byNEM
[14C]NEM bound
I
LJI'
N
LIN'
l/N
89.4
(100)
83. 8
0.944
2.2
1.75
Active enzyme (A) 94.4 Preparation A treated with GSSG in presence of isocitrate Mg2+ (E) 32 Preparation A treated with GSSG to yield inactive enzyme (C) 5
+
27
18·4 16.2
Efficiency of bound N EM in inhibition LJl/LJN 1.07 12
0.3 1
• Difference from the value for Preparation C.
weight and insoluble in 10% trichloroacetic acid (see protocol in Table VII) they could account for the radioactivity in inactivated enzyme. If this radioactivity is subtracted from the other data to obtain the denominator in the efficiency term (LJIILJN) for preparation treated with GSSG in the presence of substrate Mg2+, the ratio becomes much higher than liN.
+
DISCUSSION
Information from the PMB titrations suggests that there is the net generation of five SH groups per molecule of isocitrate lyase during activation of the enzyme. A molecule of inactive enzyme has four SH groups which seem to be unreactive to NEM. About five SH groups in active enzyme are NEM-reactive and these are presumably identical with the thiol groups generated during activation. The thiols appearing include enzymically essential groups since reaction of active enzyme with GSSG or NEM results in inactivation, and GSH activates inactive enzyme (ref. 7. p. 264). In general accordance with the above data, maximum inhibition of active enzyme was obtained when seven to eight moles of NEM per mole enzyme were added, of which the first mole of NEM had little effect. The need for a slight excess of NEM is Biochim, Biophys. Acta. 105 (1965) 496-505
ISOCITRATE LYASE.
III
505
probably due to instability of NEM8,9 and/or to the rate of reversible loss of NEM~ reactive sulfhydryl groups of the enzyme- which may be comparable to the rate of reaction of enzyme with high dilutions of NEM. At these high dilutions of reagent, some excess may be required to approach a reaction velocity of enzymic thiol with NEM which is independent of the NEM/enzyme ratio and therefore to approach maximal inhibition. Formation of NEM-cysteine residues was confirmed. The data, then, raise the important question about the nature of the activation process. Dissociation of enzyme into separate components is unlikely since sedimentation coefficients were the same for active and inactive enzyme. Additional research will be required to define the activation process more thoroughly. One of the most significant findings of the present work is the specific protection exerted by isocitrate plus Mg2+ against enzyme inactivation by GSSG, suggesting that some (one) of the :five thiols generated by activation are components of the isocitratebinding site. It also suggests formation of a complex involving isocitrate, Mg2+ and enzymic SH, in the enzymic reaction, although Mg2+-facilit at ed binding of isocitrate may simply render the thiol(s) inaccessible to some reagents. Selective labeling of sulfhydryl at the substrate-binding site would be possible by incubating active isocitrate lyase with GSSG in the presence of isocitrate plus Mg2+ so that all SH groups except those (that) at the substrate-binding site(s) become unreactive to NEM, and then treating the enzyme with [l4CJNEM after removal of the reagents. Supporting this is the fact that the efficiency of NEM inhibition as a function of NEM bound increases about two-fold when compared with that for active enzyme, even without consideration of the large amount of radioactivity in enzyme inactivated in the absence of substrate. Were this radioactivity subtracted from that for substrateprotected enzyme, the efficiency would increase about r r-fold (see Table VII) suggesting the presence of only one SH group at the substrate binding site and one binding site per enzyme molecule. Since this increase is greater than the theoretical limiting value of a S-fold increase, the subtraction may not be wholly valid. It will be necessary to separate [l4C]NEM--eysteine from hydrolysates of treated enzyme preparations to precisely evaluate the quantity of NEM which is bound to enzymic thiol. ACKNOWLEDGEMENTS
This investigation was supported in part by Research Grant GM-ogo39 from the National Institutes of Health and a Research Career Development Award (No. I - K3-AI5268) from the Institute of Allergy and Infectious Diseases, Public Health Service. REFERENCES
1. SHUO, T. SHIIO AND B. A. McFADDEN, Biochim, Biophys. Acta, 96 (I965) I23. 1. SHUO, T. SHIIO AND B. A. McFADDEN, Biochim. Biophys. Acta, 96 (1965) II4. P. D. BOYER, J. Am. Chem, Soc., 76 (1954) 433I. D. G. SMYTH, O. O. BLUMENFELD AND vv. KONIGSBERG, Biochem, j., 91 (1964) 589. C. C. LEE AND E. R. SAMUELS, Can. J. Chem., 42 (I964) I64. C. C. LEE AND E. R. SAMUELS, Can. J. Chern: 40 (I962) I040. 7 H. FRAENKEL-CONRAT, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 4, Academic Press, New York, 1957, p. ~56. 8 D. G. SMYTH, A. NAGAMATSU AND J. S. FRUTON, l- Am. Chem. Soc., 82 (1960) 4600. 9 J. D. GREGORY, J. Am. Chern. Soc., 77 (1955) 3922.
I 2 3 4 5 6
Biocbim. Biophys. Acta, 105 (1905) 496-505