Biochemistry 1981, 20, 5183-5187
5183
Modification and Inactivation of CoA Transferase by 2-Nitro-5 -(t hiocy anato)benzoatet L. Allen Kindman and William P. Jencks*
ABSTRACT:
Succinyl-CoA:3-ketoacidcoenzyme A transferase undergoes a biphasic reaction with 2-nitro-5-(thiocyanato)benzoate, giving -70% loss of activity in the initial phase. Active-site titration shows that this inactivation represents the complete loss of activity of 75 f 5% of the enzyme molecules. The remaining 25 f 5% of the active sites is protected against inactivation by methyl methanethiosulfonate and 5,5‘-dithiobis(2-nitrobenmate); this protection is removed upon treatment of the modified enzyme with dithiothreitol. Values of k,,/K,,, for the two half-reactions catalyzed by the enzyme are the same for the native and modified enzymes on the basis of
number of remaining active sites. The modified enzyme shows a smaller decrease in activity with increasing pH in the range pH 7.5-8.7 than the native enzyme. It is concluded that the “essential” thiol group of the enzyme is not involved directly in catalysis and that it reacts with 2-nitro-5-(thiocyanato)benzoate by two pathways, to form active and inactive enzymes. This can be explained by the attack of the thiol on carbon to form active enzyme-SCN and the attack on sulfur to form enzyme-SSAr, which is blocked at the active site and rapidly undergoes irreversible inactivation.
Succinyl-CoA:3-ketoacid coenzyme A transferase from pig heart is inactivated by the thiol reagents DTNB,’ N-ethylmaleimide, and p-(hydroxymercuri)benzoate (Hersh, 1966; White et al., 1976). The initial reaction of one thiol group per subunit with DTNB causes loss of activity to >99%, is blocked by the presence of both substrates, is accelerated 3-100-fold in enzyme-CoA, and presumably occurs at or near the active site; this is followed by a concentration-dependent, irreversible denaturation of the enzyme and reaction of additional thiol groups with DTNB (White et al., 1976). The enzyme from Escherichia coli contains six thiol groups that react at an increased rate in enzyme-CoA to give partial inactivation (Sramek et al., 1977). The experiments described here were carried out to determine whether the active-site thiol group of the pig heart enzyme is required for catalytic activity of the enzyme or whether the loss of activity with bulky thiol reagents is simply due to blocking of the active site. We have therefore examined the reaction of the enzyme with NTCB,’ which is expected to
Materials and Methods Materials. CoA transferase, with a specific activity of 280 pmol of acetoacetyl-CoA consumed min-’ mg-’ under standard conditions (White et al., 1976), was kindly donated by Susan Moore; it was prepared from pig heart by a slight modification of a published procedure (White & Jencks, 1976a).2 NEthylmaleimide, 5,5’-dithiobis(2-nitrobenzoic acid), acetohydroxamic acid, disodium ethylenediaminetetraacetate, succinic acid, and boric acid were recrystallized before use. Tris and magnesium sulfate were Mann Ultrapure. Diketene was distilled just prior to use for the synthesis of acetoacetyl-coA (White & Jencks, 1976b). Methyl methanethiosulfonate was kindly donated by Professor Henry Mautner. 2-Nitro-5-(thiocyanato)benzoic acid, half-potassium salt (mp 242-245 “C), was prepared according to Degani & Patchornik (1971). Water was deionized and glass distilled. Stock solutions of NTCB, DTNB, dithiothreitol, and N-ethylmaleimide were prepared shortly before use in solutions containing 0.0 1 M EDTA, pH 7, that had been deaerated by bubbling with argon for 30 min. Methods. Enzyme activity was determined by following the disappearance of the absorbance of acetoacetyl-CoA at 3 10 nm in the presence of 10 mM potassium succinate, 5 mM magnesium sulfate, and 0.067 M Trissulfate buffer, pH 8.10, at 25 OC. Incubations with NTCB were generally carried out in 0.2 M boric acid-potassium borate buffer with a final pH of 8.1; Tris buffer was generally avoided because of its slow reaction with NTCB. Phosphate buffer (0.23 M) at pH 8.1 was found to inactivate enzyme-CoA (Hersh & Jencks, 1967). Active-site titration with acetoacetyl-CoA and acetohydroxamic acid was carried out as described by Pickart & Jencks (1979). The enzyme was incubated with acetohydroxamic acid for 5-10 min after formation of enzyme-CoA by addition of acetoacetyl-CoA. It was shown that NTCBmodified enzyme that was converted to the hydroxamic acid
coo-
/
NTCB
.transfer its cyano group to the active-site thiol to form enzyme-SCN (Degani et al., 1970). The small cyano group and the thiomethyl group, which is attached to thiols by reaction with methyl methanethiosulfonate, can block thiol groups without causing the loss of enzyme activity that is found with larger thiol reagents when the thiol group is not required for activity (Vanaman & Stark, 1970; Smith & Kenyon, 1974). The results show that NTCB reacts with CoA transferase by two pathways to form irreversibly inactivated enzyme and a modified, fully active enzyme that is protected against inhibition by thiol reagents, presumably by transfer of the -SAr and -CN groups, respectively, to‘enzyme-SH. From the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254. Received February 3, 1981. Publication No. 1371. This research was supported in part by grants from the National Science Foundation (PCM 77-08369) and the National Institutes of Health (GM 20888).
0006-296018 110420-5183$01.25/0
Abbreviations used: DTNB, 5,5’-dithiobis(2-nitrobenzoic acid); EDTA, ethylenediaminetetraacetic acid; NTCB, 2-nitro-5-(thiocyanato)benzoic acid; Tris, tris(hydroxymethy1)aminomethane. The enzyme preparation examined here, while of higher specific activity than that studied previously (White & Jencks, 1976a), was found to have a break in its primary structure that gives two fragments on gel electrophoresis in sodium dodecyl sulfate (A. Cheung, unpublished experiments).
0 1981 American Chemical Society
5184
BIOCHEM I STRY
K I N D M A N AND JENCKS
Table I: Effect of NTCB on the Inactivation of CoA Transferase by Thiol ReagentsD
acetoacetylCoA (pM) methyl methanethiosulfonate'
DTNB~ N-ethylmaleimide e
1.8 mM NTCB no NTCB
0 min
120 min
80 min
0.69 1.73
0 10 0 0 10 13
170 min
0.0028
0.0039 0.0025c
0.69
2.0
0.26 0.59
0.019
0.009
0.59
0.004
Rate constants for loss of enzyme activity were determined before and after incubation of 0.2-0.4 pM enzyme with 1.7-2.0 mM NTCB for 80-170 rnin at 25 "C. Methyl methanethiosulfonate (0.4 mM), 8 mM potassium phosphate, 0.8 mM EDTA, and 0.25 M borate buffer, pH 8.3. Methyl methanethiosulfonate omitted. DTNB (0.33 mM), 6 mM potassium phosphate, 1.0 mM EDTA, and 0.18 M borate buffer, PH 8.1. 'N-Ethylmaleimide (0.86 mM), 7 mM potassium phosuhate, 0.9 mM EDTA, and 0.17 M borate buffer, pH 8.1.
0 1 ' 0
'
"
120
"
" '
" '
240
360
"
' 480
Minutes
FIGURE 1: Inactivation of 0.3 pM enzyme (0)or enzyme-CoA (0) in the presence of 1.0 mM NTCB, 5.7 mM potassium phosphate, and 1.0 mM EDTA in 0.2 M potassium borate buffer, pH 8.1, at 25 OC. Acetoacetyl-CoA, 6 pM, was added to form enzyme-CoA (0).
derivative gave 83-95% reactivation in the presence of 1.9 mM coenzyme A and that the modified enzyme gave < 10% inactivation in the presence of acetohydroxamic acid for 40 rnin under the same conditions, but in the absence of acetoacetyl-coA. Pseudo-first-order rate constants for the initial phase of the biphasic enzyme inactivation with NTCB were obtained by subtracting the extrapolated activity for the slow phase of inactivation from the observed activity and plotting the difference semilogarithmically against time; the rate constant was obtained from 0.693/t1/,. Results In the presence of 1 mM NTCB at pH 8.1, CoA transferase is inactivated with a biphasic time course (Figure 1, squares). The initial, faster phase gives approximately 70% inactivation and is followed by a slower phase; in the presence of 1.8 mM NTCB,
