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BIOCHEMICAL SOCIETY TRANSACTIONSplexes generates an extra thiol in the dithiolane ring, which is then susceptible to a stable modification with maleimides, resulting in loss of catalytic function (Brown & Perham, 1976).PDC and O G D C were purified from fresh ox heart (De Marcucci et ul.. 1985). T h e activities of both these complexes were not affected by incubation at room temperature with 0.5 mM-N-ethylmaleimide for 1 h. Addition of. respectively, 2 mwpyruvate and 2 m~-2-oxoglutarate resulted in rapid, irreversible inhibition complete within 10 min. Use o f N-ethyl-[ 2,3-"C]maleimide in this reaction and resolution of the inhibited complexes by SDS/polyacrylamide-gel electrophoresis and fluorography confirmed that the lipoate acetyltransferase and component X subunits of PDC and the lipoate succinyltransferase of OGDC were modified by the maleimide. However, when PDC was preincubated at room temperature with pyruvate before addition of Ncthylmaleimide, there was a time-dependent protection against inhibition of overall activity by the maleimide. After SO min incubation of PDC with pyruvate, addition of Ncthylmaleimide could only produce approx. 10% inhibition o f activity. Prior addition of acetyl-CoA to NADH-reduced PDC also prevented the inhibitory effect of N-ethylmaleimide but more rapidly, with 90% protection occurring 1 0 min after addition of acetyl-CoA. Incubation of PDC with either substrate in the absence of N-ethylmaleimide had no effect on ovcrall activity. There was greatly reduced incorporation of N-ethyl [ 2,3-1'C]maleimide into E 2 and component X subunits if PDC was preincubated with pyruvate or acetyl-CoA. These results suggested that prolonged incubation of PDC with pyruvate in the absence of acetyl-CoA or with acctyl-CoA plus N A D H led to the second thiol on the E 2 lipoyl group becoming unamenable to modification with N-ethylmaleimidc. When PDC was incubated with 12-1JC]pyruvate it was found that acquisition of resistance to N-ethylmaleimide inhibition over 60 rnin was accompanied by a slow increase in ["C]acetyl group incorporation into the complex after the initial rapid phase. Table 1 shows that incorporation of [ lJC]acetyI groups from [2-"C]pyruvate into both E2 and component X was approximately doubled in the absence of N-ethylmaleimide. One explanation of these observations is that a prolonged incubation with acetylating substrate under the conditions employed produced S", Sx-(diacetylj-lipoyl groups. T h e formation of Sh, Sx-(diacetyljlipoamide by mammalian PDC has been detected previously by '?C-n.m.r. spectroscopy ( O C o n n o r et al., 1982). Another interpretation is that acetylation of a second lipoate residue permitted interaction of two acetyl-lipoyl groups such that neither might react with N-ethylmaleimide, perhaps by Table 1. Iticmprutiori o~/Z-"('ll?vrirviite inlo I S titid X siihirriits PDC was incubated with [2-"C]pyruvate in the absence or presence of 0.5 mM-N-cthylmaleimidc (NEM). After 30 rnin incubation, approx. 22 pg of protein was resolved on a 10'%, (w/v)...