8022 zyxwvutsrqpon Biochemistry zyxwvut 1988, 27. 8022-8028 Krebs, H. A. (1973) Symp. SOC. Exp. Biol. 27, 299-318. Krumdieck, C. L., zyxwvutsrqp & Baugh, C. M. (1969) Biochemistry 8, Krumdieck, C. L., & Baugh, C. M. (1980) Methods Enzymol. Kutzbach, C., & Stokstad, E. L. R. (1971) Biochim. Biophys. Lu, Y.-Z., Aiello, P. A., & Matthews, R. G. (1984) Bio- MacKenzie, R. E., & Baugh, C. M. (1980) Biochim. Biophys. MacKenzie, R. E., & Tan, L. U. L. (1980) Methods Enzymol. Matthews, R. G. (1986) Methods. Enzymol. 122, 333-339. Matthews, R. G., & Baugh, C. M. (1980) Biochemistry 19, Matthews, R. zyxwvutsrqp G., Ross, J., Baugh, C. M., Cook, J. D., & Davis, L. (1982) Biochemistry 21, 1230-1238. Matthews, R. G., Lu. Y.-Z., Green, J. M., & MacKenzie, R. E. (1985) in Proceedings zyxwvutsr of the Second Workshop zyxwvutsr on Folyl and Antifolyl Polyglutamates (Goldman, I. D., Ed.) pp 65-75, Praeger, New York. Matthews, R. G., Ghose, C., Green, J. M., Matthews, K. D., & Dunlap, R. B. (1987) in Adv. Enzyme Regul. 26, 1568-1572. 66, 523-529. Acta 250, 459-477. chemistry 23, 6870-6876. Acta 611, 187-195. 66, 609-615. 2040-2045. 157-171. McGuire, J. J., & Coward, J. J. (1984) in Folates and Pterins, Vol. 1, Chemistry and Biochemistry of Folates (Blakley, R. L., & Benkovic, S. J., Eds.) pp 135-190, Wiley, New York. Mejia, N. R., & MacKenzie, R. E. (1985) J. Biol. Chem. 260, Melander, L., & Saunders, W. H. (1980) Reaction Rates of Isotopic Molecules, Wiley, New York. Richardson, R. E., Healy, M. J., & Nixon, P. F. (1979) Biochim. Biophys. Acta 585, 128-134. Ross, J., Green, J., Baugh, C. M., MacKenzie, R. E., & Matthews, R. G. (1984) Biochemistry 23, 1796-1801. Segel, I. H. (1977) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, Wiley, New York. Sliecker, L. J., & Benkovic, S. J. (1984) J. Am. Chem. SOC. Smith, S. L., Patrick, P., Stone, D., Phillips, A. W., & Burchall, J. J. (1979) J. Biol. Chem. 254, 11475-11484. Strong, W., Joshi, G., Lura, R., Muthukumaraswamy, N., & Schirch, V. (1987) J. Biol. Chem. 262, 12519-12525. Tan, L. U. L., Drury, E. J., & MacKenzie, R. E. (1977) J. Biol. Chem. 252, 1117-1 122. Vanoni, M. A., Ballou, D. P., & Matthews, R. G. (1983) J. Biol. Chem. 258, 11510-11514. 14616-1 4620. 106, 1833-1838. 2-Octynoyl Coenzyme A Is a Mechanism-Based Inhibitor of Pig Kidney Medium-Chain Acyl Coenzyme A Dehydrogenase: Isolation of the Target Peptide? Patricia J. Powell and Colin Thorpe* Received March 30, 1988; Revised Manuscript Received June 1, 1988 Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 ABSTRACT: Pig kidney medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3) is irreversibly and stoichio- metrically inactivated by [l-14C]-2-octynoylcoenzyme A. The linkage is stable at pH 2-6, but labile under basic conditions. The inhibitor labels a unique tryptic peptide, zyxwv Ile-Tyr-Gln-Ile-Tyr-Glu-Gly-Thr-Ala- Gln-Ile-Gln-Arg, close to the C-terminus of the protein. The peptide is labeled at Glu-401 with the acyl moiety of the inhibitor but does not contain detectable coenzyme A. Both the inactivation of the de- hydrogenase and the appearance of an absorption band at 800 nm show large primary deuterium isotope effects using 4,4’-dideuterio-2-octynoyl-CoA (7.3 and 6.3, respectively). Thus, 2-octynoyl-CoA is a mechanism-based inactivator of the dehydrogenase and is activated by rate-limiting y-proton abstraction. Glutamate-401 may be the base that abstracts the pro-R a-proton during the dehydrogenation of normal substrates. T e short-, medium-, and long-chain acyl-CoAl de- hydrogenases are immunologically distinct (Ikeda et al., 1985a) flavoproteins that catalyze the first oxidative step of 0-oxi- dation (Beinert, 1963). These enzymes introduce a trans double bond between C-2 and C-3 of their acyl-CoA substrates (Beinert, 1963) and are subsequently reoxidized by electron- transferring flavoprotein (ETF; Crane & Beinert, 1956): dH,,.RCH,CH,COSCoA dH,d*RCH=CHCOSCoA dH,d.RCH=CHCOSCoA + 2ETF,, + dH,,*RCH=CHCOSCoA + 2ETF,,d This work was supported in part by a grant from the US. Public Health Service (GM 26643). The most thoroughly studied of these flavoproteins is the mammalian medium-chain acyl-CoA dehydrogenase. Re- cently the gene sequences of both the human and rat liver enzymes have been reported (Kelly et al., 1987; Matsubara et al., 1987, respectively). A deficiency of this enzyme in humans leads to sometimes fatal organic acidurias, and this ’ Abbreviations: CoA(SH), coenzyme A; HPLC, high-performance liquid chromatography; ETF, electron-transferring flavoprotein; TFA, trifluoroacetic acid; TPCK, L-1 -(tosylamino)-2-phenylethyl chloromethyl ketone; PTH, phenylthiohydantoin; TCA, trichloroacetic acid; dH, de- hydrogenase; DTT, dithiothreitol; Tris, tris(hydroxymethy1)amino- methane. 0006-2960/88/0427-8022$01.50/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1988 American Chemical Society