ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 262, No. 1, April, pp. 293-297, 1988 The Nature of Enzyme-Substrate Complexes in Acyl-Coenzyme A Dehydrogenases’ SZE-ME1 LAU AND COLIN THORPE2 Department of Chemiatrg and Biochem&rg, University of Delaware, Newark, Delaware 19716 Received October 19,1987 The nature of the purple complex formed upon the addition of octanoyl-CoA to the medium chain acyl-CoA dehydrogenase from pig kidney has been addressed by chemical quenching studies. Previous work, using quenching in 0.1 M KOH, suggested that the dehydrogenation product, trans-2-octenoyl-CoA, was not a participant in reduced rat liver enzyme complexes because no octenoic acid was detected after denaturation (Y. Ikeda, D. G. Hine, K. Okamura-Ikeda and K. Tanaka (1985) J. Biol. Chem. 260, 1326-1337). However, when the octanoyl-CoA-reduced pig kidney enzyme is quenched rapidly in 2 M HCl, the ratio of trans-2-octenoyl-CoA/octanoyl-CoA released is 9/l. A milder acid denaturation procedure yields the corresponding ratio of 0.4/l, i.e., now with an excess of the saturated substrate. Similarly, quenching the pig kidney dehydro- genase in 0.1. M KOH reveals only minor levels of octenoyl chains released into the supernatant. When quenching is insufficiently rapid compared to the internal equili- bration of oxidized enzyme. octanoyl-CoA and reduced enzyme - octenoyl-CoA forms, the outcome is decided by the greater kinetic lability.of the oxidized enzyme species. These data are fully consistent with the original ascription that the purple species observed upon reduction of the acyl-CoA dehydrogenases with substrate represents a charge transfer complex between reduced flavin as the.:donor and trans-2-octenoyl-CoA as the acceptor. 0 1988 Academic Press. Inc. Medium chain acyl-CoA dehydrogenase (EC 1.3.99.3) is a flavoprotein which cata- lyzes the oxidation of acyl-CoA thioesters to their corresponding trans-2-enoyl-CoA derivatives (1). Octanoyl-CoA, a preferred substrate, binds very tightly to the dehy- drogenase (2-4), leading to rapid and sub- stantial bleaching of the bound flavin chromophore. In contrast, equimolar amounts of poorer substrates, such as bu- tyryl- and palmitoyl-CoA, bleach the fla- vin to a lesser degree (1,3,5-7). These data have been usually interpreted in terms of an internal equilibrium (K,) in the reduc- tive half-reaction which is chain-length dependent (3, 6, 8) (Scheme I): 1 This work was supported in part by USPHS Grant GM 26643. ’ To whom correspondence should be addressed. E l FAD,,.+ SHa g E.FAD,,.SH2 !% E.FAD2,aP. The purple color observed upon reduction of enzyme with preferred substrates has been ascribed to a charge transfer com- plex between the reduced flavin as the donor and tightly bound enoyl-CoA as ac- ceptor (E l FAD2,* P: Scheme I; (8-11)). Support for this assignment comes from the variation of the peak position upon re- placement of the natural substrate (11,12) or the flavin prosthetic group (8) with ap- propriate analogs. However, Ikeda et al. (13) have recently suggested that this as- cription is incorrect. Thus, after quench- ing the reduced enzyme complexes in base and methylating the liberated fatty acids, only octanoate (and not octenoate) methyl 293 0003-9861/88 $3.00 Copyrighr 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.