Desaturation, Chain Scission, and Register-Shift of Oxygen-Substituted Fatty Acids during Reaction with Stearoyl-ACP Desaturase † Corina E. Rogge and Brian G. Fox* Department of Biochemistry, College of Agricultural and Life Sciences, UniVersity of Wisconsin, Madison, Wisconsin 53706 ReceiVed April 24, 2002; ReVised Manuscript ReceiVed June 5, 2002 ABSTRACT: Stearoyl acyl carrier protein Δ 9 desaturase catalyzes the NADPH- and O 2 -dependent insertion of a cis double bond between the C-9 and C-10 positions of the acyl chain in the kinetically preferred natural substrate 18:0-ACP. In this work, substrate analogues with an oxygen atom singly replacing the methylene groups at the 8, 9, 10, and 11 positions of the stearoyl chain were synthesized, converted to acyloxy-ACPs, and used as probes of desaturase reactivity. Evidence for desaturation, acyloxy chain scission, and register-shift in binding prior to chain scission was obtained. Reactions with acyloxy-ACPs having either O-8 or O-11 substitutions gave a single desaturation product consistent with the insertion of a cis double bond between C-9 and C-10. The k cat /K M values for the O-8- and O-11-substituted acyloxy- ACPs were comparable to that of the natural substrate, indicating that the presence of an ether group adjacent to the site of reactivity did not significantly interfere either with the desaturation reaction or with the binding of substrate in the proper register for desaturation between C-9 and C-10. For reactions with the O-9 and O-10 acyloxy-ACPs, the k cat values were decreased to ∼3% of that observed for 18:0-ACP, and upon reaction, the acyloxy chain was broken to yield an ω-hydroxy fatty alkanoyl-ACP and a volatile long-chain aldehyde. For the O-9 substitution, 8-hydroxyoctanoate and 1-nonanal were obtained, corresponding to the anticipated binding register and subsequent reaction between the O-9 and C-10 positions. In contrast, the O-10 substitution yielded 9-hydroxynonanoyl-ACP and 1-octanal, corresponding to an obligate “register-shift” of acyloxy chain binding prior to reaction between the O-10 and C-11 positions. Register-shift is thus defined as a mechanistically relevant misalignment of acyl chain binding that results in reaction at positions other than between C-9 and C-10. The inability of the O-10 acyloxy probe to undergo reaction between the C-9 and O-10 positions provides evidence that the Δ9D-catalyzed desaturation of stearoyl-ACP may initiate at C-10. Possible mechanisms of the acyl chain scission and implications of these results for the desaturation mechanism are considered. The soluble acyl-ACP 1 desaturases catalyze the NADPH- and O 2 -dependent insertion of cis double bonds into fatty acyl chains that are covalently attached to ACP (1). These desaturases are members of a structurally related family of diiron enzymes that also includes the R2 component of ribonucleotide diphosphate reductase (2-4) and numerous bacterial hydrocarbon monooxygenases (5, 6). All of the above diiron enzymes bind and activate O 2 during catalysis, and a paradigm for reaction involving the generation of a high-valent iron oxidizing state has emerged from extensive studies of biological systems and chemical synthetic ana- logues (5-11). For Δ9D, protein-acyl chain interactions are recognized as important catalytic determinants (10, 12-14). This enzyme exhibits remarkably high fidelity for the insertion of a cis double bond between the C-9 and C-10 positions of a long- chain acyl-ACP, including nonnatural acyl-ACP analogues (14, 15). These results imply that an accurate, reproducible generation of the proper binding register for an otherwise nondescript acyl chain is an important aspect of catalysis. An X-ray structure revealed that the diiron center was buried within the protein and alongside a tunnel that might serve as the fatty acyl chain binding site (16). It is reasonable that the depth of the diiron center within the protein subunit and the protein-protein interaction surface provided by the ACP portion of the substrate provide important constraints to the positional specificity of double bond formation. Moreover, the exclusive cis stereochemistry observed for the introduced double bonds apparently corresponds to the presence of a prominent bend in this tunnel adjacent to the diiron center (16, 17). In addition to positional and stereochemical constraints on double bond formation provided by protein structure, the acyl-ACP desaturases exhibit a high degree of selectivity † This work was supported by National Institutes of Health Grant GM-50853. * Correspondence should be addressed to this author at 141B New Biochemistry, 433 Babcock Dr., University of Wisconsin, Madison, WI 53706. Phone: (608) 262-9708, fax: (608) 262-3453, e-mail: bgfox@biochem.wisc.edu. 1 Abbreviations: ACP, holo-acyl carrier protein; n:0-ACP, ACP with an n-carbon saturated fatty acid covalently attached to ACP through a phosphopantetheine thioester bond; n:1-ACP, acyl-ACP with a co- valently attached n-carbon monounsaturated fatty acid; Δ9D, 18:0-ACP Δ 9 desaturase; GC/EI, gas chromatography/electron ionization; ESI, electron spray ionization; MALDI, matrix-assisted laser desorption; MS, mass spectrometry; MSTFA, N-methyl-N-trimethylsilyl trifluoroaceta- mide; O-8, 7-(decyloxy)heptanoic acid; O-9, 8-(nonyloxy)octanoic acid; O-10, 9-(octyloxy)nonanoic acid; O-11, 10-(heptyloxy)decanoic acid; TMS, trimethylsilyl. 10141 Biochemistry 2002, 41, 10141-10148 10.1021/bi020306d CCC: $22.00 © 2002 American Chemical Society Published on Web 07/12/2002