Rapid-Mix and Chemical Quench Studies of Ferredoxin-Reduced Stearoyl-Acyl Carrier Protein Desaturase

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Abstract
Stearoyl-ACP Δ9 desaturase (Δ9D) catalyzes the NADPH- and O2-dependent insertion of a cis double bond between the C9 and C10 positions of stearoyl-ACP (18:0-ACP) to produce oleoyl-ACP (18:1-ACP). This work revealed the ability of reduced [2Fe-2S] ferredoxin (Fd) to act as a catalytically competent electron donor during the rapid conversion of 18:0-ACP into 18:1-ACP. Experiments on the order of addition for substrate and reduced Fd showed high conversion of 18:0-ACP to 18:1-ACP (∼95% per Δ9D active site in a single turnover) when 18:0-ACP was added prior to reduced Fd. Reactions of the prereduced enzyme−substrate complex with O2 and the oxidized enzyme−substrate complex with reduced Fd were studied by rapid-mix and chemical quench methods. For reaction of the prereduced enzyme−substrate complex, an exponential burst phase (kburst = 95 s-1) of product formation accounted for ∼90% of the turnover expected for one subunit in the dimeric protein. This rapid phase was followed by a slower phase (klinear = 4.0 s-1) of product formation corresponding to the turnover expected from the second subunit. For reaction of the oxidized enzyme−substrate complex with excess reduced Fd, a slower, linear rate (kobsd = 3.4 s-1) of product formation was observed over ∼1.5 turnovers per Δ9D active site potentially corresponding to a third phase of reaction. An analysis of the deuterium isotope effect on the two rapid-mix reaction sequences revealed only a modest effect on kburst (Dkburst ∼ 1.5) and klinear(Dklinear ∼ 1.4), indicating C−H bond cleavage does not contribute significantly to the rate-limiting steps of pre-steady-state catalysis. These results were used to assemble and evaluate a minimal kinetic model for Δ9D catalysis.