Kinetic and Chemical Mechanisms of the fabG-Encoded Streptococcus pneumoniae -Ketoacyl-ACP Reductase

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• 作者：Mehul P. Patel ; Wu-Schyong Liu ; Joshua West ; David Tew ; Thomas D. Meek ; and Sara H. Thrall
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：December 20, 2005
• 年：2005
• 卷：44
• 期：50
• 页码：16753 - 16765
• 全文大小：178K
• 年卷期：v.44,no.50(December 20, 2005)
• ISSN：1520-4995

文摘

DER=0 ALIGN="middle">-Ketoacyl-acyl carrier protein reductase (KACPR) catalyzes the NADPH-dependent reductionof ddle">-ketoacyl-acyl carrier protein (AcAc-ACP) to generate (3S)-ddle">-hydroxyacyl-ACP during the chain-elongation reaction of bacterial fatty acid biosynthesis. We report the evaluation of the kinetic and chemicalmechanisms of KACPR using acetoacetyl-CoA (AcAc-CoA) as a substrate. Initial velocity, productinhibition, and deuterium kinetic isotope effect studies were consistent with a random bi-bi rapid-equilibriumkinetic mechanism of KACPR with formation of an enzyme-NADP⁺-AcAc-CoA dead-end complex.Plots of log V/K_NADPH and log V/K_AcAc-CoA indicated the presence of a single basic group (pK = 5.0-5.8)and a single acidic group (pK = 8.0-8.8) involved in catalysis, while the plot of log V vs pH indicatedthat at high pH an unprotonated form of the ternary enzyme complex was able to undergo catalysis.Significant and identical primary deuterium kinetic isotope effects were observed for V (2.6 ± 0.4),V/K_NADPH (2.6 ± 0.1), and V/K_AcAc-CoA (2.6 ± 0.1) at pH 7.6, but all three values attenuated to values ofnear unity (1.1 ± 0.03 or 0.91 ± 0.02) at pH 10. Similarly, the large -secondary deuterium kineticisotope effect of 1.15 ± 0.02 observed for [4R-²H]NADPH on V/K_AcAc-CoA at pH 7.6 was reduced to avalue of unity (1.00 ± 0.04) at high pH. The complete analysis of the pH profiles and the solvent, primary,secondary, and multiple deuterium isotope effects were most consistent with a chemical mechanism ofKACPR that is stepwise, wherein the hydride-transfer step is followed by protonation of the enolateintermediate. Estimations of the intrinsic primary and secondary deuterium isotope effects (^Dk = 2.7,^-Dk = 1.16) and the correspondingly negligible commitment factors suggest a nearly full expression ofthe intrinsic isotope effects on ^DV/K and ^-DV/K, and are consistent with a late transition state for thehydride transfer step. Conversely, the estimated intrinsic solvent effect (^D₂Ok) of 5.3 was poorly expressedin the experimentally derived parameters ^D₂OV/K and ^D₂OV (both = 1.2 ± 0.1), in agreement with theestimation that the catalytic commitment factor for proton transfer to the enolate intermediate is large.Such detailed knowledge of the chemical mechanism of KAPCR may now help guide the rational designof, or inform screening assay-design strategies for, potent inhibitors of this and related enzymes of theshort chain dehydrogenase enzyme class.