Fluorescence Anisotropy Studies of Enzyme−Substrate Complex Formation in Stearoyl-ACP Desaturase

• Published in: Biochemistry (Easton) Vol. 41; no. 49; pp. 14472 - 14481
• Main Authors: Haas, Jeffrey A, Fox, Brian G
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10-12-2002
• Subjects: Acyl Carrier Protein - chemistry; Acylation; Apoproteins - chemistry; Binding Sites; Dansyl Compounds - chemistry; Escherichia coli Proteins; Fatty Acid Synthase, Type II; Ferredoxins - chemistry; Fluorescein-5-isothiocyanate - chemistry; Fluorescence Polarization - methods; Fluorescent Dyes - chemistry; Kinetics; Macromolecular Substances; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - metabolism; Osmolar Concentration; Pantetheine - analogs & derivatives; Pantetheine - chemistry; Protein Binding; Spectrometry, Fluorescence - methods; Static Electricity; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi020340s

Stearoyl-acyl carrier protein Δ 9-desaturase (Δ9D) catalyzes regio- and stereospecific insertion of cis double bonds into acyl chains attached to acyl carrier protein. Steady-state and stopped-flow fluorescence anisotropy measurements using acylated forms of dansyl- and fluoresceinyl-ACPs revealed equilibrium dissociation constants and dissociation rate constants for 16:0-, 17:0-, and 18:0-ACPs with resting and chemically 4e- reduced Δ9D. Binding of 1 nM 18:0-fluoresceinyl-ACP to one subunit of the dimeric resting Δ9D was observed with K D1 = 13 ± 3 nM. No significant difference in the K D1 value was observed for 4e- Δ9D. An ∼4-fold increase in K D1 per methylene group was observed upon shortening the acyl chain from 18:0 to 17:0 and then 16:0. In different experiments performed with 850 nM 18:0-dansyl-ACP, binding to the second subunit of resting Δ9D was estimated to have K D2 ≈ 350 ± 40 nM. The K D2 values exhibited a similar dependence on acyl chain length as observed for the K D1 values. The k off values measured by stopped-flow anisotropy measurements for reversal of the enzyme−substrate complex were also acyl-chain length dependent and increased 130-fold for 16:0-ACP (130 s-1) relative to 18:0-ACP (1 s-1). Increases in acyl chain length are thus associated with the presently reported increases in the K D and k off values. These results indicate that acyl chain length selectivity derives in major part from partition of the enzyme−substrate complex between substrate release and subsequent steps in catalysis.