title: Role of hydrophobic partitioning in substrate selectivity and turnover of the ricinus communis stearoyl acyl carrier protein delta(9) desaturase.
abstract: Stearoyl acyl carrier protein Delta(9) desaturase (Delta9D) uses a diiron center to catalyze the NADPH- and O(2)-dependent desaturation of stearoyl acyl carrier protein (ACP) to form oleoyl-ACP. The reaction of recombinant Ricinus communis Delta9D with natural and nonnatural chain length acyl-ACPs was used to examine the coupling of the reconstituted enzyme complex, the specificity for position of double-bond insertion, the kinetic parameters for the desaturation reaction, and the selectivity for acyl chain length. The coupling of NADPH and O(2) consumption and olefin production was found to be maximal for 18:0-ACP, and the loss of coupling observed for the more slowly desaturated acyl-ACPs was attributed to autoxidation of the electron-transfer chain. Analysis of steady-state kinetic parameters for desaturation of acyl-ACPs having various acyl chain lengths revealed that the K(M) values were similar ( approximately 2.5-fold difference) for 15:0-18:0-ACP, while the k(cat) values increased by approximately 26-fold for the same range of acyl chain lengths. A linear increase in log (k(cat)/K(M)) was observed upon lengthening of the acyl chain from 15:0- to 18:0-ACP, while no further increase was observed for 19:0-ACP. The similarity of the k(cat)/K(M) values for 18:0- and 19:0-ACPs and the retained preference for double-bond insertion at the Delta(9) position with 19:0-ACP (>98% desaturation at the Delta(9) position) suggest that the active-site channel past the diiron center can accommodate at least one more methylene group than is found in the natural substrate. The DeltaDeltaG(binding) estimated from the change in k(cat)/K(M) for increasing substrate acyl-chain length was -3 kJ/mol per methylene group, similar to the value of -3.5 kJ/mol estimated for the hydrophobic partition of long-chain fatty acids (C-7 to C-21) from water to heptane [Smith, R. , and Tanford, C. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 289-293]. Since the K(M) values are overall similar for all acyl-ACPs tested, the progressive increase in hydrophobic binding energy available from increased chain length is apparently utilized to enhance catalytic steps, which thus provides the underlying physical mechanism for acyl chain selectivity observed with Delta9D.
authors.affiliation: The Institute for Enzyme Research, Graduate School, University of Wisconsin, Madison, Wisconsin 53705, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Haas
authors.forename: B G
authors.initials: BG
authors.lastname: Fox
mesh_terms: D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012278:Ricinus / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C018747:acyl-(acyl-carrier-protein)desaturase