Role of hydrophobic partitioning in substrate selectivity and turnover of the Ricinus communis stearoyl acyl carrier protein triangle(9) desaturase

Stearoyl acyl carrier protein Delta(9) desaturase (Delta 9D) uses a diiron center to catalyze the NADPH- and O-2-dependent desaturation of stearoyl ac yl carrier protein (ACP) to form oleoyl-ACP. The reaction of recombinant Ri cinus communis Delta 9D with natural and nonnatural chain length acyl-ACPs was used to examine the coupling of the reconstituted enzyme complex, the s pecificity for position of double-bond insertion, the kinetic parameters fo r the desaturation reaction, and the selectivity for acyl chain length. The coupling of NADPH and O-2 consumption and olefin production was found to b e maximal for 18: 0-ACP, and the loss of coupling observed for the more slo wly desaturated acyl-ACPs was attributed to autoxidation of the electron-tr ansfer chain. Analysis of steady-state kinetic parameters for desaturation of acyl-ACPs having various acyl chain lengths revealed that the KM values were similar (similar to 2.5-fold difference) for 15:0-18:0-ACP, while the k(cat) values increased by similar to 26-fold for the same range of acyl ch ain lengths. A linear increase in log (k(cat)/K-M) was observed upon length ening of the acyl chain from 15:0- to 18:0-ACP, while no further increase w as 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) positio n) suggest that the active-site channel past the diiron center can accommod ate at least one more methylene group than is found in the natural substrat e. The Delta Delta G(binding) estimated from the change in k(cat)/K-M for i ncreasing substrate acyl-chain length was -3 kJ/mol per methylene group, si milar to the value of -3.5 kJ/mol estimated for the hydrophobic partition o f long-chain fatty acids (C-7 to C-21) from water to heptane [Smith, R., an d Tanford, C. (1973) PI-oc. Natl. Acad. Sci. U.S.A. 70, 289-293], Since the KM values are overall similar for all acyl-ACPs tested, the progressive in crease 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 Delt a 9D.