Roles of tyrosine 158 and lysine 165 in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis

The role of tyrosine 158 (Y158) and lysine 165 (K165) in the catalytic mech anism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, has been investigated. These residues have been identified as putative catalyt ic residues on the basis of structural and sequence homology with the short chain alcohol dehydrogenase family of enzymes. Replacement of Y158 with ph enylalanine (Y158F) and with alanine (Y158A) results in 24- and 1500-fold d ecreases in k(cat), respectively, while leaving K-m for the substrate, tran s-2-dodecenoyl-CoA, unaffected. Remarkably, however, replacement of Y158 wi th serine (Y158S) results in an enzyme with wild-type activity. Kinetic iso tope effect studies indicate that the transfer of a solvent-exchangeable pr oton is partially rate-limiting for the wild-type and Y158S enzymes, but no t for the Y158A enzyme. These data indicate that Y158 does not function for mally as a proton donor in the reaction but likely functions as an electrop hilic catalyst, stabilizing the transition state for hydride transfer by hy drogen bonding to the substrate carbonyl. A conformational change involving rotation of the Y158 side chain upon binding of the enoyl substrate to the enzyme is proposed as an explanation for the inverse solvent isotope effec t observed on V/KDD-CoA when either NADH or NADD is used as the reductant. These data are consistent with the recently published structure of a C16 fa tty acid substrate bound to InhA that shows Y158 hydrogen bonded to the sub strate carbonyl group and rotated from the position it occupies in the InhA -NADH binary complex [Rozwarski, D. A., Vilcheze, C., Sugantino, M., Bittma n, R., and Sacchettini, J. C. (1999) J. Biol. Chem. 274, 15582-15589]. Fina lly, the role of K165 has been analyzed using site-directed mutagenesis. Re placement of K165 with glutamine (K165Q) and arginine (K165R) has no effect on the enzyme's catalytic ability or on its ability to bind NADH. However, the K165A and K165M enzymes are unable to bind NADH, indicating that K165 has a primary role in cofactor binding.