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

Biochemistry
S ParikhP J Tonge

Abstract

The role of tyrosine 158 (Y158) and lysine 165 (K165) in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, has been investigated. These residues have been identified as putative catalytic residues on the basis of structural and sequence homology with the short chain alcohol dehydrogenase family of enzymes. Replacement of Y158 with phenylalanine (Y158F) and with alanine (Y158A) results in 24- and 1500-fold decreases in k(cat), respectively, while leaving K(m) for the substrate, trans-2-dodecenoyl-CoA, unaffected. Remarkably, however, replacement of Y158 with serine (Y158S) results in an enzyme with wild-type activity. Kinetic isotope effect studies indicate that the transfer of a solvent-exchangeable proton is partially rate-limiting for the wild-type and Y158S enzymes, but not for the Y158A enzyme. These data indicate that Y158 does not function formally as a proton donor in the reaction but likely functions as an electrophilic catalyst, stabilizing the transition state for hydride transfer by hydrogen 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 ...Continue Reading

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