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Rate constants for C5-proR/proS-hydron exchange from l-dihydroorotate were determined by 1H NMR and detritiation in various oxygen-containing and amine buffers at 37 °C and ionic strength 1.0 M in aqueous solution. Thermodynamically unfavorable proton transfer from the C5-proR and -proS positions (pKa = 20−21) to oxygen-containing and amine bases shows general-base catalysis with a Bronsted β value of 0.84 ± 0.05 and 0.81 ± 0.08, respectively, which is consistent with a late, enolate-like transition state. General-base catalysis is detectable because there is a 160- or 85-fold negative deviation for the C5-proR and -proS protons, respectively, from this correlation for deuterioxide ion. Deviations of (kH/kT)obsd and (kD/kT)obsd from the Swain−Schaad equation are consistent with internal return of the transferred hydron to a free C5-carbanion/enolate intermediate from the conjugate general acid. This corresponds to an Eigen-type mechanism for hydron transfer, in which both proton transfer and diffusional s...

This report describes the first demonstration of slow-onset feedback inhibition of an enzyme that catalyzes the first committed step in a biosynthetic pathway. alpha-Isopropylmalate synthase (IPMS) catalyzes the first committed step of the l-leucine biosynthetic pathway and is feedback-inhibited by l-leucine. Initial velocity experiments on the Mycobacterium tuberculosis IPMS indicate that inhibition by l-leucine is linearly noncompetitive versus alpha-ketoisovalerate. Time-courses displayed a burst of product formation followed by a linear steady-state rate when reactions were initiated by the addition of enzyme. The burst rate showed a hyperbolic dependence on the concentration of l-leucine indicating that inhibition proceeds in two steps, an initial rapid binding step followed by slow isomerization to a more tightly bound complex.

The Mycobacterium tuberculosis inhA-encoded enoyl-ACP reductase is inhibited by isonicotinoylated-NADP (INH-NADP) with a Ki of 130 nM. The crystal structure of the InhA:INH-NADP complex was solved. The structure revealed that it is the acyclic 4S isomer of INH-NADP that binds to the enzyme in a conformation similar to the InhA:INH-NAD complex solved previously. The in vivo implications of the mechanism of action of and resistance to isoniazid resulting from the dual ability of InhA to bind to INH-NAD and INH-NADP with high affinity are discussed.