RESUMO
o-Carbonyl arylboronic acids such as 2-formylphenylboronic acid (2-FPBA) are employed in biocompatible conjugation reactions with the resulting iminoboronate adduct stabilized by an intramolecular N-B interaction. However, few studies have utilized these reagents as active site-directed enzyme inhibitors. We show that 2-FPBA is a potent reversible, slow-onset inhibitor of mandelate racemase (MR), an enzyme that has served as a valuable paradigm for understanding enzyme-catalyzed abstraction of an α-proton from a carbon acid substrate with a high pKa. Kinetic analysis of the progress curves for the slow onset of inhibition of wild-type MR using a two-step kinetic mechanism gave Ki and Ki* values of 5.1 ± 1.8 and 0.26 ± 0.08 µM, respectively. Hence, wild-type MR binds 2-FPBA with an affinity that exceeds that for the substrate by â¼3000-fold. K164R MR was inhibited by 2-FPBA, while K166R MR was not inhibited, indicating that Lys 166 was essential for inhibition. Unexpectedly, mass spectrometric analysis of the NaCNBH3-treated enzyme-inhibitor complex did not yield evidence of an iminoboronate adduct. 11B nuclear magnetic resonance spectroscopy of the MR·2-FPBA complex indicated that the boron atom was sp3-hybridized (δ 6.0), consistent with dative bond formation. Surprisingly, X-ray crystallography revealed the formation of an Nζ-B dative bond between Lys 166 and 2-FPBA with intramolecular cyclization to form a benzoxaborole, rather than the expected iminoboronate. Thus, when o-carbonyl arylboronic acid reagents are employed to modify proteins, the structure of the resulting product depends on the protein architecture at the site of modification.
RESUMO
Rational approaches for the design of enzyme inhibitors furnish powerful strategies for developing pharmaceutical agents and tools for probing biological mechanisms. A new strategy for the development of gem-disubstituted substrate-product analogues as inhibitors of racemases and epimerases is elaborated using α-methylacyl-coenzyme A racemase from Mycobacterium tuberculosis (MtMCR) as a model enzyme. MtMCR catalyzes the epimerization at C2 of acyl-CoA substrates, a key step in the metabolism of branched-chain fatty acids. Moreover, the human enzyme is a potential target for the development of therapeutic agents directed against prostate cancer. We show that rationally designed, N,N-dialkylcarbamoyl-CoA substrate-product analogues inactivate MtMCR. Binding greatly exceeds that of the substrate, (S)-ibuprofenoyl-CoA, up to â¼250-fold and is proportional to the alkyl chain length (4-12 carbons) with the N,N-didecyl and N,N-didodecyl species having competitive inhibition constants with values of 1.9⯱â¯0.2⯵M and 0.42⯱â¯0.04⯵M, respectively. The presence of two decyl chains enhanced binding over a single decyl chain by â¼204-fold. Overall, the results reveal that gem-disubstituted substrate-product analogues can yield extremely potent inhibitors of an epimerase with a capacious active site.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Racemases e Epimerases/antagonistas & inibidores , Relação Dose-Resposta a Droga , Difusão Dinâmica da Luz , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Cinética , Espectrometria de Massas , Estrutura Molecular , Racemases e Epimerases/metabolismo , Relação Estrutura-AtividadeRESUMO
l-Talarate/galactarate dehydratase (TGD) is a member of the enolase superfamily of enzymes and catalyzes the dehydration of either meso-galactarate or l-talarate to form 5-keto-4-deoxy-d-glucarate (5-KDG). To facilitate study of this enzyme and other galactarate dehydratases, a continuous circular dichroism-based assay has been developed. Using recombinant enzyme from Salmonella typhimurium (StTGD), the rates of StTGD-catalyzed conversion of m-galactarate to 5-KDG were determined by following the change in ellipticity at 323â¯nm. The apparent molar ellipticity ([θ]323) for the 5-KDG formed was determined to be 202⯱â¯2 deg cm2 dmol-1, which was used to convert observed rates (Δθ/Δt) into concentration-dependent rates (Δc/Δt). The kinetic parameters Km, kcat, and kcat/Km were 0.38⯱â¯0.05â¯mM, 4.8⯱â¯0.1 s-1, and 1.3 (±0.2)â¯×â¯104â¯M-1s-1, respectively. These values are in excellent agreement with those published previously [Yew, W.S. et al. (2007) Biochemistry46, 9564-9577] using a coupled assay system. To demonstrate the utility of the assay, the inhibition constant (Kiâ¯=â¯10.7⯱â¯0.4â¯mM) was determined for the competitive inhibitor tartronate. The continuous CD-based assay offers a practical and efficient alternative method to the coupled assay that requires access to 5-KDG aldolase, and to the labor-intensive, fixed-time assays.