The mechanism of N-methyltryptophan oxidase, a flavin-dependent amine oxidase from Escherichia coli, was studied using a combination of kinetic isotope effects and theoretical calculations. The 15(kcat/Km) kinetic isotope effect for sarcosine oxidation is pH-dependent with a limiting value of 0.994-0.995 at high pH. Density functional theory calculations on model systems were used to interpret these isotope effects. The isotope effects are inconsistent with proposed mechanisms involving covalent amine-flavin adducts but cannot by themselves conclusively distinguish between some discrete electron-transfer mechanisms and a direct hydride-transfer mechanism, although the latter mechanism is more consistent with the energetics of the reaction.
A new class of amidine-oxime reactivators of organophosphate (OP)-inhibited cholinesterases (ChE) were designed, synthesized, and tested. These compounds represent a novel group of oximes with enhanced capabilities of crossing the blood-brain barrier. Lack of brain penetration is a major limitation for currently used oximes as antidotes of OP poisoning. The concept described herein relies on a combination of an amidine residue and oxime functionality whereby the amidine increases the binding affinity to the ChE and the oxime is responsible for reactivation. Amidine-oximes were tested in vitro and reactivation rates for OP-BuChE were greater than pralidoxime (2-PAM) or monoisonitrosoacetone (MINA). Amidine-oxime reactivation rates for OP-AChE were lower compared to 2-PAM but greater compared with MINA. After pretreatment for 30 min with oximes 15c and 15d (145 μmol/kg, ip) mice were challenged with a soman model compound. In addition, 15d was tested in a post-treatment experiment (145 μmol/kg, ip, administration 5 min after sarin model compound exposure). In both cases, amidine-oximes afforded 100% 24 h survival in an animal model of OP exposure.
Cyclic GMP‐AMP synthase (cGAS) is activated by ds‐DNA binding to produce the secondary messenger 2′,3′‐cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds‐DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2′,3′‐cGAMP formation, and interestingly, describe a catalytic mechanism where 2′,3′‐cGAMP may be a minor product of cGAS compared with linear nucleotides.
Tryptophan 2-monooxygenase (TMO) from Pseudomonas savastanoi catalyzes the oxidative decarboxylation of L-tryptophan during the biosynthesis of indoleacetic acid. Structurally and mechanistically the enzyme is a member of the family of L-amino acid oxidases. Deuterium and 15 N kinetic isotope effects were used to probe the chemical mechanism of L-alanine oxidation by TMO. The primary deuterium kinetic isotope effect was pH-independent over the pH range 6.5-10, with an average value of 6.0 ± 0.5, consistent with this being the intrinsic value. The deuterium isotope effect on the rate constant for flavin reduction by alanine was 6.3 ± 0.9; no intermediate flavin species were observed during flavin reduction. The 15 V/K alanine value was 1.0145 ± 0.0007 at pH 8. NMR analyses give an equilibrium 15 N isotope effect for deprotonation of the alanine amino group of 1.0233 ± 0.0004, allowing calculation of the 15 N isotope effect on the CH bond cleavage step of 0.9917 ± 0.0006. The results are consistent with TMO oxidation of alanine occurring through a hydride transfer mechanism.The flavoenzyme tryptophan 2-monooxygenase (TMO1) from Pseudomonas savastanoi catalyzes the oxidative decarboxylation of L-tryptophan (Scheme 1) in the first step of a twostep biosynthetic pathway for the plant hormone indoleacetic acid (10-12). The kinetic mechanism of TMO has been determined with its fastest substrate L-tryptophan (13), and can be divided into two half-reactions (Scheme 2). The reductive half-reaction involves cleavage of the α-CH bond of the amino acid (AA) and transfer of a hydride equivalent to the FAD to form the enzyme-bound imino acid. This is identical to the reaction of the flavoprotein L-amino acid oxidases and similar to the general reaction of flavoprotein amine oxidases. In the oxidative half-reaction of TMO, the reduced cofactor reacts with oxygen to produce hydrogen peroxide (14). Decarboxylation of the imine acid to the amide is thought to occur through the reaction of the hydrogen peroxide with the imino acid still bound to the enzyme (pathway a), analogously to the mechanism proposed by Lockridge et al. (15) for the decarboxylation of pyruvate by lactate oxidase. Although indoleacetamide is the only product of tryptophan turnover by wild-type TMO, amino acid oxidation can be uncoupled from decarboxylation to yield a keto acid in mutant enzymes (pathway b) (16,17).Despite their ubiquity and functional diversity, all flavin-dependent amine oxidases have thus far fallen into two structural families. One family includes D-amino acid oxidase (18), monomeric sarcosine oxidase (19) and glycine oxidase (20), while monoamine oxidase (21), *Address correspondence to:Paul F. Fitzpatrick,.Department of Biochemistry and Biophysics, 2128 TAMU, College Station, TX 77843-2128, Ph: 979-845-5487, Fax: 979-845-4946, Email: fitzpat@tamu.edu † This work was supported by NIH grants R01 GM58698 (PFF), R01 GM18938 (WWC), and T32 GM08523 (ECR) NIH Public Access (22), and L-amino acid oxidase (23) represent a separate f...
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