D. Shyamali Wimalasena

Evidence That Histidine Protonation of Receptor-Bound Anthrax Protective Antigen Is a Trigger for Pore Formation

The protective antigen (PA) component of the anthrax toxin forms pores within the low pH environment of host endosomes through mechanisms that are poorly understood. It has been proposed that pore formation is dependent on histidine protonation. In previous work, we biosynthetically incorporated 2-fluorohistidine (2-FHis), an isosteric analogue of histidine with a significantly reduced pK(a) ( approximately 1), into PA and showed that the pH-dependent conversion from the soluble prepore to a pore was unchanged. However, we also observed that 2-FHisPA was nonfunctional in the ability to mediate cytotoxicity of CHO-K1 cells by LF(N)-DTA and was defective in translocation through planar lipid bilayers. Here, we show that the defect in cytotoxicity is due to both a defect in translocation and, when bound to the host cellular receptor, an inability to undergo low pH-induced pore formation. Combining X-ray crystallography with hydrogen-deuterium (H-D) exchange mass spectrometry, our studies lead to a model in which hydrogen bonds to the histidine ring are strengthened by receptor binding. The combination of both fluorination and receptor binding is sufficient to block low pH-induced pore formation.

Copper ions disrupt dopamine metabolism via inhibition of V-H+-ATPase: a possible contributing factor to neurotoxicity.

The involvement of copper in the pathophysiology of neurodegeneration has been well documented but is not fully understood. Commonly, the effects are attributed to increased reactive oxygen species (ROS) production due to inherent redox properties of copper ions. Here we show copper can have physiological effects distinct from direct ROS production. First, we show that extragranular free copper inhibits the vesicular H+‐ATPase of resealed chromaffin granule ghosts. Extragranular ascorbate potentiates this inhibition. The inhibition is mixed type with Kis = 6.8 ± 2.8 µmol/L and Kii = 3.8 ± 0.6 µmol/L, with respect to ATP. Second, extracellular copper causes an inhibition of the generation of a pH‐gradient and rapid dissipation of pre‐generated pH and catecholamine gradients. Copper chelators and the ß‐amyloid peptide 1–42 were found to effectively prevent the inhibition. The inhibition is reversible and time‐independent suggesting the effects of extracellular copper on H+‐ATPase is direct, and not due to ROS. The physiological significance of these observations was shown by the demonstration that extracellular copper causes a dramatic perturbation of dopamine metabolism in SH‐SY5Y cells. Thus, we propose that the direct inhibition of the vesicular H+‐ATPase may also contribute to the neurotoxic effects of copper.

N,N,N′,N′-Tetramethyl-1,4-phenylenediamine: a facile electron donor and chromophoric substrate for dopamine β-monooxygenase

Abstract Dopamine β-monooxygenase is shown to catalyze the oxidation of N,N,N′,N′-tetramethyl-1,4-phenylenediamine (TMPD) to its cation radical in the presence of a regular substrate and molecular oxygen. The enzyme-mediated oxidation of TMPD is stoichiometrically coupled with the hydoxylation of the substrate to the corresponding enzymatic product. TMPD is kinetically well behaved as an alternate electron donor for the enzyme with a potency comparable to that of the most efficient electron donor, ascorbate. Dopamine β-monooxygenase mediated oxidation of TMPD has been employed to design a convenient and sensitive spectrophotometric assay for the enzyme. The finding that TMPD is a well behaved facile alternate electron donor for dopamine β-monooxygenase raises some interesting novel questions regarding the specificity and chemistry of the reduction site, which may have important implications on the reduction of active site coppers of the enzyme.

Reduction of Dopamine β-Monooxygenase A UNIFIED MODEL FOR APPARENT NEGATIVE COOPERATIVITY AND FUMARATE ACTIVATION

The interactions of reductants with dopamine β-monooxygenase (DβM) were examined using two novel classes of reductants. The steady-state kinetics of the previously characterized DβM reductant, N,N-dimethyl-1,4-p-phenylenediamine (DMPD), were parallel to the ascorbic acid-supported reaction with respect to pH dependence and fumarate activation. DMPD also displayed pH and fumarate-dependent apparent negative cooperativity demonstrating that the previously reported cooperative behavior of DβM toward the reductant is not unique to ascorbic acid. The 6-OH phenyl and alkylphenyl-substituted ascorbic acid derivatives were more efficient reductants for the enzyme than ascorbic acid. Kinetic studies suggested that these derivatives behave as pseudo bisubstrates with respect to ascorbic acid and the amine substrate. The lack of apparent cooperative behavior with these derivatives suggests that this behavior of DβM is not common for all the reductants. Based on these findings and additional kinetic evidence, the proposal that the apparent negative cooperativity in the interaction of ascorbic acid with DβM was due to the presence of a distinct allosteric regulatory site has been ruled out. In contrast to previous models, where fumarate was proposed to interact with a distinct anion binding site, the effect of fumarate on the steady-state kinetics of these novel reductants suggests that fumarate and the reductant may interact with the same site of the enzyme. In accordance with these observations and mathematical analysis of the experimental data, a unified model for the apparent negative cooperativity and fumarate activation of DβM in which both fumarate and the reductant interact with the same site of all forms of the enzyme with varying affinities under steady-state turnover conditions has been proposed.

Plausible molecular mechanism for activation by fumarate and electron transfer of the dopamine beta-mono-oxygenase reaction.

A series of fumarate analogues has been used to explore the molecular mechanism of the activation of dopamine beta-mono-oxygenase by fumarate. Mesaconic acid (MA) and trans -glutaconic acid (TGA) both activate the enzyme at low concentrations, similar to fumarate. However, unlike fumarate, TGA and MA interact effectively with the oxidized enzyme to inhibit it at concentrations of 1-5 mM. Monoethylfumarate (EFum) does not activate the enzyme, but inhibits it. In contrast with TGA and MA, however, EFum inhibits the enzyme by interacting with the reduced form. The saturated dicarboxylic acid analogues, the geometric isomer and the diamide of fumaric acid do not either activate or inhibit the enzyme. The phenylethylamine-fumarate conjugate, N -(2-phenylethyl)fumaramide (PEA-Fum), is an approximately 600-fold more potent inhibitor than EFum and behaves as a bi-substrate inhibitor for the reduced enzyme. The amide of PEA-Fum behaves similarly, but with an inhibition potency approximately 20-fold less than that of PEA-Fum. The phenylethylamine conjugates of saturated or geometric isomers of fumarate do not inhibit the enzyme. Based on these findings and on steady-state kinetic analysis, an electrostatic model involving an interaction between the amine group of the enzyme-bound substrate and a carboxylate group of fumarate is proposed to account for enzyme activation by fumarate. Furthermore, in light of the recently proposed model for the similar copper enzyme, peptidylglycine alpha-hydroxylating mono-oxygenase, the above electrostatic model suggests that fumarate may also play a role in efficient electron transfer between the active-site copper centres of dopamine beta-mono-oxygenase.