Reactive and potentially toxic cofactors such as copper ions are imported into eukaryotic cells and incorporated into target proteins by unknown mechanisms. Atx1, a prototypical copper chaperone protein from yeast, has now been shown to act as a soluble cytoplasmic copper(I) receptor that can adopt either a two- or three-coordinate metal center in the active site. Atx1 also associated directly with the Atx1-like cytosolic domains of Ccc2, a vesicular protein defined in genetic studies as a member of the copper-trafficking pathway. The unusual structure and dynamics of Atx1 suggest a copper exchange function for this protein and related domains in the Menkes and Wilson disease proteins.
Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine. Previous work had shown the existence of a reactive thiol group, cysteine 726, whose alkylation led to loss of all detectable enzymatic activity [González, J.C., et al. (1992) Biochemistry 31, 6045-6056]. A site-directed mutation of MetE, Cys726Ser, was constructed to investigate the possible role of this cysteine. The Cys726Ser protein was purified to homogeneity, affording a protein with no detectable activity. To assess the possibility that cysteine726 functions as a metal ligand, inductively coupled plasma-atomic emission spectrometry was performed. The wild-type enzyme contains 1.02 equiv of zinc per subunit; the Cys726Ser mutant does not contain zinc, supporting the view that cysteine726 is required for metal binding. A loss of enzymatic activity is observed upon removal of zinc from the wild-type MetE by incubation in urea and EDTA; activity can subsequently be restored by zinc reconstitution, suggesting that zinc is required for catalysis. Circular dichroism measurements further suggest that there are no major differences in the secondary structures of the wild-type and the Cys726Ser mutant enzymes. Extended X-ray absorption fine structure analysis has established that the average zinc environment is different in the presence of homocysteine than in its absence and is consistent with the changes expected for displacement of an oxygen or nitrogen ligand by the sulfur of homocysteine. A possible model for zinc-dependent activation of homocysteine by MetE is presented.
X-ray absorption spectroscopy, using the analytical methodology described in the previous paper, has been used to determine the ligation of the essential zinc ions in cobalamin-dependent (MetH) and cobalamin-independent (MetE) methionine synthases from Escherichia coli and to probe directly the changes in zinc ligation that occur upon addition of the thiol substrate, homocysteine, to each enzyme. Extended X-ray absorption fine structure (EXAFS) spectra of native MetE and a truncated fragment of MetH containing the substrate-binding sites are consistent with ZnS2(N/O)2 and ZnS3(N/O) ligation, respectively. Previous mutagenesis studies of the homocysteine binding region of MetH had identified two putative thiolate zinc ligands, Cys310 and Cys311. Since the EXAFS spectra indicate that the zinc is coordinated to three sulfur ligands derived from the protein, a third conserved cysteine, Cys247, was mutated to alanine, resulting in a MetH fragment that binds only 0.09 equiv of zinc per mol of protein and exhibits no methylcobalamin-homocysteine methyltransferase activity. Upon addition of l-homocysteine, the X-ray absorption near edge structure changes for both enzymes, and the EXAFS spectra show changes consistent with the coordination of a sulfur, giving a ZnS3(N/O) site for MetE and a ZnS4 site for MetH. Only the l-homocysteine enantiomer causes these effects; the addition of d-homocysteine to MetH(2-649) gives no detectable changes in the EXAFS or the near edge regions. These results are consistent with a mechanism in which the homocysteine is ligated to zinc. Homocysteine is then able to initiate nucleophilic attack on the methyl group needed for methionine formation, with the methyl group bound either to methylcobalamin in MetH or to a polyglutamate derivative of methyltetrahydrofolate in MetE.
Objective: To describe the time elapsed from onset of pediatric convulsive status epilepticus (SE) to administration of antiepileptic drug (AED).Methods: This was a prospective observational cohort study performed from June 2011 to June 2013. Pediatric patients (1 month-21 years) with convulsive SE were enrolled. In order to study timing of AED administration during all stages of SE, we restricted our study population to patients who failed 2 or more AED classes or needed continuous infusions to terminate convulsive SE. Results:We enrolled 81 patients (44 male) with a median age of 3.6 years. The first, second, and third AED doses were administered at a median (p 25 -p 75 ) time of 28 (6-67) minutes, 40 (20-85) minutes, and 59 (30-120) minutes after SE onset. Considering AED classes, the initial AED was a benzodiazepine in 78 (96.3%) patients and 2 (2-3) doses of benzodiazepines were administered before switching to nonbenzodiazepine AEDs. The first and second doses of nonbenzodiazepine AEDs were administered at 69 (40-120) minutes and 120 (75-296) minutes. In the 64 patients with out-of-hospital SE onset, 40 (62.5%) patients did not receive any AED before hospital arrival. In the hospital setting, the first and second in-hospital AED doses were given at 8 (5-15) minutes and 16 (10-40) minutes after SE onset (for patients with in-hospital SE onset) or after hospital arrival (for patients with out-of-hospital SE onset). Conclusions:The time elapsed from SE onset to AED administration and escalation from one class of AED to another is delayed, both in the prehospital and in-hospital settings. Status epilepticus (SE) is one of the most common pediatric neurologic emergencies.1 It has a mortality of 0%-3% 2-7 and morbidity that includes cognitive and neurodevelopmental impairments, epilepsy, and recurrent SE.2,8-10 SE is often refractory to the initial antiepileptic drugs (AEDs), 11,12 and refractory SE is associated with poor outcome. 12 Patient age, etiology, and SE duration all affect outcome, 5,9,13 but only SE duration is a potentially modifiable factor by rapid AED treatment. By convention, the treatment of convulsive SE is a sequence of AEDs, typically
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