Mutation of His465 Alters the pH-dependent Spectroscopic Properties of Escherichia coli Glutamate Decarboxylase and Broadens the Range of Its Activity toward More Alkaline pH

Glutamate decarboxylase (GadB) from Escherichia coli is a hexameric, pyridoxal 5′-phosphate-dependent enzyme catalyzing CO₂ release from the α-carboxyl group of l-glutamate to yield γ-aminobutyrate. GadB exhibits an acidic pH optimum and undergoes a spectroscopically detectable and strongly cooperative pH-dependent conformational change involving at least six protons. Crystallographic studies showed that at mildly alkaline pH GadB is inactive because all active sites are locked by the C termini and that the 340 nm absorbance is an aldamine formed by the pyridoxal 5′-phosphate-Lys²⁷⁶ Schiff base with the distal nitrogen of His⁴⁶⁵, the penultimate residue in the GadB sequence. Herein we show that His⁴⁶⁵ has a massive influence on the equilibrium between active and inactive forms, the former being favored when this residue is absent. His⁴⁶⁵ contributes with n ≈ 2.5 to the overall cooperativity of the system. The residual cooperativity (n ≈ 3) is associated with the conformational changes still occurring at the N-terminal ends regardless of the mutation. His⁴⁶⁵, dispensable for the cooperativity that affects enzyme activity, is essential to include the conformational change of the N termini into the cooperativity of the whole system. In the absence of His⁴⁶⁵, a 330-nm absorbing species appears, with fluorescence emission spectra more complex than model compounds and consisting of two maxima at 390 and 510 nm. Because His⁴⁶⁵ mutants are active at pH well above 5.7, they appear to be suitable for biotechnological applications.     

Halogenation and Oxidation Reactions with Haloperoxidases

Haloperoxidases are enzymes which catalyze the incorporation of halogen atoms into organic molecules. They are found throughout nature, playing a major role in the defence system of many organisms. Their reaction mechanisms as well as their use as catalysts for halogenation and oxidation reactions on laboratory and industrial scales are discussed. Up to now, selective halogenation reactions have only been reported for the chloroperoxidase from Pseudomonas pyrrocinia. The usefulness of the other enzymes is based on their ability to produce hypohalous acid (HOX) in a controllable way, allowing the smooth (yet nonselective) halogenation of electron-rich substrates. On the other hand, it has been shown recently that some haloperoxidases can stereoselectively convert sulfides and alkenes into their corresponding homochiral oxides. Therefore, these enzymes will undoubtedly gain importance in the near future.     

Note Sui Grani a Spighe Ramificate

Resumé

Chez les Blés branchus de l'espèce «turgidum» (Triticum turgidum compositum), il existe des sortes à thermostade plutôt froid (Blés d'hiver ou de semi-hiver) et des sortes à thermostade chaud, tièe ou «indifférent» (Blés de printemps).

Les Blés branchus de l'espèce «turgidum» apparaissent comme étant des plantes à photostade de jour long.

La plus ou moins grande rapidité de l'accomplissement du photostade, par rapport à la rapidité de l'assimilation des matières plastiques, détermine la structure, — non ramifiée ou ramifiée — de l'épi.     

Why Are Sensory Axons More Vulnerable for Ischemia than Motor Axons?

Objective

In common peripheral neuropathies, sensory symptoms usually prevail over motor symptoms. This predominance of sensory symptoms may result from higher sensitivity of sensory axons to ischemia.

Methods

We measured median nerve compound sensory action potentials (CSAPs), compound muscle action potentials (CMAPs), and excitability indices in five healthy subjects during forearm ischemia lasting up to disappearance of both CSAPs and CMAPs.

Results

Ischemia induced: (1) earlier disappearance of CSAPs than CMAPs (mean ± standard deviation 30±5 vs. 46±6 minutes), (2) initial changes compatible with axonal depolarization on excitability testing (decrease in threshold, increase in strength duration time constant (SDTC) and refractory period, and decrease in absolute superexcitability) which were all more prominent in sensory than in motor axons, and (3) a subsequent decrease of SDTC reflecting a decrease in persistent Na⁺ conductance during continuing depolarisation.

Interpretation

Our study shows that peripheral sensory axons are more vulnerable for ischemia than motor axons, with faster inexcitability during ischemia. Excitability studies during ischemia showed that this was associated with faster depolarization and faster persistent Na⁺ channel inactivation in sensory than in motor axons. These findings might be attributed to differences in ion channel composition between sensory and motor axons and may contribute to the predominance of sensory over motor symptoms in common peripheral neuropathies.