Temperature-dependent structural changes in complexes of tRNA(adenine-1)-methyltransferase from Thermus thermophilus HB8 and tRNA studied by optical and electron microscope methods

The complexation of tRNA (adenine-1-)-methyltransferase from Thermus thermophilus HB8 (E.C.2.1.1.36) with Escherichia coli tRNA(Phe) and yeast tRNA1(Val) was investigated in a temperature range from 20 to 90 degrees C. The quantity of methylase subunits bounded with tRNA and the association constant (Ka) were determined by means of fluorescence quenching of the enzyme tryptophane residues by tRNA molecules. The number of enzyme subunits bounded with one tRNA molecule at temperatures 20-70 degrees C is equal to 8 +/- 2. The Ka values increase from (2 divided by 3).10(7) at 20 degrees C up to 8.5.10(7) M-1 at 70 degrees C. The temperature increase from 70 to 90 degrees C causes a decrease in the enzyme specific activity and in Ka values. In the temperature range from 75 to 90 degrees C a cooperative transition of methylase macromolecules into associates was observed. This association is accompanied by an increase of UV-light scattering and of fluorescence polarization coefficient of methylase tryptophane residues. In the absence of tRNA the size of enzyme associates (d) is evaluated to be more than 320 nm (d greater than or equal to lambda-320 nm), in the presence of tRNA-less than 320 nm (d much less than lambda-320 nm). An electron microscopic investigation of methylase and its complexes with tRNA at 20 degrees C revealed disk-like particles with a diameter and height of 8-11 nm and 4-5 nm, respectively. These disk-like methylase preparations dialized against distilled water form flexible polymeric rods with a diameter of 10-12 nm and the length of about several hundreds nm. During complexation of methylase with tRNA, in the same conditions as the dializes was carried out, large associates were not revealed.     

Tautomeric changes in guanfacine and its copper(II) complex as revealed by X-ray crystallography; synthesis and EPR

Obtention and crystal structure of guanfacine (guaH) together with synthesis and crystal structure of its copper complex [Cu^II(gua)₂] · DMF were reported. The free molecule guaH exhibits one tautomeric form (B) in contrast to the form (A) which was reported in the Merck index. In the copper(II) complex, the anionic form gua⁻ exhibits the third tautomeric form (C). This complex is characterized by a CuN₂O₂ coordination. The EPR spectrum is in agreement with a Cu(II) ion in a square planar configuration.     

Purification and some properties of (1R,2S)-1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase from Comamonas testosteroni T-2

Abstract Inducible (1 R ,2 S )-1,2-dihydroxy-3,5-cyclohexadiene-l,4-dicarboxylate (diene-diol) dehydrogenase was found in extracts of Comamonas testosteroni T-2 grown in p -toluate-or terephthalate-salts medium and it was purified using anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is a homodimer with subunit M r 39000. It had a specific activity of 500 mkat/kg of protein and was activated by the addition of Fe²⁺. The dehydrogenase converted 1 mol diene-diol and 1 mol NAD⁺ to 1 mol protocatechuic acid, 1 mol NADH and 1 mol CO₂. Apparent K _m-values of 43 μM (NAD⁺) and about 90 μM (diene-diol) were determined. The hydride ion was transferred to the si face of NAD⁺.     

Synthesis and evaluation of (E)-2-(acrylamido)cyclohex-1-enecarboxylic acid derivatives as HCA1, HCA2, and HCA3 receptor agonists

2-(3-(Naphthalen-2-yl)propanamido)cyclohex-1-enecarboxylic acid and its 6-hydroxynaphthalen-2-yl analogue are well-known hydroxyl-carboxylic acid (HCA) receptor HCA2 agonists. A series of novel aryl derivatives of 2-amidocyclohex-1-ene carboxylic acid that contained rigidity elements, such as an E-double bond, triple bond, and trans or cis-substituted cyclopropane rings, instead of the saturated ethane linker in the amide part of the molecules were designed and synthesized, and the derivatives’ potency for the activation of HCA1, HCA2, and HCA3 receptors by 3′–5′-cyclic adenosine monophosphate (cAMP) assay were evaluated. The SAR studies revealed that the rigidifying of appropriate molecules enabled modulation of the potency and selectivity of the HCA2 receptor activation.