Formyltetrahydrofolate synthase from Clostridium thermoaceticum. An electron microscopic study and specific interaction of the enzyme with ATP and ADP.
Formyltetrahydrofolate synthetase (EC 6.3.4.3) from Clostridium thermoaceticum is a 240 000-molecular-weight, tetrameric enzyme composed of identical subunits. When negatively stained with uranyl acetate or sodium phosphotungstate the enzyme appears as two distinct projections when viewed in the electron microscope. One of these, resembling the letter "H", exhibits four flexible arms, while the other, called a "twin", has a compact projection. The twin projection appears to represent the native enzyme in solution. It consists of two outstanding intensity maxima separated by a narrow bridge of dense stain, with each of these intensity maxima being divided into the two submaxima by a second bridge of stain running perpendicular to the first one. The outstanding intensity maxima represent dimers, whereas the submaxima represent a single subunit. The volume calculated from the electron microscopy projections for the dimer is 157 nm3, which corresponds to a Mr of 127 000 or 254 000 for the tetrameric enzyme. The H projections reflect enzyme particles artificially flattened and "opened" during the drying of the negatively stained enzyme. The four arms of the H each represent a subunit. The appearance of H projections is prevented by either using a deep-stain procedure, which offers a mechanical preservation of the native enzyme structure, or by the binding of ATP, ADP or adenosine 5'-[beta, gamma-methylene]triphosphate. These nucleotides, when binding to the enzyme, cause a conformational change. This was observed by ultraviolet and visible circular dichroism and absorption spectrometry with the native enzyme and an enzyme labeled with fluorescein mercuric acetate attached to a sulfhydryl group of each subunit.
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D003013:Clostridium / Q000201:enzymology*; D005574:Formate-Tetrahydrofolate Ligase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008854:Microscopy, Electron; D008970:Molecular Weight; D011485:Protein Binding
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008025:Ligases; D005574:Formate-Tetrahydrofolate Ligase