title: Unlike the quaternary structure transition, the tertiary structure change of the 240s loop in allosteric aspartate transcarbamylase requires active site saturation by substrate for completion.
abstract: The quaternary structural change associated with the homotropic cooperative interactions in Escherichia coli aspartate transcarbamylase (ATCase) is accompanied by various tertiary structural modifications; the most notable one involves the 240s loop formed by residues 230--245 of the catalytic chain. In order to monitor local conformational changes in this region by fluorescence spectroscopy, Tyr-240 has been replaced by a Trp residue, in a mutant enzyme, in which both naturally occurring Trp residues in positions 209 and 284 of the catalytic chains had previously been substituted by Phe residues. This F209F284W240-ATCase still displays homotropic cooperativity for aspartate and undergoes the same T to R quaternary structure change as does the wild-type enzyme. Upon binding of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate, the fluorescence emission spectrum of this mutant shows a red shift directly proportional to the fraction of catalytic sites occupied by this compound, a maximum value of 4 nm being attained when all six active sites are ligated. An identical shift is observed with the catalytic subunits of this modified enzyme, when all three active sites are occupied. In contrast, the quaternary structural change of the F209F284W240-ATCase, monitored by small-angle X-ray scattering, is complete when only four out of six catalytic sites are occupied. Thus, the 240s loop adopts its final conformation only when the neighboring active site is bound.
mesh_terms: D000495:Allosteric Site; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014965:X-Rays
chemical_list: D005456:Fluorescent Dyes; D001221:Aspartate Carbamoyltransferase