Resolving the Fluorescence Response of Escherichia coli Carbamoyl Phosphate Synthetase: Mapping Intra- and Intersubunit Conformational Changes

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• 作者：Jason L. Johnson ; Joseph K. West ; Andrew D. L. Nelson ; Gregory D. Reinhart
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：January 16, 2007
• 年：2007
• 卷：46
• 期：2
• 页码：387 - 397
• 全文大小：443K
• 年卷期：v.46,no.2(January 16, 2007)
• ISSN：1520-4995

文摘

Carbamoyl phosphate synthetase (CPS) from Escherichia coli is potentially overlaid with anetwork of allosterism, interconnecting active sites, effector binding sites, and aggregate interfaces tocontrol its mechanisms of catalytic synchronization, regulation, and oligomerization, respectively. Tocharacterize these conformational changes, a tryptophan-free variant of CPS was genetically engineeredby substituting six native tryptophans with tyrosines. Each tryptophan was then reinserted, singly, as aspecific fluorescence probe of its corresponding microenvironment. The amino acid substitutions themselvesresult in little apparent disruption of the protein; variants maintain catalytic and allosteric functionality,and the fluorescence properties of each tryptophan, while unique, are additive to wild-type CPS. Whereasthe collective, intrinsic fluorescence response of E. coli CPS is largely insensitive to ligand binding, changesof the individual probes in intensity, lifetime, anisotropy, and accessibility to acrylamide quenching highlightthe dynamic interplay between several protein domains, as well as between subunits. W213 within thecarboxy phosphate domain, for example, exhibits an almost 40% increase in intensity upon saturationwith ATP; W437 of the oligomerization domain, in contrast, is essentially silent in its fluorescence to thebinding of ligands. Nucleotide and bicarbonate association within the large subunit induces fluorescencechanges in both W170 and W175 of the small subunit, indicative of the type of long-range interactionspurportedly synchronizing the carboxy phosphate and amidotransferase domains of the enzyme to initiatecatalysis. ATP and ADP engender different fluorescence responses in most tryptophans, perhaps reflectingcoordinating, conformational changes accompanying the cycling of reactants and products during catalysis.