Dissociation of the octameric bifunctional enzyme formiminotransferase-cyclodeaminase in urea. Isolation of two monofunctional dimers

Abstract

Partial denaturation of the circular octameric bifunctional enzyme formiminotransferase-cyclodeaminase in increasing urea concentrations leads to sequential dissociation via dimers to inactive monomers. In potassium phosphate buffer, dissociation to dimers in 3 M urea coincides with loss of both activities and a major decrease in intensity of intrinsic tryptophan fluorescence. In the presence of folic acid, these dimers retain the deaminase activity, but with folylpolyglutamates, both activities are protected and the native octameric structure is retained. The protection profiles with polyglutamates are cooperative with a Hill coefficient greater than 2, suggesting that binding of more than one folylpolyglutamate per octamer is required to stabilize the native structure. In triethanolamine hydrochloride buffer, transferase-active dimers that retain the intrinsic tryptophan fluorescence can be obtained in 1 M urea and stabilized at higher urea concentration by the addition of glutamate. Deaminase-active dimers are obtained by the protection of folate in 3 M urea. Proteolysis of the two kinds of dimers by chymotrypsin leads to very different fragmentation patterns, indicating that they are structurally different. We propose that the two dimers retain different subunit-subunit interfaces, one of which is required for each activity. This suggests that the native octameric structure is required for expression of both activities and therefore for "channeling" of intermediates.

A Formiminotransferase Cyclodeaminase Isoform Is Localized to the Golgi Complex and Can Mediate Interaction of Trans-Golgi Network-derived Vesicles with Microtubules

“…It has been shown that each subunit of the octameric FTCD consists of an N-terminal transferase and a C-terminal deaminase active site, separated by a short linker sequence (51). Both domains self-dimerize, confirming that FTCD octamers include two different types of subunit interfaces which are most likely maintained for expression of both activities (52,53). It has been suggested that the polyglutamate-binding site resides at the subunit interface formed between the deaminase domains (51).…”

Molecular Cloning, Characterization, and Dynamics of Rat Formiminotransferase Cyclodeaminase, a Golgi-associated 58-kDa Protein

“…Biochemical properties of porcine FTCD have been extensively studied, and the enzyme exists as a planar ring (ϳ12 nm in diameter) of eight identical subunits (23), relatively resistant to chymotryptic digestion (37). To examine the proteolytic behavior of rat FTCD, the SG fraction was subjected to proteinase K treatments in the absence or presence of 6.5 M urea as a denaturing reagent.…”

“…Previous analysis has indicated that porcine FTCD exists as a dimer organized into higher order structures, tetramers and octamers (37). To determine whether rat FTCD shows similar structural characteristics, rat liver cytosolic fraction was either untreated or subjected to cross-linking, analyzed by SDS-PAGE in the presence or absence of reducing agent, and, after transfer to nitrocellulose filters, immunoblotted with mAb 58K-9.…”

Moonlighting Glutamate Formiminotransferases Can Functionally Replace 5-Formyltetrahydrofolate Cycloligase

“…Given that moonlighting functions can depend on differences in protein-protein interactions (48), it seems possible that running these opposing reactions concurrently is facilitated by FT joining/not joining a protein complex related to histidine degradation. In this connection, it is relevant that mammalian FT-CD is an octamer that can dissociate into two types of monofunctional dimers, and that while the FT and CD activities of the octamer function separately with monoglutamylated folates, polyglutamates are channeled from the FT to the CD active site (34,53). Analogous behavior of prokaryote FT oligomers, or perhaps FT-CD complexes, together with polyglutamate-mediated channeling between FT and CD reactions, could result in the functional partitioning, i.e.…”