Channeling between the active sites of formiminotransferase-cyclodeaminase. Binding and kinetic studies.
Formiminotransferase-cyclodeaminase, a circular tetramer of dimers, binds four tetrahydropteroylpolyglutamates/octamer, which indicates that these polyglutamate sites are formed by one type of subunit interface. The transferase and deaminase are separate catalytic sites as determined by inhibition studies with (6R)-tetrahydropteroylglutamate and by the observation that the activities can operate simultaneously. Under conditions where the transferase is saturated with tetrahydropteroyl(glutamate)n substrate, exogenously added formimino intermediate is utilized by the deaminase only if at least one of the substrate/intermediate pair is a monoglutamate. These properties indicate the existence of only one polyglutamate site/pair of catalytic sites. Kinetic specificity for each activity as measured by Vm/Km increases for longer polyglutamates, but does not differentiate among 4, 5, 6, and 7 glutamates. The enzyme shows distinct preference for hexaglutamate based on Kd as well as on Km values. With all substrates, Vm of the deaminase is greater than that of the transferase, allowing for potential channeling of the intermediate between active sites. Efficiency of channeling, optimal with pentaglutamate, does not correspond with affinity for binding. This demonstrates that a steric requirement predominates over simple sequestering of intermediates on the enzyme surface as the fundamental mechanism for channeling.
D000642:Ammonia-Lyases / Q000378:metabolism*; D001665:Binding Sites; D051042:Glutamate Formimidoyltransferase; D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D046911:Macromolecular Substances; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism; D013997:Time Factors; D014166:Transferases / Q000378:metabolism*
D046911:Macromolecular Substances; D011624:Pteroylpolyglutamic Acids; C033064:tetrahydropteroylpentaglutamate; D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; D051042:Glutamate Formimidoyltransferase; D000642:Ammonia-Lyases; C020148:formiminotetrahydrofolate cyclodeaminase