Formiminotransferase-cyclodeaminase is stabilized and activated approx. 40% in the presence of low concentrations (equal or less than 0.2%) of Triton X-100, possibly because the average hydrophobicity (1.10 kcal per residue) and the frequency of large non-polar side-chains (0.34) of this protein are both somewhat higher than average. This stabilization enabled us to develop a new purification procedure for the enzyme using chromatography on Matrex Gel Orange A and heparin-Sepharose columns in the presence of Triton X-100. This procedure is easier, much more reproducible, and gives slightly higher yield than the previous method described by Drury, et al. Further investigations of the role of tetrahydropteroylpolyglutamates with formiminotransferase-cyclodeaminase reveal that the use of polyglutamylated folate substrates does not change the mechanism of the transferase reaction, but decreases the K(m) for formininoglutamate, the second substrate, more than 10-fold, bringing it closer to the expected physiological concentration.
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http://dx.doi.org/10.1016/0167-4838(89)90029-0 | DOI Listing |
Biochim Biophys Acta
November 1989
Department of Biochemistry, McGill University, Montreal, Canada.
Formiminotransferase-cyclodeaminase is stabilized and activated approx. 40% in the presence of low concentrations (equal or less than 0.2%) of Triton X-100, possibly because the average hydrophobicity (1.
View Article and Find Full Text PDFFormiminotransferase-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.
View Article and Find Full Text PDFAdv Exp Med Biol
October 1983
The naturally occurring pteroylpolyglutamate derivatives are substrates for the folate-mediated reactions in cells, including the reactions catalyzed by two multifunctional folate dependent enzymes in eucaryotes. The appropriate derivatives of tetrahydropteroyl (glutamate)n where n = 1, 3, 5, or 7 were used to determine the specificity for, and kinetic advantages of the extra glutamyl residues with two multifunctional proteins from pig liver: methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase, and formiminotransferase-formininotetrahydrofolate cyclodeaminase. Specificity for the polyglutamate derivatives ranged from 10- to 70-fold as indicated from Km values or from the ability to inhibit the five different enzyme activities.
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