Site-directed mutagenesis of a potential catalytic and formyl phosphate binding site and substrate inhibition of N10-formyltetrahydrofolate synthetase.
Structural studies of N(10)-formyltetrahydrofolate synthetase (FTHFS) have indicated the involvement of Arg 97 in the binding of the formyl phosphate intermediate. Two site-directed mutants were constructed to test this hypothesis: R97S (Ser substitution) and R97E (Glu substitution). The k(cat) of R97S was approximately 60% that of the wild-type enzyme and had K(m) for ATP and formate twofold higher than those of wild type. R97E was completely inactive and had a K(m) for ATP nearly six times that of wild type. Substrate inhibition by tetrahydrofolate was shown to occur in wild-type and R97S enzymes using both steady-state and transient-state kinetic approaches. These results lend greater insight into the mechanistic function of FTHFS by confirming the interaction of both ATP and formate with Arg 97 and introducing the aspect of substrate inhibition by tetrahydrofolate with regard to substrate binding and dissociation.
Leaphart
Adam B
AB
Department of Biological Sciences, University of South Carolina, 715 Sumter Street, Columbia, SC 29208, USA.
Trent Spencer
H
H
Lovell
Charles R
CR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D005574:Formate-Tetrahydrofolate Ligase / Q000737:chemistry* / Q000378:metabolism*; D005561:Formates / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009943:Organophosphorus Compounds / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism
D005561:Formates; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; C030544:formic acid; C053858:formyl phosphate; C030371:5,6,7,8-tetrahydrofolic acid; D000255:Adenosine Triphosphate; D005574:Formate-Tetrahydrofolate Ligase