Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme.
C1-tetrahydrofolate (THF) synthase is a trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. The current model divides this protein into two functionally independent domains with dehydrogenase/cyclohydrolase activities sharing an overlapping active site on the N-terminal domain and synthetase activity associated with the C-terminal domain. Previous chemical modification studies on C1-THF synthase from the yeast Saccharomyces cerevisiae indicated at least two cysteinyl residues involved in the dehydrogenase/cyclohydrolase reactions [Appling, D. R., & Rabinowitz, J. C. (1985) Biochemistry 24, 3540-3547]. In the present work, site-directed mutagenesis of the S. cerevisiae ADE3 gene, which encodes C1-THF synthase, was used to individually change each cysteine contained within the dehydrogenase/cyclohydrolase domain (Cys-11, Cys-144, and Cys-257) to serine. The resulting proteins were overexpressed in yeast and purified for kinetic analysis. Site-specific mutations in the dehydrogenase/cyclohydrolase domain did not affect synthetase activity, consistent with the proposed domain structure. The C144S and C257S mutations result in 7- and 2-fold increases, respectively, in the dehydrogenase Km for NADP+. C144S lowers the dehydrogenase maximal velocity roughly 50% while C257S has a maximal velocity similar to that of the wild type. Cyclohydrolase catalytic activity is reduced 20-fold by the C144S mutation but increased 2-fold by the C257S mutation. Conversion of Cys-11 to serine has a negligible effect on dehydrogenase/cyclohydrolase activity. A double mutant, C144S/C257S, results in catalytic properties roughly multiplicative of the individual mutations.(ABSTRACT TRUNCATED AT 250 WORDS)
Barlowe
C K
CK
Clayton Foundation Biochemical Institute, Department of Chemistry, University of Texas, Austin 78712.
Williams
M E
ME
Rabinowitz
J C
JC
Appling
D R
DR
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D004720:Endonucleases; D005574:Formate-Tetrahydrofolate Ligase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D045064:Methenyltetrahydrofolate Cyclohydrolase; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism*; D010957:Plasmids; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D010088:Oxidoreductases; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D000619:Aminohydrolases; D045064:Methenyltetrahydrofolate Cyclohydrolase; D008025:Ligases; D005574:Formate-Tetrahydrofolate Ligase; D003545:Cysteine