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Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme.
Biochemistry. 1989 Mar 07; 28(5):2099-106.B

Abstract

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)

Authors+Show Affiliations

Clayton Foundation Biochemical Institute, Department of Chemistry, University of Texas, Austin 78712.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

2541774

Citation

Barlowe, C K., et al. "Site-directed Mutagenesis of Yeast C1-tetrahydrofolate Synthase: Analysis of an Overlapping Active Site in a Multifunctional Enzyme." Biochemistry, vol. 28, no. 5, 1989, pp. 2099-106.
Barlowe CK, Williams ME, Rabinowitz JC, et al. Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme. Biochemistry. 1989;28(5):2099-106.
Barlowe, C. K., Williams, M. E., Rabinowitz, J. C., & Appling, D. R. (1989). Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme. Biochemistry, 28(5), 2099-106.
Barlowe CK, et al. Site-directed Mutagenesis of Yeast C1-tetrahydrofolate Synthase: Analysis of an Overlapping Active Site in a Multifunctional Enzyme. Biochemistry. 1989 Mar 7;28(5):2099-106. PubMed PMID: 2541774.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme. AU - Barlowe,C K, AU - Williams,M E, AU - Rabinowitz,J C, AU - Appling,D R, PY - 1989/3/7/pubmed PY - 1989/3/7/medline PY - 1989/3/7/entrez SP - 2099 EP - 106 JF - Biochemistry JO - Biochemistry VL - 28 IS - 5 N2 - 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) SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/2541774/Site_directed_mutagenesis_of_yeast_C1_tetrahydrofolate_synthase:_analysis_of_an_overlapping_active_site_in_a_multifunctional_enzyme_ L2 - https://biocyc.org/gene?orgid=PDIF272563&id=G12WB-831 DB - PRIME DP - Unbound Medicine ER -