Probing the role of an active site aspartic acid in dihydrofolate reductase

Vladimir A. Karginov, Sergey V. Mamaev, Haoyun An, Mark D. Van Cleve, Sidney Hecht, George A. Komatsoulis, John N. Abelson

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

Analogues of E. coli dihydrofolate reductase (DHFR) containing modified amino acids at single, predetermined sites have been prepared. This was accomplished by the use of the DHFR gene containing an engineered nonsense codon (TAG) at the positions corresponding to Val-10 and Asp-27. Misacylated suppressor tRNAs activated with the modified amino acids of interest were employed for the suppression of the nonsense codons in a cell free protein biosynthesizing system, thereby permitting the elaboration of the desired protein analogues. In this fashion, the aspartic acid analogues erythro-carboxyproline, cysteic acid, β,β-dimethylaspartic acid, α-methylaspartic acid, erythro- and threo-β-methylaspartic acid, N-methylaspartic acid, and phosphonoalanine were incorporated into one or both of the aformentioned positions. Although a number of these analogues were incorporated only in low yield, a modification of the strategy has suggested how this might be improved significantly. The derived proteins were purified and then characterized by their mobility on polyacrylamide gels in comparison with wild-type DHFR. Representative DHFRs modified at position 10 were also degraded by defined proteolysis with Glu-C endoproteinase; the fragments containing the modified amino acids were shown to have the same chromatographic properties on reverse phase HPLC as authentic synthetic standards. Individual analogues were assayed for their abilities to bind to the substrate analogue methotrexate and to convert dihydrofolate to tetrahydrofolate. DHFR analogues containing erythro- and threo-β-methylaspartic acid and β,β-dimethylaspartic acid were all shown to mediate tetrahydrofolate production 74-86% as efficiently as wild-type DHFR under conditions of multiple substrate turnover. Analysis of the rates of tetrahydrofolate production in the presence of NADPH and NADPH at two pH values suggests that this was due to rate-limiting hydride transfer from NADPH bound to DHFR analogues whose active site had been altered structurally.

Original languageEnglish (US)
Pages (from-to)8166-8176
Number of pages11
JournalJournal of the American Chemical Society
Volume119
Issue number35
DOIs
StatePublished - Sep 3 1997
Externally publishedYes

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Tetrahydrofolate Dehydrogenase
Aspartic Acid
Catalytic Domain
Acids
NADP
Nonsense Codon
Amino acids
Amino Acids
Cysteic Acid
Proteins
Proteolysis
Substrates
Transfer RNA
Methotrexate
Hydrides
Escherichia coli
Oxidoreductases
Genes
Polyacrylates
High Pressure Liquid Chromatography

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Karginov, V. A., Mamaev, S. V., An, H., Van Cleve, M. D., Hecht, S., Komatsoulis, G. A., & Abelson, J. N. (1997). Probing the role of an active site aspartic acid in dihydrofolate reductase. Journal of the American Chemical Society, 119(35), 8166-8176. https://doi.org/10.1021/ja971099l

Probing the role of an active site aspartic acid in dihydrofolate reductase. / Karginov, Vladimir A.; Mamaev, Sergey V.; An, Haoyun; Van Cleve, Mark D.; Hecht, Sidney; Komatsoulis, George A.; Abelson, John N.

In: Journal of the American Chemical Society, Vol. 119, No. 35, 03.09.1997, p. 8166-8176.

Research output: Contribution to journalArticle

Karginov, VA, Mamaev, SV, An, H, Van Cleve, MD, Hecht, S, Komatsoulis, GA & Abelson, JN 1997, 'Probing the role of an active site aspartic acid in dihydrofolate reductase', Journal of the American Chemical Society, vol. 119, no. 35, pp. 8166-8176. https://doi.org/10.1021/ja971099l
Karginov, Vladimir A. ; Mamaev, Sergey V. ; An, Haoyun ; Van Cleve, Mark D. ; Hecht, Sidney ; Komatsoulis, George A. ; Abelson, John N. / Probing the role of an active site aspartic acid in dihydrofolate reductase. In: Journal of the American Chemical Society. 1997 ; Vol. 119, No. 35. pp. 8166-8176.
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