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Dihydrofolate reductase from soybean seedlings: A fluorescence and circular dichroism study

Reddy, V Anthony and Rao, N Appaji (1977) Dihydrofolate reductase from soybean seedlings: A fluorescence and circular dichroism study. In: Archives of Biochemistry and Biophysics, 183 (1). pp. 90-97.

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The fluorescence emission spectrum of soybean dihydrofolate reductase suggests that the emitting tryptophan residues are situated in a hydrophobic microenvironment. The dissociation constants determined from fluorescence and circular dichroism data reveal that the soybean enzyme has a lower affinity for substrates and substrate analogs than that determined for dihydrofolate reductases isolated from other sources. The binding of methotrexate to the soybean enzyme does not affect the binding of NADPH. Similarly, the binding of NADPH has no effect on subsequent methotrexate binding. Polarimetric study indicates that the enzyme has a low (ca. 5%) α-helical content. Addition of dihydrofolate to the soybean enzyme results in the generation of a positive ellipticity band at 298 nm with a molar ellipticity, [θ], of 186,000, whereas the binding of folate induces a negative ellipticity band at 280 nm with [θ] of −181,000. The qualitative and quantitative differences in the circular dichroism of the enzyme-dihydrofolate and enzyme-folate complexes indicate that the mode of binding of these ligands may be different. The formation of an enzyme-NADPH complex is accompanied by a negative Cotton effect at 270 nm. These studies indicate that the binding of substrates or inhibitors causes significant conformational changes in the enzyme and also leads to the formation of a number of spectroscopically identifiable complexes.

Item Type: Journal Article
Publication: Archives of Biochemistry and Biophysics
Publisher: Elsevier Science
Additional Information: Copyright of this article belongs to Elsevier Science.
Department/Centre: Division of Biological Sciences > Biochemistry
Date Deposited: 03 Feb 2010 09:03
Last Modified: 19 Sep 2010 05:50
URI: http://eprints.iisc.ac.in/id/eprint/24395

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