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

Anthony Reddy, V. ; Appaji Rao, N. (1977) Dihydrofolate reductase from soybean seedlings: a fluorescence and circular dichroism study Archives of Biochemistry and Biophysics, 183 (1). pp. 90-97. ISSN 0003-9861

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Official URL: http://linkinghub.elsevier.com/retrieve/pii/000398...

Related URL: http://dx.doi.org/10.1016/0003-9861(77)90422-2


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 280nm 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 270nm. 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.

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