13C NMR studies of complexes of Escherichia coli dihydrofolate reductase formed with methotrexate and with folic acid

FEBS Letters

Birdsall, B.¹  Feeney, J.¹  Cheung, H.T.A.¹ 

[1] Laboratory of Molecular Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK

关键词: Dihydrofolate reductase;    Methotrexate;    Folic acid;    13C NMR;    Multiple conformations;   

DOI  :  10.1016/0014-5793(92)80923-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

¹³C NMR studies of ¹³C-labelled ligands bound to dihydrofolate reductase provide (DHFR) a powerful means of detecting and characterizing multiple bound conformations. Such studies of complexes of Escherichia coli DHFR with [4,7,8a,9-¹³C]- and [2,4a,6-¹³C]methotrexate (MTX) and [4,6.8a-¹³C]- and [2,4a,7,9-¹³C]folic acid confirm that in the binary complexes, MTX binds in two conformational forms and folate binds as a single conformation. Earlier studies on the corresponding complexes with Lactobacillus casei DHFR indicated that, in this case, MTX binds as a single conformation whereas folate binds in multiple conformational forms (both in its binary complex and ternary complex with NADP⁺); two of the bound conformational states for the folate complexes are very different from each other in that there is a 180° difference in their pteridine ring orientation. In contrast, the two different conformational states observed for MTX bound to E. coli DHFR do not show such a major difference in ring orientation and bind with N1 protonated in both forms. The major difference appears to involve the manner in which the 4-NH₂ group of MTX binds to the enzyme (although the same protein residues are probably involved in both interactions). Addition of either NADP⁺ or NADPH to the E. coli DHFR-MTX complex results in a single set of ¹³C signals for bound methotrexate consistent with only one conformational form in the ternary complexes.

【 授权许可】

Unknown