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13 C NMR studies of complexes of Escherichia coli dihydrofolate reductase formed with methotrexate and with folic acid
Author(s) -
Cheung H.T.A.,
Birdsall B.,
Feeney J.
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)80923-5
Subject(s) - dihydrofolate reductase , escherichia coli , folic acid , chemistry , methotrexate , antifolate , biochemistry , stereochemistry , enzyme , organic chemistry , biology , antimetabolite , medicine , toxicity , gene , immunology
13 C NMR studies of 13 C‐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‐ 13 C]‐ and [2,4a,6‐ 13 C]methotrexate (MTX) and [4,6.8a‐ 13 C]‐ and [2,4a,7,9‐ 13 C]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‐NH 2 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 13 C signals for bound methotrexate consistent with only one conformational form in the ternary complexes.