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The influence of aspartate 26 on the tautomeric forms of folate bound to Lactobacillus casei dihydrofolate reductase
Author(s) -
Birdsall Berry,
Casarotto Marco G,
Cheung H.T.Andrew,
Basran Jaswir,
Roberts Gordon C.K,
Feeney James
Publication year - 1997
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/s0014-5793(96)01519-0
Subject(s) - dihydrofolate reductase , protonation , chemistry , lactobacillus casei , deprotonation , tautomer , ternary complex , stereochemistry , aspartic acid , residue (chemistry) , dissociation constant , enzyme , biochemistry , amino acid , organic chemistry , receptor , ion , fermentation
The ternary complex of Lactobacillus casei dihydrofolate reductase (DHFR) with folate and NADP + exists as a mixture of three interconverting forms (I, IIa and IIb) whose relative populations are pH dependent, with an effective p K of approx. 6. To investigate the role of Asp 26 in this pH dependence we have measured the 13 C chemical shifts of [2,4 a ,7,9‐ 13 C 4 ]folate in its complex with the mutant DHFR Asp 26 →Asn and NADP + . Only a single form of the complex is detected and this has the characteristics of form I, an enol form with its N1 unprotonated. A study of the pH dependence of the 13 C chemical shifts of DHFR selectively labelled with [4‐ 13 C]aspartic acid in its complex with folate and NADP + indicates that no Asp residue has a p K value greater than 5.4. Two of the Asp CO − 2 signals appear as non‐integral signals with chemical shifts typical of non‐ionised COOH groups and with a pH dependence characteristic of the slow exchange equilibria previously characterised for signals in forms I and IIb (or IIa). It is proposed that the protonation/deprotonation controlling the equilibria involves the O4 position of the folate and that Asp 26 influences this indirectly by binding in its CO − 2 form to the protonated N1 group of folate in forms I and IIa thus reducing the p K involving protonation at the O4 position to approx. 6. These findings indicate that, in forms I and IIa of the ternary complex, folate binds to DHFR in a very similar way to methotrexate.