Abstract
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 13C chemical shifts of [2,4 a,7,9- 13C 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 13C chemical shifts of DHFR selectively labelled with [4- 13C]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.
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