THE INFLUENCE OF ASPARTATE-26 ON THE TAUTOMERIC FORMS OF FOLATE BOUNDTO LACTOBACILLUS-CASEI DIHYDROFOLATE-REDUCTASE

The ternary complex of Lactobacillus casei dihydrofolate reductase (DH PR) with folate and NADP(+) exists as a mixture of three interconverti ng forms (I, IIa and IIb) whose relative populations are pH dependent, with an effective pK of approx. 6. To investigate the role of Asp(26) in this pH dependence we have measured the C-13 chemical shifts of [2 ,4a,7,9-C-13(4)]folate in its complex with the mutant DHFR Asp(26) --> Asn and NADP(+). Only a single form of the complex is detected and th is has the characteristics of form I, an enol form with its N1 unproto nated. A study of the pH dependence of the C-13 chemical shifts of DHF R selectively labelled with [4-C-13]aspartic acid in its complex: with folate and NADP(+) indicates that no Asp residue has a pK value great er than 5.4. Two of the Asp CO2- signals appear as non-integral signal s with chemical shifts typical of non-ionised COOH groups and with a p H 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 involve s the O4 position of the folate and that Asp(26) influences this indir ectly by binding in its CO2- form to the protonated N1 group of folate in forms I and IIa thus reducing the pK 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 wa y to methotrexate.