The influence of aspartate 26 on the tautomeric forms of folate bound to Lactobacillus casei dihydrofolate reductase.
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 pK of approx. 6. To investigate the role of Asp26 in this pH dependence we have measured the 13C chemical shifts of [2,4a,7,9-(13)C4]folate in its complex with the mutant DHFR Asp26 --> 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-(13)C]aspartic acid in its complex with folate and NADP+ indicates that no Asp residue has a pK value greater than 5.4. Two of the Asp CO2 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 Asp26 influences this indirectly 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 way to methotrexate.
Birdsall
B
B
Molecular Structure Division, National Institute for Medical Research, London, UK.
Casarotto
M G
MG
Cheung
H T
HT
Basran
J
J
Roberts
G C
GC
Feeney
J
J
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001665:Binding Sites; D005492:Folic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D009249:NADP / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*
D011994:Recombinant Proteins; D001224:Aspartic Acid; D009249:NADP; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase