The influence of aspartate 26 on the tautomeric forms of folate bound to Lactobacillus casei dihydrofolate reductase

FEBS Letters

Roberts, Gordon C.K¹  Basran, Jaswir¹  Casarotto, Marco G¹  Birdsall, Berry²  Feeney, James²  Cheung, H.T.Andrew³ 

[1] Biological NMR Centre and Department of Biochemistry, University of Leicester, University Road, Leicester LE1 9HN, UK;Molecular Structure Division, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK;Department of Pharmacy, University of Sydney, Sydney, New South Wales 2006, Australia

关键词: Dihydrofolate reductase;    13C-NMR;    Folate;    Tautomeric form;    Ionisation state;   

DOI  :  10.1016/S0014-5793(96)01519-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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,7,9-¹³C₄]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 [4-¹³C]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 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 Asp²⁶ 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 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.

【 授权许可】

Unknown