NMR Studies of the Interaction of a Type II Dihydrofolate Reductase with Pyridine Nucleotides Reveal Unexpected Phosphatase and Reductase Activity

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• 作者：Wayne H. Pitcher ; III ; Eugene F. DeRose ; Geoffrey A. Mueller ; Elizabeth E. Howell ; and Robert E. London
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：September 30, 2003
• 年：2003
• 卷：42
• 期：38
• 页码：11150 - 11160
• 全文大小：259K
• 年卷期：v.42,no.38(September 30, 2003)
• ISSN：1520-4995

文摘

The interaction of type II R67 dihydrofolate reductase (DHFR) with its cofactor nicotinamideadenine dinucleotide phosphate (NADP⁺) has been studied using nuclear magnetic resonance (NMR).Doubly labeled [U-¹³C,¹⁵N]DHFR was obtained from Escherichia coli grown on a medium containing[U-¹³C]-D-glucose and ¹⁵NH₄Cl, and the 16 disordered N-terminal amino acids were removed by treatmentwith chymotrypsin. Backbone and side chain NMR assignments were made using triple-resonanceexperiments. The degeneracy of the amide ¹H and ¹⁵N shifts of the tetrameric DHFR was preserved uponaddition of NADP⁺, consistent with kinetic averaging among equivalent binding sites. Analysis of themore titration-sensitive DHFR amide resonances as a function of added NADP⁺ gave a K_D of 131 ± 50ages/entities/mgr.gif">M, consistent with previous determinations using other methodology. We have found that the ¹H spectrumof NADP⁺ in the presence of the R67 DHFR changes as a function of time. Comparison with standardsamples and mass spectrometric analysis indicates a slow conversion of NADP⁺ to NAD⁺, i.e., an apparentNADP⁺ phosphatase activity. Studies of this activity in the presence of folate and a folate analogue supportthe conclusion that this activity results from an interaction with the DHFR rather than a contaminatingphosphatase. ¹H NMR studies of a mixture of NADP⁺ and NADPH in the presence of the enzyme revealthat a ternary complex forms in which the N-4A and N-4B nuclei of the NADPH are in the proximity ofthe N-4 and N-5 nuclei of NADP⁺. Studies using the NADP⁺ analogue acetylpyridine adenosinedinucleotide phosphate (APADP⁺) demonstrated a low level of enzyme-catalyzed hydride transfer fromNADPH. Analysis of DHFR backbone dynamics revealed little change upon binding of NADP⁺. Theseadditional catalytic activities and dynamic behavior are in marked contrast to those of type I DHFR.