NMR Studies of Ligand Binding to Dihydrofolate Reductase

Feeney, J.

doi:10.1002/(sici)1521-3773(20000117)39:2<290::aid-anie290>3.0.co;2-1

Cited by 51 publications

(16 citation statements)

References 101 publications

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“…These antifolate drugs often bind to the protein with large positive cooperativity in the presence of the coenzyme NADPH. 30 In the case of the antibacterial drug trimethoprim (TMP; trimethoprim(2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine)), the cooperative binding has been implicated directly in the specificity of drug binding to bacterial DHFRs when compared to mammalian DHFRs. 31 Another ligand, the tetrahydrofolate analogue 5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid), binds to DHFR with strong negative co-operativity in the presence of NADPH: 32 similar negative co-operativity binding effects have important implications in the control of product release in the enzyme reaction mechanism.…”

Section: Introductionmentioning

confidence: 99%

Effects of Co-operative Ligand Binding on Protein Amide NH Hydrogen Exchange

Polshakov¹,

Birdsall²,

Feeney³

2006

Journal of Molecular Biology

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Section: Introductionmentioning

confidence: 99%

Effects of Co-operative Ligand Binding on Protein Amide NH Hydrogen Exchange

Polshakov¹,

Birdsall²,

Feeney³

2006

Journal of Molecular Biology

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“…Such drugs act by inhibiting the enzyme in parasitic or malignant cells. There have been several studies aimed at investigating the speci®city of the ligand binding (see for example Roth & Cheng, 1982;Baccanari & Kuyper, 1993) and extensive structural information has been obtained on a number of DHFR complexes from different species using NMR and X-ray crystallography (reviewed by Blakley, 1985;Freisheim & Matthews, 1984;Feeney, 1990Feeney, , 1996. For the enzyme from Escherichia coli, there are high-resolution crystal structures available on the enzyme alone (Bystroff & Kraut, 1991) several binary complexes Kuyper et al, 1982;Matthews et al, 1985a,b;Bystroff et al, 1990;Warren et al, 1991;Reyes et al, 1995;Lee et al, 1996;Sawaya & Kraut, 1997) and ternary complexes (Bystroff et al, 1990;Sawaya & Kraut, 1997).…”

Section: Introductionmentioning

confidence: 99%

The solution structure of the complex of Lactobacillus casei dihydrofolate reductase with methotrexate

Gargaro¹,

Soteriou²,

Frenkiel

et al. 1998

Journal of Molecular Biology

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“…All structurally determined, chromosomally encoded DHFRs are single domain proteins containing an eight‐stranded mixed β‐sheet, which is flanked by two α‐helices on either side (Fig. 1) (Sawaya and Kraut 1997; Feeney 2000). DHFRs are organized into two subdomains, the adenosine‐binding domain, which binds the adenosine portion of NADPH, and the loop domain, which is dominated by three loops, namely the M20 loop, the βF–βG loop, and the βG–βH loop.…”

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Role of water in the catalytic cycle of E. coli dihydrofolate reductase

Shrimpton

Allemann

2002

Protein Science

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Dihydrofolate reductase (DHFR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of 7,8-dihydrofolate (H 2 F) to 5,6,7,8-tetrahydrofolate (H 4 F). Because of the absence of any ionizable group in the vicinity of N5 of dihydrofolate it has been proposed that N5 could be protonated directly by a water molecule at the active site in the ternary complex of the Escherichia coli enzyme with cofactor and substrate. However, in the X-ray structures representing the Michaelis complex of the E. coli enzyme, a water molecule has never been observed in a position that could allow protonation of N5. In fact, the side chain of Met 20 blocks access to N5. Energy minimization reported here revealed that water could be placed in hydrogen bonding distance of N5 with only minor conformational changes. The r.m.s. deviation between the conformation of the M20 loop observed in the crystal structures of the ternary complexes and the conformation adopted after energy minimization was only 0.79 Å. We performed molecular dynamics simulations to determine the accessibility by water of the active site of the Michaelis complex of DHFR. Water could access N5 relatively freely after an equilibration time of approximately 300 psec during which the side chain of Met 20 blocked water access. Protonation of N5 did not increase the accessibility by water. Surprisingly the number of near-attack conformations, in which the distance between the pro-R hydrogen of NADPH and C6 of dihydrofolate was less than 3.5 Å and the angle between C4 and the pro-R hydrogen of NADPH and C6 of dihydrofolate was greater than 120 degrees, did not increase after protonation. However, when the hydride was transferred from NADPH to C6 of dihydrofolate before protonation, the side chain of Met 20 moved away from N5 after approximately 100 psec thereby providing water access. The average time during which water was found in hydrogen bonding distance to N5 was significantly increased. These results suggest that hydride transfer might occur early to midway through the reaction followed by protonation. Such a mechanism is supported by the very close contact between C4 of NADP + and C6 of folate observed in the crystal structures of the ternary enzyme complexes, when the M20 loop is in its closed conformation.

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NMR Studies of Ligand Binding to Dihydrofolate Reductase

Cited by 51 publications

References 101 publications

Effects of Co-operative Ligand Binding on Protein Amide NH Hydrogen Exchange

Effects of Co-operative Ligand Binding on Protein Amide NH Hydrogen Exchange

The solution structure of the complex of Lactobacillus casei dihydrofolate reductase with methotrexate

Role of water in the catalytic cycle of E. coli dihydrofolate reductase

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