Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase

Cocco, Lennie; Roth, Barbara; Temple, Carroll; Montgomery, John A.; London, Robert E.; Blakley, Raymond L.

doi:10.1016/0003-9861(83)90326-0

Cited by 76 publications

(51 citation statements)

References 16 publications

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“…We observe directly a proton at N1 of MTX, imparting it with a positive charge. This result agrees with nearly all available biochemical and complementary structural data for E. coli and L. casei DHFRs: that the MTX⅐D27 interaction is ionic in nature (45)(46)(47)(48)(49)(50)(51). However, it is in disagreement with the theoretical calculations of Cannon et al (52), who proposed that the MTX N1⅐D27 pair involves a neutral dipole-dipole interaction.…”

Section: Discussionsupporting

confidence: 80%

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate

Bennett

Langan

Coates

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Section: Discussionsupporting

confidence: 80%

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate

Bennett

Langan

Coates

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…This results in salt bridge formation with the Type I enzyme, in which the N1-protonated inhibitor is salt bridged with the active site carboxyl group (64,65). A similar effect occurs for trimethoprim complexes with bacterial DHFR (66). However, as discussed above, the Type II DHFR lacks an active site carboxyl group and does not appear to utilize a mechanism that strongly favors N5-protonated DHF, and hence will have lower affinity for high-pK analogs such as trimethoprim or aminopterin.…”

Section: Insensitivity To Trimethoprimmentioning

confidence: 87%

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

et al. 2007

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Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the Dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR•DHF•NADP + catalytic complex, resolved to1.26 Å. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67′-Y69′ residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationallychallenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation. KeywordsR67 DHFR; Dihydrofolate Reductase; X-ray crystallography; ternary complex; Type II DHFR Antifolate drug therapy plays a critical role in the treatment of pathogenic and neoplastic diseases. The evolution of a plasmid-encoded, Type II dihydrofolate reductase (DHFR) provides one mechanism for bacterial evasion of drugs such as trimethoprim that target the bacterial dihydrofolate reductase enzyme (1-4). Type II DHFR is an extremely unusual enzyme that exhibits no apparent structural or evolutionary relationship with the type I (chromosomal) enzyme. It is one of the smallest enzymes known to self-assemble into an active quaternary structure, forming a homotetramer consisting of four 78-residue peptides * To whom correspondence should be addressed. This type of active site structure also creates substantial evolutionary and mutational challenges to the enzyme. Since each mutation will alter four active site residues at a time, most of the evolutionary pressure that would normally optimize enzyme function is compromised by the need to balance the effects of substitutions at all four symmetry-related sites. For example, a residue substitution on one monomer, which might promote folate N5 protonation, may also interfere with NADPH binding when it is prese...

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“…Among a number of methods designed to determine the MTX concentrations in biological fluids, a commercially available MTX assay kit is based on its inhibition of dihydrofolate reductase (DHFR) (Wang et al 1976) ; bacterial DHFR is conveniently measured by following NADPH consumption spectrometrically. An important pitfall in the use of this method for determination of MTX in body fluids is interference by trimethoprim [2, 4-diamino-5 (3, 4, 5-trimethoxybenzyl) pyrimidine], which is a potent inhibitor of bacterial DHFR (Bock and Pierce 1980;Hande et al 1980;Cocco et al 1983). Recently, we had a patient with central nervous system (CNS) leukemia who showed apparent concentrations of MTX in CSF for several weeks after its intrathecal injection, while he was receiving trimethoprim/ sulfamethoxazole (T 5) treatment.…”

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confidence: 99%

“…Recently, we had a patient with central nervous system (CNS) leukemia who showed apparent concentrations of MTX in CSF for several weeks after its intrathecal injection, while he was receiving trimethoprim/ sulfamethoxazole (T 5) treatment. This experience prompted us to examine the interference of TMP in an assay system for MTX which is based on its inhibition of DHFR (Cocco et al 1983).Spurious MTX levels were estimated using an MTX assay Kit (Nippon Ledery, Tokyo) in 7 CSFs and 5 sera from CNS leukemia patients who were on T/S treatment but not on MTX. Out of them, 5 CSFs and 2 sera were subjected simultaneously to TMP determination by spectrofluorometry (Schwartz et al 1969).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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Trimethoprim interference in methotrexate assay by an enzyme inhibition assay kit.

Kitaoka

Terasawa

Goto

et al. 1986

Tohoku J. Exp. Med.

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Spurious methotrexate (MTX) concentrations in sera and cerebrospinal fluids from leukemia patients who were given trimethoprim (TMP) were estimated using an MTX assay kit which is based on its inhibition of dihydrofolate reductase. The interference of TMP with MTX was confirmed in the assay system. A concentration of 0.171ag/ml of TMP gave a value for an apparent MTX of 10 pM. methotrexate ; trimethoprim ; methotrexate assay kitThe clinical use of methotrexate (4-amino-4-deoxy-l0-methylfolic acid, MTX) requires careful monitoring of its serum and cerebrospinal fluid (CSF) concentrations to facilitate early detection of patients who are at high risk of toxicity (Stoller et al. 1977). Among a number of methods designed to determine the MTX concentrations in biological fluids, a commercially available MTX assay kit is based on its inhibition of dihydrofolate reductase (DHFR) (Wang et al. 1976) ; bacterial DHFR is conveniently measured by following NADPH consumption spectrometrically. An important pitfall in the use of this method for determination of MTX in body fluids is interference by trimethoprim [2, 4-diamino-5 (3, 4, 5-trimethoxybenzyl) pyrimidine], which is a potent inhibitor of bacterial DHFR (Bock and Pierce 1980;Hande et al. 1980;Cocco et al. 1983). Recently, we had a patient with central nervous system (CNS) leukemia who showed apparent concentrations of MTX in CSF for several weeks after its intrathecal injection, while he was receiving trimethoprim/ sulfamethoxazole (T 5) treatment. This experience prompted us to examine the interference of TMP in an assay system for MTX which is based on its inhibition of DHFR (Cocco et al. 1983).Spurious MTX levels were estimated using an MTX assay Kit (Nippon Ledery, Tokyo) in 7 CSFs and 5 sera from CNS leukemia patients who were on T/S treatment but not on MTX. Out of them, 5 CSFs and 2 sera were subjected simultaneously to TMP determination by spectrofluorometry (Schwartz et al. 1969).As shown in Fig. 1, spurious MTX concentrations were found to be present in CSFs and sera examined. Such "MTX" levels were correlated with TMP levels (r0.96). = To examine the direct interference of TMP with MTX in the assay system used, various concentrations of TMP were added to the assay system and expressed in terms of apparent MTX concentrations. As shown in Fig. 2, a linear relation was obtained between the concentrations of TMP added and apparent MTX estimated. An increment of 0.5pg/ml

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Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase

Cited by 76 publications

References 16 publications

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

Trimethoprim interference in methotrexate assay by an enzyme inhibition assay kit.

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