TY - JOUR
T1 - Mechanism-based inhibition of human folylpolyglutamate synthetase
T2 - Design, synthesis, and biochemical characterization of a phosphapeptide mimic of the tetrahedral intermediate
AU - Tsukamoto, Takashi
AU - Haile, William H.
AU - McGuire, John J.
AU - Coward, James K.
N1 - Funding Information:
1 This research was supported by grants from the National Cancer Institute, CA28097 (JKC), CA43500 (JJM), and Roswell Park Cancer Institute Core Grant CA 16056 (JJM).
PY - 1998/7/1
Y1 - 1998/7/1
N2 - Folylpolyglutamate synthetase (FPGS) catalyzes an ATP-dependent ligation reaction that results in the synthesis of poly(γ-glutamate) metabolites of folates and some antifolates. We have synthesized and characterized the prototype of a new class of mechanism-based FPGS inhibitor in which a phosphonate moiety mimics the tetrahedral intermediate formed during the ligation reaction. This phosphonate, 4-amino-4-deoxy-10-methyl-pteroyl-L- glutamyl-γ-[Ψ{P(O) (OH)-O}]glutarate (4-NH2-10-CH3-Pte-L-Glu-γ- [Ψ{P(O)(OH)-O}]glutarate), is not a substrate for human FPGS, but is a linear, competitive inhibitor (K(in) = 46 nM) with respect to methotrexate as the variable substrate. Inhibition is not time-dependent and preincubation of FPGS with this phosphonate does not increase the degree of inhibition, suggesting that it is not a slow, tight-binding inhibitor involving a time- dependent isomerization, EI → EI*. Substructures containing the phosphonate moiety but lacking the pterin are much less inhibitory to FPGS, indicating that a significant portion of the inhibitor binding energy is derived from the pterin moiety, a feature also observed in substrate binding. 4-NH2-10- CH3-Pte-L-Glu-γ-[Ψ{P(O)(OH)-O}]glutarate is also an analog of a proposed tetrahedral intermediate in the reaction catalyzed by γ-glutamyl hydrolase (γ-GH), another enzyme of importance in controlling folate homeostasis in cells. This intermediate would arise from direct attack of H2O on the dipeptide, 4-NH2-10-CH3-Pte-L-Glu-γ-L-Glu. The fact that 4-NH2-10-CH3- Pte-L-Glu-γ-[Ψ{P(O)(OH)-O}]glutarate is not an inhibitor of γ-GH strongly suggests that hydrolysis of poly-γ-glutamates catalyzed by γ-GH does not involve the direct attack of water at the scissile amide bond. Methotrexate, its γ-glutamyl dipeptide metabolite, and 4-NH2-10-CH3-Pte- L-Glu-γ-[Ψ{P(O)(OH)-O}]glutarate are equipotent as inhibitors of human dihydrofolate reductase (the primary target of methotrexate), but the phosphonate does not significantly inhibit another important folate-dependent enzyme, thymidylate synthase. Thus, the phosphonate moiety in this analog represents an important new lead in the development of FPGS inhibitors.
AB - Folylpolyglutamate synthetase (FPGS) catalyzes an ATP-dependent ligation reaction that results in the synthesis of poly(γ-glutamate) metabolites of folates and some antifolates. We have synthesized and characterized the prototype of a new class of mechanism-based FPGS inhibitor in which a phosphonate moiety mimics the tetrahedral intermediate formed during the ligation reaction. This phosphonate, 4-amino-4-deoxy-10-methyl-pteroyl-L- glutamyl-γ-[Ψ{P(O) (OH)-O}]glutarate (4-NH2-10-CH3-Pte-L-Glu-γ- [Ψ{P(O)(OH)-O}]glutarate), is not a substrate for human FPGS, but is a linear, competitive inhibitor (K(in) = 46 nM) with respect to methotrexate as the variable substrate. Inhibition is not time-dependent and preincubation of FPGS with this phosphonate does not increase the degree of inhibition, suggesting that it is not a slow, tight-binding inhibitor involving a time- dependent isomerization, EI → EI*. Substructures containing the phosphonate moiety but lacking the pterin are much less inhibitory to FPGS, indicating that a significant portion of the inhibitor binding energy is derived from the pterin moiety, a feature also observed in substrate binding. 4-NH2-10- CH3-Pte-L-Glu-γ-[Ψ{P(O)(OH)-O}]glutarate is also an analog of a proposed tetrahedral intermediate in the reaction catalyzed by γ-glutamyl hydrolase (γ-GH), another enzyme of importance in controlling folate homeostasis in cells. This intermediate would arise from direct attack of H2O on the dipeptide, 4-NH2-10-CH3-Pte-L-Glu-γ-L-Glu. The fact that 4-NH2-10-CH3- Pte-L-Glu-γ-[Ψ{P(O)(OH)-O}]glutarate is not an inhibitor of γ-GH strongly suggests that hydrolysis of poly-γ-glutamates catalyzed by γ-GH does not involve the direct attack of water at the scissile amide bond. Methotrexate, its γ-glutamyl dipeptide metabolite, and 4-NH2-10-CH3-Pte- L-Glu-γ-[Ψ{P(O)(OH)-O}]glutarate are equipotent as inhibitors of human dihydrofolate reductase (the primary target of methotrexate), but the phosphonate does not significantly inhibit another important folate-dependent enzyme, thymidylate synthase. Thus, the phosphonate moiety in this analog represents an important new lead in the development of FPGS inhibitors.
KW - ATP-dependent ligase
KW - Folylpolyglutamates
KW - Phosphapeptide
KW - Tetrahedral mimic
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U2 - 10.1006/abbi.1998.0703
DO - 10.1006/abbi.1998.0703
M3 - Article
C2 - 9647673
AN - SCOPUS:0032126162
SN - 0003-9861
VL - 355
SP - 109
EP - 118
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -