INHIBITION OF PIG-LIVER METHYLENETETRAHYDROFOLATE REDUCTASE BY DIHYDROFOLATE - SOME MECHANISTIC AND REGULATORY IMPLICATIONS

被引:62
作者
MATTHEWS, RG [1 ]
HAYWOOD, BJ [1 ]
机构
[1] UNIV MICHIGAN,DEPT BIOL CHEM,ANN ARBOR,MI 48109
关键词
D O I
10.1021/bi00589a012
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Methylenetetrahydrofolate reductase has been purified 3400-fold from pig liver. The enzyme exhibits ping-pong bi-bi kinetics during catalysis of NADPH-methylenetetrahydrofolate oxidoreduction. Dihydrofolate is an inhibitor of this reaction and is competitive with respect to methylenetetrahydrofolate. The K1 for dihydrofolate is 6μM. The inhibition of NADPH-methylenetetrahydrofolate reductase activity is greatest when dihydrofolate is present, and similar concentrations of tetrahydrofolate, methyltetra-hydrofolate, folic acid, methenyltetrahydrofolate, or 5-methyl-5,6-dihydrofolate are 2.5-10-fold less effective. These observations suggest that the enzyme-catalyzed reduction of methylenetetrahydrofolate may proceed by tautomerization to form an N-methyldihydrofolate derivative. However, the formation of N-methyl-7,8-dihydrofolate is precluded by our failure to see loss of tritium from C-6 of the pteridine ring of methylenetetrahydrofolate during reduction. Proton magnetic resonance spectroscopy studies on the methyltetrahydrofolate formed during enzymatic reduction of methylenetetrahydrofolate in deuterium oxide demonstrate that two atoms of deuterium are incorporated into the methyl group. We suggest that inhibition of NADPH-methylenetetrahydrofolate oxidoreductase activity by dihydrofolate may be important in the regulation of the flux of methylenetetrahydrofolate in vivo. Both thymidylate synthetase and methylenetetrahydrofolate reductase are found in the cytoplasm, as is dihydrofolate reductase. When thymidylate biosynthesis is occurring, the resulting increase in steady-state levels of dihydropteroyl-glutamate derivatives may serve to inhibit methylenetetrahydrofolate reductase, thus sparing methylenetetrahydro-pteroylglutamates for purine and pyrimidine biosynthesis. © 1979, American Chemical Society. All rights reserved.
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页码:4845 / 4851
页数:7
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