Formation and utilization of formyl phosphate by N10-formyltetrahydrofolate synthetase: evidence for formyl phosphate as an intermediate in the reaction.
Formation and utilization of formyl phosphate by N10-formyltetrahydrofolate synthetase: evidence for formyl phosphate as an intermediate in the reaction.
复制标题
N10-甲酰四氢叶酸合成酶形成和利用甲酰磷酸：甲酰磷酸作为反应中间体的证据。
DOI：
10.1021/bi00438a034
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Himes,RH
中科院分区：
文献类型：
--
作者：
Mejillano,MR;Jahansouz,H;Matsunaga,TO;Kenyon,GL;Himes,RH
Department of Biochemistry, University of Kansas, Lawrence, Kansas 66045, andDepartment of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 Received January 31, 1989; Revised Manuscript Received March 14, 1989 abstract: iV10-Formyltetrahydrofolate synthetase from bacteria and yeast catalyzes a slow formate-dependent ADP formation in the absence of H4folate. The synthesis of formyl phosphate by the enzyme was detected by trapping the intermediate as formyl hydroxamate. That the “formate kinase” activity was part of the catalytic center of iV10-formyltetrahydrofolate synthetase was shown by demonstrating coordinateinactivation of the “kinase” and synthetase activities by heat and a sulfhydryl reagent, similar effects of monovalent cations, similar Km values for substrates, and similar K¡ values for the inhibitor phosphonoacetaldehyde for both activities. The relative rates of the kinase activities for the bacterial and yeast enzymes are about 10" 4 and 4 X 10" 6 of their respective synthetase activities. These slow rates for the kinase reaction can be explained by the slow dissociation of ADP and formyl phosphate from the enzyme. This conclusion is supported by rapid-quench studies where a “burst” of ADP formation (6.4 s'1) was observed that is considerably faster than the steady-state rate (0.024 s'1). The demonstration of enzyme-bound products by a micropartition assay and the lack of a significant formate-stimulated exchange between ADP and ATP provide further evidence for the slow release of the products from the enzyme. The synthesis of 7V10-CHO-H4folate when H4folate was added to the E-formyl phosphate-ADP complex is also characterized by a “burst” of product formation. The rate of this burst phase at 5 C occurs with a rate constant of 18 s'1 compared to 14 s'1 for the overall reaction at the same temperature. These results provide further evidence for formyl phosphate as an intermediate in the reaction and are consistent with the sequential mechanism of the normalcatalytic pathway. Positional isotope exchange experiments using [/?, 7-180] ATP showed no evidence for exchange during turnover experiments in the presence of either H4folate or the competitive inhibitor pteroyltriglutamate. The absence of scrambling of the I80 label as observed by 31P NMR suggests that the central complex may impose restraints to limit free rotation of the P^ oxygens of the productADP.
