New Insights into Inhibitor Design from the Crystal Structure and NMR Studies of Escherichia coli GAR Transformylase in Complex with 
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- 作者：Samantha E. Greasley ; Mason M. Yamashita ; Hui Cai ; Stephen J. Benkovic ; Dale L. Boger ; and Ian A. Wilson
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：December 21, 1999
- 年：1999
- 卷：38
- 期：51
- 页码：16783 - 16793
- 全文大小：492K
- 年卷期：v.38,no.51(December 21, 1999)
- ISSN：1520-4995
文摘
The crystal structure of Escherichia coli GAR Tfase at 2.1 Å resolution in complex with10-formyl-5,8,10-trideazafolic acid (10-formyl-TDAF, Ki = 260 nM), an inhibitor designed to form anenzyme-assembled multisubstrate adduct with the substrate, 
-GAR, was studied to determine the exactnature of its inhibitory properties. Rather than forming the expected covalent adduct, the folate inhibitorbinds as the hydrated aldehyde (gem-diol) in the enzyme active site, in a manner that mimics the tetrahedralintermediate of the formyl transfer reaction. In this hydrated form, the inhibitor not only provides unexpectedinsights into the catalytic mechanism but also explains the 10-fold difference in inhibitor potency between10-formyl-TDAF and the corresponding alcohol, and a further 10-fold difference for inhibitors that lackthe alcohol. The presence of the hydrated aldehyde was confirmed in solution by 13C-1H NMR spectroscopyof the ternary GAR Tfase--GAR-10-formyl-TDAF complex using the 13C-labeled 10-formyl-TDAF.This insight into the behavior of the inhibitor, which is analogous to protease or transaminase inhibitors,provides a novel and previously unrecognized basis for the design of more potent inhibitors of the folate-dependent formyl transfer enzymes of the purine biosynthetic pathway and development of anti-neoplasticagents.
-GAR, was studied to determine the exactnature of its inhibitory properties. Rather than forming the expected covalent adduct, the folate inhibitorbinds as the hydrated aldehyde (gem-diol) in the enzyme active site, in a manner that mimics the tetrahedralintermediate of the formyl transfer reaction. In this hydrated form, the inhibitor not only provides unexpectedinsights into the catalytic mechanism but also explains the 10-fold difference in inhibitor potency between10-formyl-TDAF and the corresponding alcohol, and a further 10-fold difference for inhibitors that lackthe alcohol. The presence of the hydrated aldehyde was confirmed in solution by 13C-1H NMR spectroscopyof the ternary GAR Tfase--GAR-10-formyl-TDAF complex using the 13C-labeled 10-formyl-TDAF.This insight into the behavior of the inhibitor, which is analogous to protease or transaminase inhibitors,provides a novel and previously unrecognized basis for the design of more potent inhibitors of the folate-dependent formyl transfer enzymes of the purine biosynthetic pathway and development of anti-neoplasticagents.