PMID- 11695901
OWN - NLM
STAT- MEDLINE
DCOM- 20011207
LR  - 20190613
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 40
IP  - 45
DP  - 2001 Nov 13
TI  - Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the 
      active site of GAR transformylase.
PG  - 13538-47
AB  - Multisubstrate adduct inhibitors (MAI) of glycinamide ribonucleotide 
      transformylase (GAR Tfase), which incorporate key features of the folate cofactor 
      and the beta-GAR substrate, typically exhibit K(i)'s in the picomolar range. 
      However, these compounds have reduced bioavailability due to the incorporation of 
      a negatively charged phosphate moiety that prevents effective cellular uptake. 
      Thus, a folate analogue that is capable of adduct formation with the substrate on 
      the enzyme active site could lead to a potent GAR Tfase inhibitor that takes 
      advantage of the cellular folate transport systems. We synthesized a dibromide 
      folate analogue, 10-bromo-10-bromomethyl-5,8,10-trideazafolic acid, that was an 
      intermediate designed to assemble with the substrate beta-GAR on the enzyme 
      active site. We have now determined the crystal structure of the Escherichia coli 
      GAR Tfase/MAI complex at 1.6 A resolution to ascertain the nature and mechanism 
      of its time-dependent inhibition. The high-resolution crystal structure clearly 
      revealed the existence of a covalent adduct between the substrate beta-GAR and 
      the folate analogue (K(i) = 20 microM). However, the electron density map 
      surprisingly indicated a C10 hydroxyl in the adduct rather than a bromide and 
      suggested that the multisubstrate adduct is not formed directly from the 
      dibromide but proceeds via an epoxide. Subsequently, we demonstrated the in situ 
      conversion of the dibromide to the epoxide. Moreover, synthesis of the authentic 
      epoxide confirmed that its inhibitory, time-dependent, and cytotoxic properties 
      are comparable to those of the dibromide. Further, inhibition was strongest when 
      the dibromide or epoxide is preincubated with both enzyme and substrate, 
      indicating that inhibition occurs via the enzyme-dependent formation of the 
      multisubstrate adduct. Thus, the crystal structure revealed the successful 
      formation of an enzyme-assembled multisubstrate adduct and highlighted a 
      potential application for epoxides, and perhaps aziridines, in the design of 
      efficacious GAR Tfase inhibitors.
FAU - Greasley, S E
AU  - Greasley SE
AD  - Department of Molecular Biology, The Scripps Research Institute, 10550 North 
      Torrey Pines Road, La Jolla, California 92037, USA.
FAU - Marsilje, T H
AU  - Marsilje TH
FAU - Cai, H
AU  - Cai H
FAU - Baker, S
AU  - Baker S
FAU - Benkovic, S J
AU  - Benkovic SJ
FAU - Boger, D L
AU  - Boger DL
FAU - Wilson, I A
AU  - Wilson IA
LA  - eng
SI  - PDB/1JKX
GR  - CA63536/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Epoxy Compounds)
RN  - 0 (Ligands)
RN  - EC 2.1.2.- (Hydroxymethyl and Formyl Transferases)
RN  - EC 2.1.2.2 (Phosphoribosylglycinamide Formyltransferase)
SB  - IM
MH  - Binding Sites
MH  - Crystallization
MH  - Crystallography, X-Ray
MH  - Enzyme Inhibitors/chemical synthesis/chemistry/pharmacology
MH  - Epoxy Compounds/chemical synthesis/*chemistry/pharmacology
MH  - Escherichia coli/enzymology
MH  - Hydroxymethyl and Formyl Transferases/antagonists & 
      inhibitors/*chemistry/metabolism
MH  - Ligands
MH  - Models, Molecular
MH  - Molecular Conformation
MH  - Phosphoribosylglycinamide Formyltransferase
MH  - Protein Conformation
EDAT- 2001/11/07 10:00
MHDA- 2002/01/05 10:01
CRDT- 2001/11/07 10:00
PHST- 2001/11/07 10:00 [pubmed]
PHST- 2002/01/05 10:01 [medline]
PHST- 2001/11/07 10:00 [entrez]
AID - bi011482+ [pii]
AID - 10.1021/bi011482+ [doi]
PST - ppublish
SO  - Biochemistry. 2001 Nov 13;40(45):13538-47. doi: 10.1021/bi011482+.