Type II isopentenyl diphosphate isomerase: irreversible inactivation by covalent modification of flavin.
Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic building blocks of isoprenoid molecules. Two structurally unrelated classes of IDI are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction; whereas, the type II enzyme (IDI-2) requires reduced flavin. Epoxy, diene, and fluorinated substrate analogues, irreversible inhibitors of IDI-1, were analyzed as mechanistic probes for IDI-2. 3,4-Oxido-3-methyl-1-butyl diphosphate (eIPP), 3-methylene-4-penten-1-yl diphosphate (vIPP), and 3-(fluoromethyl)-3-buten-1-yl diphosphate (fmIPP) inactivate IDI-2 through formation of covalent adducts with the reduced flavin. UV-visible spectra of the inactivated complexes are consistent with modification of the isoalloxazine ring at position N5. vIPP and fmIPP are also alternate substrates with isomerization competing with alkylation of the flavin cofactor. (Z)-3-(Fluoromethyl)-2-buten-1-yl diphosphate ((Z)-fmDMAPP) and (Z)-3-(difluoromethyl)-2-buten-1-yl diphosphate ((Z)-dfmDMAPP) are alternate substrates, which are isomerized to the corresponding IPP derivatives. The rates of isomerization of fmIPP and (Z)-fmDMAPP are approximately 50-fold less than IPP and DMAPP, respectively. dfmIPP is not an irreversible inhibitor. These studies indicate that the irreversible inhibitors inactivate the reduced flavin required for catalysis by electrophilic alkylation and are consistent with a protonation-deprotonation mechanism for the isomerization catalyzed by IDI-2.
Rothman
Steven C
SC
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Johnston
Jonathan B
JB
Lee
Sungwon
S
Walker
Joel R
JR
Poulter
C Dale
CD
D001667:Binding, Competitive; D019748:Carbon-Carbon Double Bond Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D045782:Hemiterpenes; D007527:Isoenzymes; D007700:Kinetics; D013056:Spectrophotometry, Ultraviolet; D016963:Thermus thermophilus / Q000201:enzymology
D045782:Hemiterpenes; D007527:Isoenzymes; D005486:Flavin Mononucleotide; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase