Evidence for the involvement of acid/base chemistry in the reaction catalyzed by the type II isopentenyl diphosphate/dimethylallyl diphosphate isomerase from Staphylococcus aureus.
The type II isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IDI-2) is a flavin mononucleotide (FMN)-dependent enzyme that catalyzes the reversible isomerization of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP), a reaction with no net change in redox state of the coenzyme or substrate. Here, UV-vis spectral analysis of the IDI-2 reaction revealed the accumulation of a reduced neutral dihydroflavin intermediate when the reduced enzyme was incubated with IPP or DMAPP. When IDI-2 was reconstituted with 1-deazaFMN and 5-deazaFMN, similar reduced neutral forms of the deazaflavin analogues were observed in the presence of IPP. Single turnover stopped-flow absorbance experiments indicated that this flavin intermediate formed and decayed at kinetically competent rates in the pre-steady-state and, thus, most likely represents a true intermediate in the catalytic cycle. UV-vis spectra of the reaction mixtures reveal trace amounts of a neutral semiquinone, but evidence for the presence of IPP-based radicals could not be obtained by EPR spectroscopy. Rapid-mix chemical quench experiments show no burst in DMAPP formation, suggesting that the rate determining step in the forward direction (IPP to DMAPP) occurs prior to DMAPP formation. A solvent deuterium kinetic isotope effect (D2OVmax = 1.5) was measured on vo in steady-state kinetic experiments at saturating substrate concentrations. A substrate deuterium kinetic isotope effect was also measured on the initital velocity (DVmax = 1.8) and on the decay rate of the flavin intermediate (Dks = 2.3) in single-turnover stopped-flow experiments using (R)-[2-2H]-IPP. Taken together, these data suggest that the C2-H bond of IPP is cleaved in the rate determining step and that general acid/base catalysis may be involved during turnover. Possible mechanisms for the IDI-2 catalyzed reaction are presented and discussed in terms of the available X-ray crystal structures.
Thibodeaux
Christopher J
CJ
Division of Medicinal Chemistry, College of Pharmacy, Department of Chemistry and Biochemistry, and Institute of Cellular and Molecular Biology, University of Texas at Austin, 1 University Station A1935, Austin, Texas 78712, USA.
Mansoorabadi
Steven O
SO
Kittleman
William
W
Chang
Wei-chen
WC
Liu
Hung-wen
HW
D000143:Acids / Q000737:chemistry*; D000588:Amines / Q000737:chemistry*; D019748:Carbon-Carbon Double Bond Isomerases / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D005486:Flavin Mononucleotide / Q000378:metabolism; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D013056:Spectrophotometry, Ultraviolet; D013211:Staphylococcus aureus / Q000201:enzymology*
D000143:Acids; D000588:Amines; D045782:Hemiterpenes; D007527:Isoenzymes; D005486:Flavin Mononucleotide; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase