Type 1 And 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase: Synthesis, Enzyme Engineering and Mechanistic Studies

摘要

Enzymes are efficient biological catalysts that enormously accelerate the rates of chemical reactions by many orders of magnitude compared to the uncatalyzed reaction. The remarkable catalytic rate enhancement derives from the enzymes three dimensional structure, folding along with the active site geometry and packing. An enzyme active site has selective affinity for the substrates and stabilizes high-energy chemical species and unstable intermediates during the catalysis. To enhance the enzyme's catalytic ability, many enzymes have also evolved coenzymes (or) cofactors for catalysis. These cofactors often provide chemical functionality and reactivity that are not accessible to the twenty canonical amino acids. Thus, cofactors mediate diverse and unique chemical reactions that are catalyzed by enzymes. Isopentenyl diphosphate:dimethylallyl diphosphate isomerase were identified in two isoforms (IDI-1 and IDI-2) that catalyze the IPP/DMAPP isomerization. The work described in this dissertation focuses on attempts towards understanding the role of flavin cofactor in the chemical mechanism of type II isopentenyl diphosphate:dimethylallyl diphosphate (IDI-2) isomerase as well as mechanistic studies of IDI-1 and IDI-2. In the first section of this dissertation, studies will focus on the engineered enzyme variant of type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) from Streptococcus pneumoniae, a crucial enzyme in the isoprenoid biosynthetic pathway that utilizes a flavin mononucleotide (FMN) cofactor for catalysis. In most enzymes, flavin cofactors mediate redox electron transfer reactions. However, the IDI-2 catalyzed reaction involves no net redox change, raising questions on the role of the flavin in IDI-2 chemical mechanism. The chemical mechanism of IDI-2 will be studied with a combination of spectroscopic studies and biochemical techniques. Others and our studies suggest that the flavin cofactor of IDI-2 may employ an unusual and novel mode of flavin-dependent catalysis involving acid/base chemistry. In the second section, attention will be focused on the site-selective synthesis of 15N- and 13C-enriched flavin isotopologues to understand the acid/base functionality of flavin in the chemical mechanism of IDI-2. Finally, mechanistic studies of IDI-1 and IDI-2 in D2O will address that the IPP/DMAPP isomerization is very likely proceeding through a step-wise mechanism via a tertiary (3o) carbocationic intermediate. Taken together, kinetic, mechanistic, and spectroscopic studies on IDI-2 demonstrate that it catalyzes the reaction utilizing a flavin coenzyme in an atypical manner where the coenzyme performs acid/base chemistry instead of redox chemistry in which the later is the typical function of flavin cofactor.