| dc.identifier.uri | http://hdl.handle.net/11401/77137 | |
| dc.description.sponsorship | This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. | en_US |
| dc.format | Monograph | |
| dc.format.medium | Electronic Resource | en_US |
| dc.language.iso | en_US | |
| dc.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dc.type | Dissertation | |
| dcterms.abstract | The enoyl-acyl-carrier-protein reductase (ENR) catalyzes the last reaction in the elongation cycle in the fatty acid biosynthesis type II (FAS-II) pathway. To date, there are four known ENR isoenzymes: FabI, FabK, FabL, and FabV. We have rigorously characterized the FabV ENR from <italic>Burkholderia mallei</italic> (BmFabV) and have shown that this enzyme catalyzes substrate reduction via an ordered bi-bi mechanism, in which NADH binds first to the enzyme followed by the enoyl substrate [Lu, H. (2010) Biochemistry 49, 1281-1289]. However, this pathogen contains both FabI and FabV ENRs, and mechanistic insights into ENR substrate recognition are lacking in pathogens that solely express the FabV ENR. Thus, we extended our mechanistic studies to the FabV ENR from <italic>Yersinia pestis</italic>(YpFabV). Here, steady-state kinetic analysis revealed that YpFabV catalyzes substrate reduction via a random bi-bi mechanism. Site-directed mutagenesis at the N-terminal end of the helical substrate binding loop revealed that residue T276 plays a key role in substrate specificity and catalytic efficiency. Kinetic analysis and X-ray crystallographic structures demonstrated that the hydroxyl side chain of T276 is essential for hydrogen bonding interactions with NADH, while the methyl group provides favorable hydrophobic interactions with the acyl-coenzyme A (CoA) substrate. Our studies also revealed that alteration of the substrate binding mechanism through site-directed mutagenesis may affect the mode of inhibition of YpFabV. Structure-activity relationship (SAR) studies on the FabI ENR isoenzymes have been used as a platform to determine how slow binding inhibitors effect the transition and ground states of the drug-target binary complex. In turn, we were able to use rational inhibitor design to translate the slow-onset inhibition mechanism from FabI to FabV. Steady-state kinetic analysis of the P142W YpFabV mutant revealed a gain of slow-onset inhibition for an inhibitor (PT156) that displays rapid-reversible binding kinetics for the wild-type enzyme. This is the first example of slow-onset inhibition of a FabV ENR. | |
| dcterms.available | 2017-09-20T16:52:03Z | |
| dcterms.contributor | Carrico, Isaac | en_US |
| dcterms.contributor | Tonge, Peter J | en_US |
| dcterms.contributor | Parker, Kathlyn | en_US |
| dcterms.contributor | Seeliger, Jessica | en_US |
| dcterms.contributor | Garcia-Diaz, Miguel. | en_US |
| dcterms.creator | Neckles, Carla Maya | |
| dcterms.dateAccepted | 2017-09-20T16:52:03Z | |
| dcterms.dateSubmitted | 2017-09-20T16:52:03Z | |
| dcterms.description | Department of Chemistry. | en_US |
| dcterms.extent | 171 pg. | en_US |
| dcterms.format | Monograph | |
| dcterms.format | Application/PDF | en_US |
| dcterms.identifier | http://hdl.handle.net/11401/77137 | |
| dcterms.issued | 2014-12-01 | |
| dcterms.language | en_US | |
| dcterms.provenance | Made available in DSpace on 2017-09-20T16:52:03Z (GMT). No. of bitstreams: 1
Neckles_grad.sunysb_0771E_12052.pdf: 2276697 bytes, checksum: 173f7e3ed7df85a6a095bb03407b3370 (MD5)
Previous issue date: 1 | en |
| dcterms.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dcterms.subject | Chemistry | |
| dcterms.subject | Chemical Biology, Enzyme Kinetics | |
| dcterms.title | Mechanism and Inhibition of the Enoyl-ACP Reductases from Biodefense and Emerging Opportunistic Pathogens | |
| dcterms.type | Dissertation | |
