Strained intermediates have been involve as playing a crucial role in the conversion of the binding energy between enzymes and substrates to lowering the activation energy of the catalyzed reaction. In spite of the central role these intermediates may play, they have rarely been spectroscopically observed or the degree of strain quantified. Strain can by identified by changes in the vibrations spactrum of the bound substrate. Attempts to identify and quantify this strain have been limited to studies where the spectroscopic background of the protein is not overwhelming. Isotope effects are also sensitive to chances in vibrational frequencies, and consequently can detect electronic charge redistribution, and/or steric strain that is placed on a molecule. By measuring the binding isotope effect at equilibrium, strain in the lowest enzyme substrate complex will be detected. Being able to determine where strain is located in a substrate molecule will aid the design of improved inhibitors designed to relieve the strain but maintain the other essential binding features of the enzyme reactions. Carbanions of CoA thioesters are believed to be strained intermediates in a wide range of biosynthetic and fatty acid metabolizing enzymes. Inhibitors and inactivators of these enzymes have diverse bioactivities, ranging from antibiotics to hypocholesteremics. The mechanistic information available on the existence of these intermediates and on the factors promoting stabilization of the enzyme bound intermediates is unknown. Dithioesters of can provide a spectroscopic probe, both ultraviolet and resonance Raman, of that nature of the intermediates and the ability of this class of enzymes to strain these substrates. Alpha-Halo CoA dithioesters are more reactive than the alpha- halothioester analogs that can potentially alkylate the enzymatic general base that promotes ionization of the CoA esters. These novel reagants will be useful in mechanistic studies of the carbanion forming enzymes and as extremely potent inhibitors or inactivators may prove to be useful pharmaceutical, themselves.

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Anderson, Vernon E (2005) Quantifying energetic contributions to ground state destabilization. Arch Biochem Biophys 433:27-33
Bahnson, Brian J; Anderson, Vernon E; Petsko, Gregory A (2002) Structural mechanism of enoyl-CoA hydratase: three atoms from a single water are added in either an E1cb stepwise or concerted fashion. Biochemistry 41:2621-9
Liu, Binqiu; Wang, Yingqiang; Fillgrove, Kerry L et al. (2002) Triclosan inhibits enoyl-reductase of type I fatty acid synthase in vitro and is cytotoxic to MCF-7 and SKBr-3 breast cancer cells. Cancer Chemother Pharmacol 49:187-93
Fillgrove, K L; Anderson, V E (2001) The mechanism of dienoyl-CoA reduction by 2,4-dienoyl-CoA reductase is stepwise: observation of a dienolate intermediate. Biochemistry 40:12412-21
Goshe, M B; Chen, Y H; Anderson, V E (2000) Identification of the sites of hydroxyl radical reaction with peptides by hydrogen/deuterium exchange: prevalence of reactions with the side chains. Biochemistry 39:1761-70
Fillgrove, K L; Anderson, V E (2000) Orientation of coenzyme A substrates, nicotinamide and active site functional groups in (Di)enoyl-coenzyme A reductases. Biochemistry 39:7001-11
Baker-Malcolm, J F; Lantz, M; Anderson, V E et al. (2000) Novel inactivation of enoyl-CoA hydratase via beta-elimination of 5, 6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA. Biochemistry 39:12007-18
Kean, E L; Wei, Z; Anderson, V E et al. (1999) Regulation of the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol, feedback and product inhibition. J Biol Chem 274:34072-82
Fillgrove, K L; Anderson, V E; Mizugaki, M (1999) Cloning, expression, and purification of the functional 2,4-dienoyl-CoA reductase from rat liver mitochondria. Protein Expr Purif 17:57-63
Fedoriw, A M; Liu, H; Anderson, V E et al. (1998) Equilibrium and kinetic parameters of the sequence-specific interaction of Escherichia coli RNA polymerase with nontemplate strand oligodeoxyribonucleotides. Biochemistry 37:11971-9

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