Research
 

        Many essential biochemical energy-transducing reactions are electron-transfer reactions; e.g. those catalyzing the fatty acid metabolism and the detoxification of aromatic substances in the human body and in the environment.  We have developed an analytical tool, the spectroelectrochemical cell, that enables us to measure the formal redox potential (Eº') and number of electrons transferred (n) for electron transferring enzymes.  With this data, we can build and test electron transfer mechanisms for the enzymes.

Figure 1 shows the b-oxidation of fatty acids

       With spectroelectrochemical studies we can ask fundamental questions about substrate- enzyme interaction. Important information comes in the form of the active site induced polarization of the substrate itself. A careful analysis of the system can relate this information to the substrate binding and finally to the enzyme catalysis process. Although the relation between substrate polarization, its binding and subsequent activation of an enzyme is complex, a suitable methodology will be to use other small molecule analogs to observe these substrate/protein polarization and its effect on sustrate binding and catalysis. We use synthetic organic chemistry to build novel molecules which are either substrate or product analogs to probe these enzymes. The redox, kinetic and spectroscopic properties of these small molecule-enzyme are analyzed and computational modeling studies are performed to see a structure-function relationship of these enzymes. We also attempt to probe the effect of mutation in the active site or the surrounding and observe its effect on the electron-transfer mechanisms.
 

Medium Chain Acyl CoA Dehydrogenase (MCAD)
Effects of Substituted Substrate Analogs on the Mechanism of Medium-Chain Acyl-CoA Dehydrogenase

       Medium-chain acyl-CoA dehydrogenase (MCAD) belongs to a group of flavin adenine dinucleotide (FAD) containing enzymes, known as the fatty acyl-CoA dehydrogenases (ACDs), which catalyze the first step of the b-oxidation cycle, converting a fatty acyl-CoA thioester to a trans-a, b-enoyl-CoA thioester via a two-electron oxidation (see Figure 1). These electrons are initially stored on the flavin of MCAD and later transferred to electron transferring flavoprotein (ETF) and are ultimately funneled into the mitochondrial respiratory chain and converted to adenosine triphosphate (ATP).  Disorders of the ACDs have been linked to Sudden Infant Death Syndrome (SIDS), Jamaican Vomiting Sickness (JVS), riboflavin deficiency, and other metabolic disorders.
       Although redox data has yielded insight into the MCAD catalyzed reaction mechanism, it is unclear if the reaction proceeds as stepwise or concerted. Studies utilizing human wild-type MCAD (hMCAD) have revealed that the dehydrogenation reaction proceeds via a proi-R-proton abstraction by the active site of the catalytic base, Glu376, and a b-hydride transfer to the N(5) position of the flavin.
        Upon binding to the enzyme, the substrate is polarized.   Partial negative charge at the a-carbon and partial positive charge at the b-carbon could form.  The polarization facilitates the removal of  a-proton and the transfer b-hydride.   We are using computation and fluorinated analogs to probe the enzyme mechanism. Recently computation has shown that the reaction exhibits a stepwise mechanism, but the potential energy height of the two steps is about equal and relatively low, so that both steps control the rate of the overall reaction. If the mechanism is truly stepwise, an enolic intermediate, after deprotonation of the a-proton,  should occur.
        Slow or inactive fluorinated substrate analogs, where the a-proton is selectively replaced with a fluorine atom, will be used to study the nature of the reaction mechanism.  The electronically perturbed substrates should slow the reaction rate and allow for the identification of intermediates.  Future work with the S-isomer as a slow substrate will be needed to identify a possible intermediate.  Stopped-flow kinetic studies and 19F NMR experiments will be designed to probe for this intermediate.  The kinetically dead R-isomer will be studied using spectroelectrochemistry and off-resonance Raman spectroscopy to investigate substrate binding and polarization.
 

Figure 2 represents the potential modulations induced by enzyme/ligand complexation
The thin lines represent data for MCAD binding to T3F-CoA. The thick lines are related to the natural substrate/product couple and its expected potential alterations
 

Lamm et al Arch. Biochem. Biophys., 2002, 404, 136-146.

Short Chain Acyl CoA Dehydrogenase (SCAD)
Investigating Thermodynamic Regulation in HumanShort-Chain Acyl-CoA Dehydrognase
 
 

        Human short-chain acyl-CoA dehydrogenase (hSCAD) is a flavoprotein that catalyzes the first step in the b-oxidation cycle, which produces up to 40% of the total human energy requirement. Previous work with acyl-CoA dehydrogenases (ACDs) has shown that these enzymes are specifically modulated upon binding of the substrate/product couple, allowing the reaction to proceed in a thermodynamically favorable manner. Deficiencies in hSCAD can lead to serious metabolic disorders, due to inhibition of energy production. Two nucleotide variations in the hSCAD gene, G625A and C511T, have been identified and associated with SCAD deficiency. We are investigating the redox properties of these mutants through spectroelectrochemistry.  A kinetically dead mutant is also being probed to examine the effects of substrate and product binding on redox potentials.

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