Bioorganic and Medicinal Chemistry p. 2157 - 2164 (1997)
Update date:2022-07-30
Topics:
Dakoji, Srikanth
Shin, Injae
Battaile, Kevin P.
Vockley, Jerry
Liu, Hung-Wen
The acyl-CoA dehydrogenases are a family of related enzymes that share high structural homolog and a common catalytic mechanism which involves abstraction of an α-proton from the substrate by an active site glutamate residue. Several lines of investigation have shown that the position of the catalytic glutamate is conserved in most of these dehydrogenases (the E2 site), but is in a different location in two other family members (the E1 site). Using site specific in vitro mutagenesis, a double mutant rat short chain acyl-CoA dehydrogenase (rSCAD) has been constructed in which the catalytic glutamate is moved from the E2 to the E1 site (Glu368Gly/Gly247Glu). This mutant enzyme is catalytically active, but utilizes substrate less efficiently than the native enzyme (K(m) = 0.6 and 2.0 μM, and V(max) = 2.8 and 0.3 s-1 for native and mutant enzyme respectively)). In this study we show that both the wild-type and mutant rSCADs display identical stereochemical preference for catalysis-abstraction of the α-H(R) from the substrate followed by transfer of the β-H(R) to the FAD coenzyme. These results, in conjunction with molecular modeling of the native and double mutant SCAD indicate that the catalytic base in the E1 and E2 sites are topologically similar and catalytically competent. However, analysis of the 1H NMR spectra of the incubation products of these two enzymes revealed that, in contrast to the wild-type rSCAD, the Gly368Glu/Gly247Glu rSCAD could not perform γ-proton exchange of the product with the solvent, a property inherent to most acyl-CoA dehydrogenases. It is evident that the base in the mutant enzyme has access to the α-H(R) but is far removed from the γ-Hs. These findings provide further support for a one base mechanism of α- and γ-reprotonation/deprotonation catalysis by acyl-CoA dehydrogenases.
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