Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 2157-2164 |
| Number of pages | 8 |
| Journal | Bioorganic and Medicinal Chemistry |
| Volume | 5 |
| Issue number | 12 |
| DOIs | |
| Publication status | Published - 1997 Dec 1 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Medicine
- Molecular Biology
- Pharmaceutical Science
- Drug Discovery
- Clinical Biochemistry
- Organic Chemistry
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Redesigning the active-site of an acyl-CoA dehydrogenase : New evidence supporting a one-base mechanism. / Dakoji, Srikanth; Shin, Injae; Battaile, Kevin P.; Vockley, Jerry; Liu, Hung Wen.
In: Bioorganic and Medicinal Chemistry, Vol. 5, No. 12, 01.12.1997, p. 2157-2164.Research output: Contribution to journal › Article
TY - JOUR
T1 - Redesigning the active-site of an acyl-CoA dehydrogenase
T2 - New evidence supporting a one-base mechanism
AU - Dakoji, Srikanth
AU - Shin, Injae
AU - Battaile, Kevin P.
AU - Vockley, Jerry
AU - Liu, Hung Wen
PY - 1997/12/1
Y1 - 1997/12/1
N2 - 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.
AB - 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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UR - http://www.scopus.com/inward/citedby.url?scp=0031438569&partnerID=8YFLogxK
U2 - 10.1016/S0968-0896(97)00159-4
DO - 10.1016/S0968-0896(97)00159-4
M3 - Article
C2 - 9459013
AN - SCOPUS:0031438569
VL - 5
SP - 2157
EP - 2164
JO - Bioorganic and Medicinal Chemistry
JF - Bioorganic and Medicinal Chemistry
SN - 0968-0896
IS - 12
ER -