Spatial arrangement of coenzyme and substrates bound to L-3-hydroxyacyl-CoA dehydrogenase as studied by spin-labeled analogues of NAD+ and CoA.
The synthesis of nitroxide spin-labeled derivatives of S-acetoacetyl-CoA, S-acetoacetylpantetheine, and S-acetoacetylcysteamine is described. These compounds are active substrates of L-3-hydroxyacyl-CoA dehydrogenase [(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.35] exhibiting vmax values from 20% to 70% of S-acetoacetyl-CoA itself. S-Acetoacetylpantetheine and S-acetoacetylcysteamine form binary complexes with the enzyme and exhibit ESR spectra typical for immobilized nitroxides. In the case of spin-labeled pantetheine, the radical is more mobile. When spin-labeled substrates are bound simultaneously to each active site of this dimeric enzyme, spin-spin interactions differentiate between two alternate orientations of the substrate [Birktoft, J.J., Holden, H.M., Hamlin, R., Xuong, N.H., & Banaszak, L.J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8262-8266]. The fatty acid moiety is thought to be located in a cleft between two domains whereas a large part of the CoA moiety probably extends into the solution. NAD+, spin-labeled at N6 of the adenine ring, is an active coenzyme of L-3-hydroxyacyl-CoA dehydrogenase (60% vmax). Complexes with the enzyme exhibit ESR spectra typical of highly immobilized nitroxides. Binding of coenzyme NAD+ causes conformational changes of the binary enzyme/substrate complex as revealed by changes in the ESR spectrum of spin-labeled S-acetoacetylpantetheine.
Hartmann
D
D
Fachbereich Chemie, Universität Kaiserslautern, Kaiserslautern, Federal Republic of Germany.
Philipp
R
R
Schmadel
K
K
Birktoft
J J
JJ
Banaszak
L J
LJ
Trommer
W E
WE
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003065:Coenzyme A / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007700:Kinetics; D008958:Models, Molecular; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels; D013552:Swine
D013113:Spin Labels; D009243:NAD; D015094:3-Hydroxyacyl CoA Dehydrogenases; D003065:Coenzyme A