Acyl-CoA Dehydrogenases : mechanistic studies on Medium Chain Acyl-CoA Dehydrogenase

Abstract

Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the a,b-dehydrogenation of fatty acid acyl-CoA thioesters. Medium chain acyl-CoA dehydrogenase (MCAD) is one of the best-studied members of this family. The a,b-dehydrogenation reaction involves the concerted C-H bonds cleavage of the substrate. First, the active site base, Glu376-COO-, removes a proton by and then a hydride is transferred to the flavin N(5) position of FAD. In my thesis MCAD several mechanistic details of the dehydrogenation reaction for MCAD were investigated. For this, among other things, a mutant of MCAD was created, which carries a C-terminal "His Tag". Addition of affinity His Tag facilitates purification of recombinant MCAD. For the investigation of the mechanism above several E376- or/and E99-MCAD mutants were used. Last one received an earlier attention since the Glu99 is located underneath of the active site of MCAD. This residue affects ionizations inside the active center cavity. Many studies were focused on E376Q-MCAD mutant. This mutant was highly inactive, because the glutamine does not play the role of the base. However its residual activity is 1/100000 of that of wtMCAD. This is a small value, but has the same order of magnitude as those found in non-catalyzed reactions. Proton inventory technique was suitable for mechanistic study of this mutant. Apart from this, it was observed that the log of rates of dehydrogenation increases linearly with the pH suggesting HO- as a reactant. A similar dependence was observed with Glu376Gln+Glu99Gly-MCAD. Thus, activity and reduction studies exclude Glu99 as a candidate for proton abstraction in the first step of dehydrogenation. E376Q-MCAD mutant reflected a large unexpected solvent isotope effect of approx. 8.5. The large isotope effects resulted from proton inventory experiments are attributed to the change in state of several H-bonds that occur during the process. A further investigation concerns the role of a special H-bond between N(5) of the flavin cofactor and Thr168-OH. However, an amino acid functional group that forms such a H-bond is strictly conserved in the ACAD familily (Thr or Ser). In the absence of this H-bond (T168A-MCAD) two effects could be observed: a) electronic influence on the substrate activation as well as on the redox potential of the flavin; b) steric - this H-bond is involved in the fine-tuning of the orientation of the flavin cofactor and ligand. Another threonine residue (Thr136) modulates the redox potential of the flavin (approx. -30 mV compared to wtMCAD 1.4 Kcal M-1). Thus e.g. with the Thr136Ala mutant the cofactor was partially reduced by the substrate, which is attributed to decrease of the redox potential. These experiments were supported by theoretical calculations, which were accomplished by Olga Dmitrenko working at Univ. of Delaware (USA) in Prof. R. Bach group.