Crystal Structures of the Wild Type and the Glu376Gly/Thr255Glu Mutant of Human Medium-Chain Acyl-CoA Dehydrogenase:  Influence of the Location of the Catalytic Base on Substrate Specificity

• Published in: Biochemistry (Easton) 1996-09, Vol.35 (38), p.12412-12420
• Main Authors: Lee, Hyun-Joo K, Wang, Ming, Paschke, Rosemary, Nandy, Andreas, Ghisla, Sandro, Kim, Jung-Ja P
• Format: Article
• Language: English
• Subjects: Acyl Coenzyme A - chemistry; Acyl Coenzyme A - metabolism; Acyl-CoA Dehydrogenase; Acyl-CoA Dehydrogenase, Long-Chain - chemistry; Acyl-CoA Dehydrogenase, Long-Chain - genetics; Acyl-CoA Dehydrogenase, Long-Chain - metabolism; Animals; Binding Sites; Crystallography, X-Ray; Glutamic Acid - metabolism; Humans; Hydrogen Bonding; Kinetics; Models, Molecular; Mutation; Oxidation-Reduction; Oxygen - metabolism; Protein Conformation; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Substrate Specificity; Swine
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9607867

Crystal structures of the wild type human medium-chain acyl-CoA dehydrogenase (MCADH) and a double mutant in which its active center base-arrangement has been altered to that of long chain acyl-CoA dehydrogenase (LCADH), Glu376Gly/Thr255Glu, have been determined by X-ray crystallography at 2.75 and 2.4 Å resolution, respectively. The catalytic base responsible for the α-proton abstraction from the thioester substrate is Glu376 in MCADH, while that in LCADH is Glu255 (MCADH numbering), located over 100 residues away in its primary amino acid sequence. The structures of the mutant complexed with C8-, C12, and C14-CoA have also been determined. The human enzyme structure is essentially the same as that of the pig enzyme. The structure of the mutant is unchanged upon ligand binding except for the conformations of a few side chains in the active site cavity. The substrate with chain length longer than C12 binds to the enzyme in multiple conformations at its ω-end. Glu255 has two conformations, “active” and “resting” forms, with the latter apparently stabilized by forming a hydrogen bond with Glu99. Both the direction in which Glu255 approaches the Cα atom of the substrate and the distance between the Glu255 carboxylate and the Cα atom are different from those of Glu376; these factors are responsible for the intrinsic differences in the kinetic properties as well as the substrate specificity. Solvent accessible space at the “midsection” of the active site cavity, where the Cα−Cβ bond of the thioester substrate and the isoalloxazine ring of the FAD are located, is larger in the mutant than in the wild type enzyme, implying greater O2 accessibility in the mutant which might account for the higher oxygen reactivity.