Probing Hydrogen-Bonding Interactions in the Active Site of Medium-Chain Acyl-CoA Dehydrogenase Using Raman Spectroscopy

• Published in: Biochemistry (Easton) Vol. 42; no. 40; pp. 11846 - 11856
• Main Authors: Wu, Jiaquan, Bell, Alasdair F, Luo, Lian, Stephens, Avery W, Stankovich, Marian T, Tonge, Peter J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 14.10.2003
• Subjects: Acyl Coenzyme A - metabolism; Acyl-CoA Dehydrogenase - chemistry; Acyl-CoA Dehydrogenase - genetics; Acyl-CoA Dehydrogenase - metabolism; Animals; Binding Sites - genetics; Catalysis; Flavin-Adenine Dinucleotide - analogs & derivatives; Flavin-Adenine Dinucleotide - metabolism; Glutamic Acid - genetics; Glutamine - genetics; Hydrogen Bonding; Kinetics; Mutagenesis, Site-Directed; Potentiometry; Proline - genetics; Protons; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Spectrophotometry, Ultraviolet; Spectrum Analysis, Raman - methods; Swine
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0344578

The role of the oxyanion hole in the reaction catalyzed by pig medium-chain acyl-CoA dehydrogenase (pMCAD) has been investigated using enzyme reconstituted with 2‘-deoxy-FAD. The k cat (18.8 ± 0.5 s-1) and K m (2.5 ± 0.4 μM) values for the oxidation of n-octanoyl-CoA (C8-CoA) by WT pMCAD recombinantly expressed in Escherichia coli are similar to those of native pMCAD isolated from pig kidney. In agreement with previous studies [Engst et al. (1999) Biochemistry 38, 257−267], reconstitution of the WT enzyme with 2‘-deoxy-FAD causes a large (400-fold) decrease in k cat but has little effect on K m. To investigate the molecular basis for the alterations in activity resulting from changes in hydrogen bonding between the substrate and the enzyme's oxyanion hole, the structure of the product analogue hexadienoyl-CoA (HD-CoA) bound to the 2‘-deoxy-FAD-reconstituted enzyme has been probed by Raman spectroscopy. Importantly, while WT pMCAD causes a 27 cm-1 decrease in the vibrational frequency of the HD enone band, from 1595 to 1568 cm-1, the enone band is only shifted 10 cm-1 upon binding HD-CoA to 2‘-deoxy-FAD pMCAD. Thus, removal of the 2‘-ribityl hydroxyl group results in a substantial reduction in the ability of the enzyme to polarize the ground state of the ES complex. On the basis of an analysis of a similar system, it is estimated that ground state destabilization is reduced by up to 17 kJ mol-1, while the activation energy for the reaction is raised 15 kJ mol-1. In addition, removal of the 2‘-ribityl hydroxyl reduces the redox potential shift that is induced by HD-CoA binding from 18 to 11 kJ mol-1. Consequently, while ligand polarization caused by hydrogen bonding in the oxyanion hole is intimately linked to substrate turnover, additional factors must be responsible for ligand-induced changes in redox potential. Finally, while replacement of the catalytic base E376 with Gln abolishes the ability of the enzyme to catalyze substrate oxidation and to catalyze the exchange of the C8-CoA α-protons with solvent deuterium, the 2‘-deoxy-FAD-reconstituted enzyme catalyzes α-proton exchange at a rate (k exc) of 0.085 s-1, which is only 4-fold slower than k exc for WT pMCAD (0.35 s-1). Thus, either the oxyanion hole plays only a minor role in stabilizing the transition state for α-proton exchange, in contrast to its role in substrate oxidation, or the value of k exc for WT pMCAD reflects a process such as exchange of the E376 COOH proton with solvent.