The Role of N299α in the Catalytic Mechanism of Clostridium sticklandii Lysine 5,6-aminomutase

• Main Authors: Yu-Chiang Pan, 潘宇將
• Other Authors: Shyue-Chu Ke
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/07066093277125318655

碩士 === 國立東華大學 === 物理學系 === 99 === Lysine 5,6-aminomutase (5,6-LAM) from Clostridium sticklandii is an adenosylcobalamin (coenzyme B12) and pyridoxal-5’-phosphate (PLP) dependent enzyme that catalyzes a 1,2 rearrangement of the terminal amino group of DL-lysine and L-β-lysine. 5, 6-LAM consists of α and β subunits and forms a α2β2-tetramer. Coenzyme B12 binds to H133β residue of 5,6-LAM and is capable of catalyzing challenging chemical reactions via homolysis of Co-C bond followed by generation of 5’-deoxyadenosyl radical which abstracts H-atom from the substrate. Coenzyme PLP binds to K144β residue of 5,6-LAM and may play a role in stabilizing a high-energy radical intermediate through a conjugated π-electron system, in which unpaired electron spin densities originate from the radical center C4 covalently transferred to the pyridine ring of PLP. This is revealed by EPR and ENDOR spectra and is supported by DFT calculation. The PLP hydroxyl group is also expected as an important participant in this reaction. In 5, 6-LAM, N299α has a hydrogen-bonding interaction with the phenolic oxygen of coenzyme PLP. Whether this interaction directly involves in the catalytic turn over or plays a role in confining the PLP stereospecifically is unclassified and is a subject of extreme interest. To address this issue, we studied a series of mutants by kinetic measurements, UV-visible absorption and EPR techniques. We found that removal of the hydrogen-bonding interaction between residue N299 and coenzyme PLP by N299D mutation results in a significant decrease in enzymatic activity, as revealed by HPLC kinetic measurements. The similarities of EPR spectra, and the derived spin-Hamiltonian parameters, between N299D and wild type 5, 6-LAM in reaction with 4-thialysine suggest that the N299D mutation does not considerably alter the relative geometrical arrangement between PLP-substrate complex and coenzyme B12. In conclusion, our work suggests that the major cause dramatic declined activity of N299D mutant is not from confining the PLP stereospecifically, but from direct involvement in the catalytic turning over of reaction.