Study of substrate-enzyme interaction between immobilized pyridoxamine and recombinant porcine pyridoxal kinase using surface plasmon resonance biosensor.

摘要: Abstract Pyridoxal kinase (PK) is an important enzyme involved in bioactivation of vitamin B6. Binding PK with its substrate the prerequisite step for subsequent catalytic phosphorylation substrate. In present study, a surface plasmon resonance biosensor (BIAcore) was employed to characterize binding interaction between wild-type porcine and immobilized substrate, pyridoxamine. Pyridoxamine modified 11-mercaptoundecanic acid on sensor chip through formation self-assembled monolayer. The pyridoxamine followed real time kinetic parameters were derived from non-linear analysis sensorgram. effects buffer pH, monovalent cations (Na+, K+) divalent (Mn2+, Zn2+, Mg2+) kinetics determined. Optimal pH PK–pyridoxamine absence ions at around 7.4. While K+ increased Na+ decreased affinity (KA) pyridoxamine, all KA Solution phase measurement based competitive assay used determine affinities different B6 analogues. order analogues is: pyridoxal-oxime>pyridoxine>pyridoxamine>pyridoxal>pyridoxal phosphate. This first study demonstrate that conditions such as concentration and/or can directly alter substrates. quantitative thermodynamic obtained by SPR provide insight information into activity this enzyme.