Study of substrate-enzyme interaction between immobilized pyridoxamine and recombinant porcine pyridoxal kinase using surface plasmon resonance biosensor.
Pyridoxal kinase (PK) is an important enzyme involved in bioactivation of vitamin B(6). Binding of PK with its substrate is the prerequisite step for the subsequent catalytic phosphorylation of the substrate. In the present study, a surface plasmon resonance biosensor (BIAcore) was employed to characterize the binding interaction between wild-type porcine PK and an immobilized substrate, pyridoxamine. Pyridoxamine was modified with 11-mercaptoundecanic acid and immobilized on a sensor chip through the formation of a self-assembled monolayer. The binding of PK to the immobilized pyridoxamine was followed in real time and the kinetic parameters were derived from non-linear analysis of the sensorgram. The effects of buffer pH, monovalent cations (Na(+), K(+)) and divalent cations (Mn(2+), Zn(2+), Mg(2+)) on the binding kinetics were determined. Optimal pH for PK-pyridoxamine interaction in the absence of divalent ions is at around 7.4. While K(+) increased and Na(+) decreased the binding affinity (K(A)) of PK to immobilized pyridoxamine, all divalent cations increased the K(A) of PK for pyridoxamine. Solution phase affinity measurement based on a competitive binding assay was used to determine the affinities of PK for different vitamin B(6) analogues. The order of affinity of PK for different analogues is: pyridoxal-oxime>pyridoxine>pyridoxamine>pyridoxal>pyridoxal phosphate. This is the first study to demonstrate that buffer conditions such as pH and concentration of monovalent and/or divalent ions can directly alter the binding of PK for its substrates. The quantitative kinetic and thermodynamic parameters obtained by SPR measurement provide the insight information into the catalytic activity of this enzyme.
Fong
Chi-Chun
CC
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong.
Lai
Wan-Ping
WP
Leung
Yun-Chung
YC
Lo
Samuel C-L
SC
Wong
Man-Sau
MS
Yang
Mengsu
M
D000818:Animals; D001667:Binding, Competitive; D002413:Cations, Divalent; D002414:Cations, Monovalent; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011731:Pyridoxal Kinase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011733:Pyridoxamine / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013552:Swine; D013816:Thermodynamics
D002413:Cations, Divalent; D002414:Cations, Monovalent; D011994:Recombinant Proteins; D011733:Pyridoxamine; D011731:Pyridoxal Kinase