The structure of catalytic domain on pyridoxal kinase purified from pig brain was investigated by using a bifunctional synthetic inhibitor D-ATP-PM and a photoaffinity labeling reagent, N-4-azido-2-nitrophenyl-pyridoxal (NANP). D-ATP-PM is a competitive inhibitor with respect to ATP ($K_i=3;{mu}M$) and it also behaves as a strong competitive inhibitor respect to pyridoxal ($K_i=4;{mu}M$). These behavior suggests that D-ATP-PM acts as a bifunctional inhibitor which recognizes both nucleotide and pyridoxal binding site of the kinase. Thus the binding of the binary inhibitor D-ATP-PM may be discussed in reference to a model which assumes that the two substrate binding sites, located on different domains, are in close proximity. A bulky P-pyridoxamine derivative, N-4-azido-2-nitrophenyl-pyridoxal (NANP) recognizes the binding site of pyridoxal moiety of the enzyme. Upon illumination, the arylazide of NANP acts as an efficient photolabeling reagent of the kinase. A characteristic feature of the photolabeling reagent, which has ability to recognize the substrate binding site, can be exploited to ascertain the chemical nature of amino acid residues at the catalytic domain