Scholz G, Kwok F
School of Pharmacy, South Australian Institute of Technology, Adelaide.
J Biol Chem. 1989 Mar 15;264(8):4318-21.
4-Benzoylbenzoic acid inhibits pyridoxal kinase activity competitively with respect to pyridoxal. The Ki was determined to be 5 x 10(-5) M. Binding studies showed that 4-benzoylbenzoic acid bound to pyridoxal kinase at a 1:1 molar ratio and with a dissociation constant (Kd) of 5.9 x 10(-5) M. Photoirradiation of pyridoxal kinase in the presence of a 10-fold excess of 4-benzoylbenzoic acid at pH 6.5 resulted in an irreversible loss of enzymatic activity; this photoinactivation was prevented by the presence of pyridoxal. Amino acid analysis revealed that 1 tyrosine residue/subunit was modified during photoinactivation. The presence of a tyrosine residue at the active site of pyridoxal kinase was confirmed by reaction with tetranitromethane. In the presence of 1 x 10(-4) M tetranitromethane, a complete loss of the kinase activity was observed after incubation at 25 degrees C for 8 min, with modification of a total of 3 tyrosine residues. The second-order rate constant (K2) of the reaction between the tyrosine residues and tetranitromethane was determined to be 53.3 s-1 M-1.
4-苯甲酰苯甲酸相对于吡哆醛竞争性抑制吡哆醛激酶活性。测定其抑制常数（Ki）为5×10⁻⁵ M。结合研究表明，4-苯甲酰苯甲酸以1:1的摩尔比与吡哆醛激酶结合，解离常数（Kd）为5.9×10⁻⁵ M。在pH 6.5条件下，存在10倍过量的4-苯甲酰苯甲酸时对吡哆醛激酶进行光照射，导致酶活性不可逆丧失；吡哆醛的存在可防止这种光失活。氨基酸分析表明，光失活过程中每个亚基有1个酪氨酸残基被修饰。通过与四硝基甲烷反应证实了吡哆醛激酶活性位点存在酪氨酸残基。在存在1×10⁻⁴ M四硝基甲烷的情况下，25℃孵育8分钟后观察到激酶活性完全丧失，共修饰了3个酪氨酸残基。酪氨酸残基与四硝基甲烷之间反应的二级速率常数（K2）测定为53.3 s⁻¹ M⁻¹。
