Kwok F, Churchich J E
J Biol Chem. 1979 Jul 25;254(14):6489-95.
Pyridoxal kinase has been purified 2,000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Pyridoxal kinase, 60,000 molecular weight, catalyzes the phosphorylation of pyridoxal (Km = 2.5 x 10(-5) M) and pyridoxine (Km = 1.7 x 10(-5) M). Pyridoxamine is not a substrate of the purified kinase. Irradiation of the kinase in the presence of riboflavin leads to irreversible loss of catalytic activity. Riboflavin binds to the kinase with a KD = 5 microM as shown by fluorometric titrations. Singlet excited oxygen, generated by energy transfer from the lowest triplet of riboflavin to oxygen, acts as the oxidizing agent of approximately one histidine residue per mol of enzyme. The amino acid residues tyrosine, tryptophan, and cysteine are not photooxidized by the sensitizer bound to the enzyme. It is postulated that histidine is involved in the binding of the substrate ATP to the catalytic site of pyridoxal kinase.
吡哆醛激酶已从猪脑中纯化了2000倍。该酶制剂在分析凝胶电泳上作为单一蛋白质和活性条带迁移。分子量为60,000的吡哆醛激酶催化吡哆醛（Km = 2.5×10^(-5) M）和吡哆醇（Km = 1.7×10^(-5) M）的磷酸化。吡哆胺不是纯化激酶的底物。在核黄素存在下对激酶进行辐照会导致催化活性的不可逆丧失。荧光滴定表明，核黄素与激酶结合的KD = 5 microM。通过从核黄素的最低三线态到氧的能量转移产生的单线态激发氧，作为每摩尔酶约一个组氨酸残基的氧化剂。与酶结合的敏化剂不会使酪氨酸、色氨酸和半胱氨酸氨基酸残基发生光氧化。据推测，组氨酸参与底物ATP与吡哆醛激酶催化位点的结合。
