Dominici P, Scholz G, Kwok F, Churchich J E
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
J Biol Chem. 1988 Oct 15;263(29):14712-6.
Pyridoxal kinase is inactivated by preincubation with the affinity label reagent adenosine tetraphosphate pyridoxal (AP4-PL) at a mixing molar ratio of 5:1 AP4-PL contains structural features of the substrates pyridoxal and ATP. The substrate ATP affords substantial protection against inactivation. The extent of chemical modification by the affinity label was determined by measuring the spectroscopic properties of AP4-pyridoxyl chromophores attached to the enzyme after reduction with NaBH4. The incorporation of 2 mol of the affinity label per enzyme dimer is needed for complete inactivation of the kinase. After chymotryptic digestion of the enzyme modified with AP4-PL and reduced with tritiated NaBH4, only one radioactive peptide absorbing at 325 nm was separated by reverse-phase high performance liquid chromatography. The amino acid sequence of the radioactive peptide, elucidated by Edman degradation, revealed that a specific lysyl residue of monomeric pyridoxal kinase has reacted with the affinity label reagent. It is postulated that the modified lysyl residue is involved in direct interactions with phosphoryl groups of ATP.
吡哆醛激酶与亲和标记试剂四磷酸腺苷吡哆醛（AP4-PL）以5:1的混合摩尔比预孵育后会失活。AP4-PL含有底物吡哆醛和ATP的结构特征。底物ATP可提供显著的抗失活保护。通过测量用NaBH4还原后附着在酶上的AP4-吡哆醛发色团的光谱特性，来确定亲和标记的化学修饰程度。激酶完全失活需要每个酶二聚体掺入2摩尔亲和标记。在用AP4-PL修饰并用氚化NaBH4还原的酶经胰凝乳蛋白酶消化后，通过反相高效液相色谱仅分离出一个在325nm处有吸收的放射性肽段。通过埃德曼降解法阐明的放射性肽段的氨基酸序列表明，单体吡哆醛激酶的一个特定赖氨酰残基已与亲和标记试剂发生反应。据推测，修饰的赖氨酰残基参与了与ATP磷酸基团的直接相互作用。
