Scholz G, Kwok F, Churchich J E
Department of Biochemistry, University of Tennessee, Knoxville 37996-8040.
Eur J Biochem. 1990 Oct 24;193(2):479-84. doi: 10.1111/j.1432-1033.1990.tb19362.x.
The binding of pyridoxal analogues to the structural domains of pyridoxal kinase was studied by fluorescence spectroscopy and chromatographic techniques. Two fragments of 24 and 16 kDa, arising from limited proteolysis of the native enzyme, were separated by ion-exchange chromatography and used for binding studies with pyridoxal oxime. Fluorometric titrations yielded dissociation constants of 6 and 12.4 MicroM for pyridoxal oxime bound to the native enzyme and 24-kDa fragment, respectively. 4-(4-Azido-2-nitrophenyl)-pyridoxamine, a new photolabeling reagent, binds irreversibly to the kinase with concomitant loss of catalytic activity. The modified kinase (2.1 mol label/mol dimer) yields two fragments upon limited proteolysis with chymotrypsin. The two fragments were separated by reverse-phase HPLC and SDS/polyacrylamide gel electrophoresis. Radiolabeled ligand was detected only in the 24-kDa fragment. It is postulated that the pyridoxal binding site is located in the 24-kDa structural domain.
通过荧光光谱法和色谱技术研究了吡哆醛类似物与吡哆醛激酶结构域的结合。通过离子交换色谱法分离了天然酶有限蛋白水解产生的两个分别为24 kDa和16 kDa的片段，并用于与吡哆醛肟的结合研究。荧光滴定法得出吡哆醛肟与天然酶和24 kDa片段结合的解离常数分别为6 μM和12.4 μM。一种新的光标记试剂4-(4-叠氮基-2-硝基苯基)-吡哆胺不可逆地与激酶结合，同时伴随催化活性丧失。经胰凝乳蛋白酶有限蛋白水解后，修饰后的激酶（2.1摩尔标记/摩尔二聚体）产生两个片段。通过反相高效液相色谱法和SDS/聚丙烯酰胺凝胶电泳分离了这两个片段。仅在24 kDa片段中检测到放射性标记配体。据推测，吡哆醛结合位点位于24 kDa结构域中。
