Churchich J E, Kim Y T
Department of Biochemistry, University of Tennessee, Knoxville 37916.
Ann N Y Acad Sci. 1990;585:357-67. doi: 10.1111/j.1749-6632.1990.tb28068.x.
Chymotryptic digestion of sheep brain pyridoxal kinase, a dimer of identical subunits each of 40 kDa, yields two fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were separated by HPLC and used for binding studies with ATP and pyridoxal analogues. The spectroscopic properties of trinitrophenyl-ATP bound to the 24-kDa fragment are indistinguishable from those of TNP-ATP bound to the native kinase. The small 16-kDa fragment, generated by proteolytic cleavage of the kinase, does not bind any of the analogues. The same pattern of digestion was observed when IAF pyridoxal kinase, carrying a fluorescent probe covalently bound to a specific SH residue, was preincubated with chymotrypsin. The kinetics of proteolysis of IAF-pyridoxal kinase was monitored by emission anisotropy, and the analysis of the initial rate of proteolysis at various concentrations of chymotrypsin reveals that the rate of unfolding of native pyridoxal kinase plays a dominant role in the proteolytic process.
绵羊脑吡哆醛激酶是由两个相同的40 kDa亚基组成的二聚体，经胰凝乳蛋白酶消化后，产生两个分别为24 kDa和16 kDa的片段，同时催化活性丧失。这些片段通过高效液相色谱法分离，并用于与ATP和吡哆醛类似物的结合研究。与24 kDa片段结合的三硝基苯基-ATP的光谱性质与与天然激酶结合的TNP-ATP的光谱性质无法区分。通过激酶的蛋白水解切割产生的小的16 kDa片段不结合任何类似物。当将携带共价结合到特定SH残基上的荧光探针的IAF吡哆醛激酶与胰凝乳蛋白酶预孵育时，观察到相同的消化模式。通过发射各向异性监测IAF-吡哆醛激酶的蛋白水解动力学，并且在不同浓度的胰凝乳蛋白酶下对蛋白水解初始速率的分析表明，天然吡哆醛激酶的解折叠速率在蛋白水解过程中起主导作用。
