Jakobi R, Traugh J A
Department of Biochemistry, University of California, Riverside 92521-0129, USA.
Eur J Biochem. 1995 Jun 15;230(3):1111-7. doi: 10.1111/j.1432-1033.1995.tb20662.x.
Casein kinase II exists in vivo as an active holoenzyme consisting of catalytic alpha and/or alpha' and regulatory beta subunits, which form a tetrameric structure of alpha 2 beta 2. Unlike most other protein kinases, casein kinase II uses both ATP and GTP effectively as phosphate donors. Two residues unique to the catalytic subunit of casein kinase II, Val66 and Trp176, were mutated to Ala66 and Phe176, respectively, the amino acids present in more than 95% of the identified protein kinase sequences. Using recombinant alpha subunit expressed in Escherichia coli, the mutations have been previously shown to reduce the utilization of GTP by changing Km values for ATP and GTP and to reduce the approximate fivefold stimulation observed upon addition of the regulatory subunit [Jakobi, R. & Traugh, J. A. (1992) J. Biol. Chem. 267, 23,894-23,902]. In this study, the mutations are shown to affect the catalytic activity of the reconstituted holoenzyme by changing both Km and Vmax values. The Vmax for ATP is reduced by the mutation of Trp176 to phenylalanine, but no change is observed with GTP. The Val66 to alanine and Val66/Trp176 to alanine/phenylalanine mutations reduce the Vmax values for ATP and GTP to levels comparable to those of the catalytic subunits alone, indicating that changes in the stimulation of activity by the beta subunit are due to changes in Vmax. Structural studies using ultraviolet CD spectroscopy show that changes in stimulation of Vmax by the beta subunit are correlated with changes in the secondary structure; the extent of these changes is reduced by both mutations. Correlation of changes in secondary structure and stimulation of activity by the beta subunit indicate that the formation of the wild-type holoenzyme causes conformational changes in the active site, leading to an increased rate of reaction. As shown by the mutations, Val66 and Trp176 are involved both in the conformational changes and in the selectivity of ATP and GTP.
酪蛋白激酶II在体内以一种活性全酶的形式存在，该全酶由催化性的α和/或α'亚基以及调节性的β亚基组成，形成α2β2的四聚体结构。与大多数其他蛋白激酶不同，酪蛋白激酶II能有效地将ATP和GTP都用作磷酸供体。酪蛋白激酶II催化亚基特有的两个残基，Val66和Trp176，分别被突变为Ala66和Phe176，这两种氨基酸存在于超过95%已鉴定的蛋白激酶序列中。利用在大肠杆菌中表达的重组α亚基，先前的研究表明这些突变通过改变ATP和GTP的Km值降低了GTP的利用率，并降低了添加调节亚基后观察到的约五倍的刺激作用[雅各比，R. & 特劳，J. A.（1992年）《生物化学杂志》267, 23894 - 23902]。在本研究中，这些突变被证明通过改变Km和Vmax值来影响重组全酶的催化活性。将Trp176突变为苯丙氨酸会降低ATP的Vmax，但GTP未观察到变化。将Val66突变为丙氨酸以及将Val66/Trp176突变为丙氨酸/苯丙氨酸会将ATP和GTP的Vmax值降低到与单独催化亚基相当的水平，表明β亚基对活性的刺激变化是由于Vmax的改变。使用紫外圆二色光谱的结构研究表明，β亚基对Vmax的刺激变化与二级结构的变化相关；这两种突变都降低了这些变化的程度。二级结构的变化与β亚基对活性的刺激之间的相关性表明，野生型全酶的形成会导致活性位点的构象变化，从而导致反应速率增加。如突变所示，Val66和Trp176既参与构象变化，也参与ATP和GTP的选择性。
