Azuma Y, Seino H, Seki T, Uzawa S, Klebe C, Ohba T, Wittinghofer A, Hayashi N, Nishimoto T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
J Biochem. 1996 Jul;120(1):82-91. doi: 10.1093/oxfordjournals.jbchem.a021397.
Charged amino acid residues of human RCC1 were converted to alanine and mutants which were unable to complement tsBN2 cells (a temperature-sensitive rcc1- mutant of the hamster BHK21 cell line) were selected. These RCC1 mutants were analyzed for the ability to inhibit premature chromatin condensation by microinjection into tsBN2 cells, and their steady-state kinetic parameters for guanine nucleotide exchange reaction were measured. Examined RCC1 mutants were unstable in tsBN2 cells at the restrictive temperature, yet they significantly inhibited premature chromatin condensation. Mutants located on the N-terminus of the RCC1 repeat showed an increased K(m), while their kcat values were comparable to that of wild-type RCC1. In contrast, mutants containing the conserved histidine residues in the C-terminus of the RCC1 repeat showed a value of K(m) similar to that of wild-type RCC1, while the kcat values of these mutants were reduced, depending upon the RCC1 repeats on which the mutation was located. These steady-state kinetic parameters of mutants indicate that the N-terminus and the C-terminus of RCC1 repeats play different roles in guanine nucleotide exchange on Ran. The comparison of kcat among the histidine mutants suggests that those histidine residues which are conserved in the RCC1 repeats and also through evolution comprise the catalytic site for the guanine nucleotide exchange reaction.
将人RCC1的带电荷氨基酸残基转化为丙氨酸，并筛选出无法互补tsBN2细胞（仓鼠BHK21细胞系的温度敏感型rcc1 - 突变体）的突变体。通过显微注射到tsBN2细胞中分析这些RCC1突变体抑制染色质过早凝聚的能力，并测量它们鸟嘌呤核苷酸交换反应的稳态动力学参数。所检测的RCC1突变体在限制温度下于tsBN2细胞中不稳定，但它们能显著抑制染色质过早凝聚。位于RCC1重复序列N端的突变体K(m)增加，而其kcat值与野生型RCC1相当。相比之下，在RCC1重复序列C端含有保守组氨酸残基的突变体K(m)值与野生型RCC1相似，而这些突变体的kcat值降低，这取决于发生突变的RCC重复序列。突变体的这些稳态动力学参数表明，RCC1重复序列的N端和C端在Ran上的鸟嘌呤核苷酸交换中发挥不同作用。组氨酸突变体之间kcat的比较表明，在RCC1重复序列中以及在进化过程中保守的那些组氨酸残基构成了鸟嘌呤核苷酸交换反应的催化位点。
