Nakamura Tooru, Sakai Miyo, Sadakane Yutaka, Haga Tatsuya, Goto Yuji, Kinouchi Tadatoshi, Saito Takeshi, Fujii Noriko
Research Reactor Institute, Faculty of Science, Kyoto University, Kumatori, Sennan, Osaka, Japan.
Biochim Biophys Acta. 2008 Sep;1784(9):1192-9. doi: 10.1016/j.bbapap.2008.04.008. Epub 2008 Apr 25.
Asp58 and Asp151 in alpha A-crystallin of human eye lenses become highly inverted and isomerized to d-beta-Asp residues with age. Racemization was previously shown to proceed rapidly when the residue on the carboxyl side of the Asp residue is small. Asn was also demonstrated to be more susceptible to racemization than Asp in protein. In this study, the changes of rate constants for racemization at Asp58 and Asp151 and at Asn58 and Asn151 were investigated using D58N, S59T, D151N and A152V mutants obtained through site-directed mutagenesis. The rate constant of racemization at Asn151 in D151N was found to be 1.5 times more rapid than Asp151 in the wild-type. For A152V, the rate constant at Asp151 was 1/4 that of the wild-type. There were no significant differences in the rate constants of racemization for both Asp58 and Asn58 residues. The aggregate size of D58N, S59T and D151N mutants increased or increased in polydispersity and their chaperone activities decreased. The size and chaperone activity of A152V was unchanged. These results suggest that structures close to Asp58 and Asp151 residues in the protein affect the rate constant of Asp racemization and the size and chaperone function of alpha A-crystallin.
人眼晶状体αA-晶体蛋白中的Asp58和Asp151会随着年龄的增长而高度反转并异构化为d-β-Asp残基。先前的研究表明，当Asp残基羧基侧的残基较小时，消旋化反应会迅速进行。在蛋白质中，Asn也被证明比Asp更容易发生消旋化。在本研究中，通过定点诱变获得D58N、S59T、D151N和A152V突变体，研究了Asp58和Asp151以及Asn58和Asn151消旋化反应速率常数的变化。发现D151N中Asn151的消旋化速率常数比野生型中的Asp151快1.5倍。对于A152V，Asp151处的速率常数是野生型的1/4。Asp58和Asn58残基的消旋化速率常数没有显著差异。D58N、S59T和D151N突变体的聚集体大小增加或多分散性增加，其伴侣活性降低。A152V的大小和伴侣活性没有变化。这些结果表明，蛋白质中靠近Asp58和Asp151残基的结构会影响Asp消旋化的速率常数以及αA-晶体蛋白的大小和伴侣功能。
