Estévez Marcel, Skarda Jeremy, Spencer Josh, Banaszak Leonard, Weaver Todd M
Johns Hopkins University, School of Medicine, Department of Neuroscience, Baltimore, Maryland 21205, USA.
Protein Sci. 2002 Jun;11(6):1552-7. doi: 10.1110/ps.0201502.
Fumarase catalyzes the reversible conversion of fumarate to S- malate during the operation of the ubiquitous Kreb's cycle. Previous studies have shown that the active site includes side chains from three of the four subunits within the tetrameric enzyme. We used a clinically observed human mutation to narrow our search for potential catalytic groups within the fumarase active site. Offspring homozygous for the missense mutation, a G-955-C transversion in the fumarase gene, results in the substitution of a glutamine at amino acid 319 for the normal glutamic acid. To more fully understand the implications of this mutation, a single-step site-directed mutagenesis method was used to generate the homologous substitution at position 315 within fumarase C from Escherichia coli. Subsequent kinetic and X-ray crystal structure analyses show changes in the turnover number and the cocrystal structure with bound citrate.
在普遍存在的三羧酸循环运行过程中，延胡索酸酶催化延胡索酸可逆地转化为L-苹果酸。先前的研究表明，活性位点包括四聚体酶中四个亚基中三个亚基的侧链。我们利用临床观察到的人类突变来缩小对延胡索酸酶活性位点内潜在催化基团的搜索范围。该错义突变（延胡索酸酶基因中的G955C颠换）的纯合子后代，导致第319位氨基酸的谷氨酰胺替代了正常的谷氨酸。为了更全面地理解这种突变的影响，我们使用单步定点诱变方法在大肠杆菌延胡索酸酶C的第315位产生同源替代。随后的动力学和X射线晶体结构分析显示了周转数的变化以及与结合柠檬酸盐的共晶体结构。
