Veiga-da-Cunha M, Courtois S, Michel A, Gosselain E, Van Schaftingen E
Laboratory of Physiological Chemistry, University of Louvain, Brussels, Belgium.
J Biol Chem. 1996 Mar 15;271(11):6292-7. doi: 10.1074/jbc.271.11.6292.
To delineate the regions of liver glucokinase that are involved in the binding of its regulatory protein and have therefore been conserved throughout evolution, we have cloned the cDNA of the Xenopus laevis enzyme. It contains an open reading frame of 1374 nucleotides and encodes a protein of 458 amino acids, which displays 78 and 79% overall identity to rat and human liver glucokinases, respectively. The conserved regions are predicted to be present mainly in the small domain and the hinge region of glueokinase, and the nonconserved regions in the large domain of the enzyme. We constructed five mutants of Xenopus glucokinase by replacing sets of 2-5 glucokinase-specific residues with their counterparts in the C-terminal half of rat hexokinase I. The affinity for the regulatory protein was not markedly changed for mutants B, D, and E despite a decreased affinity for glucose in mutants B and D. Two other mutants (A and C) were 9- and 250-fold less sensitive to the rat regulator and 40- and 770-fold less sensitive to the Xenopus regulator, respectively, but presented a normal affinity for glucose. The double mutant (A-C) was completely insensitive to inhibition by the regulatory protein. A control mutant (F), obtained by replacing 3 residues that were not conserved in all glucokinases, had a normal affinity for glucose and for the regulatory protein. The property of glucokinase to be inhibited by palmitoyl-CoA was not affected by the mutations described. It is concluded that His-141 to Leu-144, which are located close to the tip of the small domain, as well as Glu-51 and Glu-52, which are present in the large domain of the enzyme close to the hinge region, or nearby residues participate in the binding of the regulatory protein.
为了确定肝脏葡萄糖激酶中参与其调节蛋白结合且在整个进化过程中保守的区域，我们克隆了非洲爪蟾葡萄糖激酶的cDNA。它包含一个1374个核苷酸的开放阅读框，编码一个458个氨基酸的蛋白质，该蛋白质与大鼠和人肝脏葡萄糖激酶的总体一致性分别为78%和79%。保守区域预计主要存在于葡萄糖激酶的小结构域和铰链区，而非保守区域存在于该酶的大结构域。我们构建了五个非洲爪蟾葡萄糖激酶突变体，用大鼠己糖激酶I C端一半的对应残基替换了2 - 5个葡萄糖激酶特异性残基。尽管突变体B和D对葡萄糖的亲和力降低，但突变体B、D和E对调节蛋白的亲和力没有明显变化。另外两个突变体（A和C）对大鼠调节蛋白的敏感性分别降低了9倍和250倍，对非洲爪蟾调节蛋白的敏感性分别降低了40倍和770倍，但对葡萄糖具有正常的亲和力。双突变体（A - C）对调节蛋白的抑制作用完全不敏感。通过替换在所有葡萄糖激酶中不保守的3个残基获得的对照突变体（F）对葡萄糖和调节蛋白具有正常的亲和力。所描述的突变不影响葡萄糖激酶被棕榈酰辅酶A抑制的特性。得出的结论是，位于小结构域顶端附近的His - 141至Leu - 144，以及存在于靠近铰链区的酶大结构域中的Glu - 51和Glu - 52，或附近的残基参与调节蛋白的结合。
