Tiedge M, Richter T, Lenzen S
Institute of Clinical Biochemistry, Hannover, D-30623, Germany.
Arch Biochem Biophys. 2000 Mar 15;375(2):251-60. doi: 10.1006/abbi.1999.1666.
The low-affinity glucose phosphorylating enzyme glucokinase has the function of a physiological glucose sensor in pancreatic beta cells and in liver. In contrast to the high-affinity hexokinase types I-III glucokinase shows extraordinary sensitivity toward SH group oxidizing compounds. To characterize the function of sulfhydryl groups cysteine residues in the vicinity of the sugar binding site (Cys 213, Cys 220, Cys 230, Cys 233, and Cys 252) as well as cysteine residues a distance from the active site (Cys 364, Cys 371, and Cys 382), they were replaced in human beta cell glucokinase by serine through site-directed mutagenesis. Controlled proteolysis of wild-type glucokinase by proteinase K revealed that the SH group oxidizing agent alloxan can induce the formation of multiple intramolecular disulfide bridges corresponding to a double-band pattern of glucokinase protein in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of intramolecular disulfide bridges altered the mobility of the protein. None of the cysteine mutations could prevent the formation of the 49-kDa glucokinase conformation after alloxan treatment. The cysteine mutants Cys 233, Cys 252, and Cys 382 showed nearly complete loss of catalytic activity, whereas the V(max) values of the Cys 213, Cys 220, Cys 364, and Cys 371 mutants were decreased by 30-60%. Only the Cys 230 mutant showed kinetic characteristics comparable to those of wild-type glucokinase. The sensitivity of the Cys 213, Cys 230, Cys 364, and Cys 371 mutants toward alloxan-induced inhibition of enzyme activity was up to 10-fold lower compared with wild-type glucokinase. d-Glucose and dithiotreitol provided protection against alloxan-induced inhibition of wild-type glucokinase and all catalytically active cysteine mutants. Conclusively our data demonstrate the functional significance of the cysteine residues of beta cell glucokinase for both structural instability of the enzyme and catalytic function. Knowledge of sensitive cysteine targets may help to develop strategies that improve glucokinase enzyme function under conditions of oxidative stress.
低亲和力葡萄糖磷酸化酶葡萄糖激酶在胰腺β细胞和肝脏中具有生理葡萄糖传感器的功能。与高亲和力的己糖激酶I-III型不同，葡萄糖激酶对SH基团氧化化合物表现出非凡的敏感性。为了表征糖结合位点附近（Cys 213、Cys 220、Cys 230、Cys 233和Cys 252）以及距活性位点一定距离的半胱氨酸残基（Cys 364、Cys 371和Cys 382）的巯基功能，通过定点诱变将它们在人β细胞葡萄糖激酶中替换为丝氨酸。用蛋白酶K对野生型葡萄糖激酶进行可控的蛋白水解表明，SH基团氧化剂四氧嘧啶可诱导形成多个分子内二硫键，这与非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中葡萄糖激酶蛋白的双带模式相对应。分子内二硫键的形成改变了蛋白质的迁移率。在四氧嘧啶处理后，没有一个半胱氨酸突变体能阻止49 kDa葡萄糖激酶构象的形成。半胱氨酸突变体Cys 233、Cys 252和Cys 382的催化活性几乎完全丧失，而Cys 213、Cys 220、Cys 364和Cys 371突变体的V(max)值降低了30-60%。只有Cys 230突变体表现出与野生型葡萄糖激酶相当的动力学特征。与野生型葡萄糖激酶相比，Cys 213、Cys 230、Cys 364和Cys 371突变体对四氧嘧啶诱导的酶活性抑制的敏感性低至10倍。d-葡萄糖和二硫苏糖醇可防止四氧嘧啶对野生型葡萄糖激酶和所有具有催化活性的半胱氨酸突变体的抑制。总之，我们的数据证明了β细胞葡萄糖激酶半胱氨酸残基对酶的结构不稳定性和催化功能的功能意义。了解敏感的半胱氨酸靶点可能有助于制定在氧化应激条件下改善葡萄糖激酶酶功能的策略。
