葡萄糖激酶基因E339K突变导致酶动力学改变和蛋白产量下降并引起高血糖.docVIP

  葡萄糖激酶基因 E339K 突变导致酶动力学 改变和蛋白产量下降并引起高血糖# 沈云峰1,2，梁华1，蔡梦茵1，翁建平1** 5 10 15 20 25 30 35 40 （1. 中山大学附属第三医院，广州 510630 2. 南昌大学第二附属医院，南昌 330006） 摘要：目的 探讨葡萄糖激酶（GK）基因 E339K 突变酶动力学与青少年中的成年起病型糖尿 病 2 型的关系。 方法 通过点突变构建 GST-GK-E339K-cDNA。在 E.Coli.中分别表达野生型 和突变型 GK。亲和层析法纯化细菌裂解液，提取 GK 蛋白。酶偶联分析法分别测定野生型和 突变型 GK 的酶动力学指标。结果 GK 基因 E339K 突变导致 GK 蛋白产量下降(12.7±1.72 vs 16.2±2.65 mg/L, P0.01)，与底物--葡萄糖(S0.5: 13.96±1.89 vs 5.92±0.99 mmol/L, P0.001)和 ATP(Km: 3.27±1.14 vs 0.30±0.09 mmol/L, P0.001)的亲和力下降，催化活 性下降(1.62±0.35 vs 25.18±2.10 s-1, P0.001)。 结论 酶动力学改变和蛋白产量下降 可能是 GK 基因 E339K 突变引起高血糖的主要原因之一。 关键词：葡萄糖激酶；基因突变；酶动力学 中图分类号：R589.1 Impaired enzymatic kinetics and decreased protein yeild induced by glucokinase gene E339K mutation resulted in hyperglycemia SHEN Yunfeng1,2, LIANG Hua1, CAI Mengyin1, WENG Jianping1 (1. The Third Affiliated Hospital of Sun Yat-sen University, GuangZhou 510630; 2. The Second Affiliated Hospital of Nanchang University, NanChang 330006) Abstract: Glucokinase (GCK) acts as a glucose sensor and regulates b-cell insulin secretion. The heterozygous mutations in the gene encoding GCK cause a reduction of the enzyme activity, which results in a monogenic form of diabetes, maturity-onset diabetes of the young. In the present study, we identified and enzymatic kinetically characterized a novel missense mutation in the GCK gene, which results in a protein mutation Glu339→Lys (E339K), from a Chinese family with hyperglycemia. We expressed and affinity-purified the GCK proteins from bacterial expression system that carries mutation (E339K) and fused to glutathione S-transferase. The expressed GCK protein was subjected to the measurement of its biochemical effects of the missense mutation on GCK activity. Our results showed that the mutation reduced the GCK protein yield. The enzymatic kinetics on the recombinant GCK proteins revealed that the mutation inactivates enzyme kinetics. Keywords: Glucokinase; Gene Mutation; Enzymatic Kinetics 0 引言 葡萄糖激酶（GK）是已糖激酶的一种，又称已糖激酶 4 或 D，是脊椎动物体内四个葡