Hinson D D, Chambliss K L, Hoffmann G F, Krisans S, Keller R K, Gibson K M
Institute of Metabolic Disease, Baylor University Medical Center, Dallas, Texas 75226, USA.
J Biol Chem. 1997 Oct 17;272(42):26756-60. doi: 10.1074/jbc.272.42.26756.
Sequencing of polymerase chain reaction-amplified cDNAs from cultured cells of three patients with mevalonate kinase deficiency revealed a G --> A transversion at nucleotide 1000 of the coding region, converting alanine to threonine at position 334 (A334T). To characterize this defect, we expressed wild-type and mutant cDNAs in Escherichia coli as the glutathione S-transferase fusion proteins, with purification by affinity chromatography. SDS-polyacrylamide gel electrophoresis analysis for wild-type and mutant fusion proteins indicated an expected molecular mass of 42-43 kDa. Kinetic characterization of the wild-type fusion protein yielded Km values of 150 +/- 23 and 440 +/- 190 microM (mean +/- S.E.) for substrates (RS)-mevalonate and ATP, respectively. Expressed wild-type mevalonate kinase (MKase) had a maximum velocity of 13.6 +/- 1.4 units/mg of protein (n = 22, +/-S.E.), whereas the A334T mutation yielded an enzyme with average Vmax of 0.26 +/- 0.02 unit/mg of protein (n = 6, +/-S.E.), representing a decrease to 1.4% of control Vmax. Restriction digestion with HhaI, in conjunction with direct sequencing of cDNAs, revealed that two patients were homozygous and one heterozygous for the A334T allele, establishing autosomal recessive inheritance within families. Although the A334T enzyme had a normal Km for ATP of 680 +/- 226 microM (n = 3, +/-S.E.), the Michaelis constant for (RS)-mevalonate was increased >30-fold to 4623 +/- 1167 microM (n = 4, +/-S.E.) under standard assay conditions. Comparable kinetic results were obtained using extracts of lymphoblasts, which were homozygous for the A334T allele. Alanine 334 is invariant in MKase from bacteria to man and located in a glycine-rich region postulated to have homology with ATP-binding sequences. Our results indicate that the bacterial expression system for human MKase will provide a useful model system in which to analyze inherited mutations and identify the first active site residue in MKase associated with stabilization of mevalonate binding.
对三名甲羟戊酸激酶缺乏症患者培养细胞的聚合酶链反应扩增cDNA进行测序，结果显示编码区第1000位核苷酸发生了G→A颠换，导致第334位的丙氨酸转变为苏氨酸（A334T）。为了表征这一缺陷，我们将野生型和突变型cDNA在大肠杆菌中表达为谷胱甘肽S-转移酶融合蛋白，并通过亲和层析进行纯化。对野生型和突变型融合蛋白进行的SDS-聚丙烯酰胺凝胶电泳分析表明预期分子量为42 - 43 kDa。野生型融合蛋白的动力学表征显示，底物（RS）-甲羟戊酸和ATP的Km值分别为150±23和440±190μM（平均值±标准误）。表达的野生型甲羟戊酸激酶（MKase）的最大速度为13.6±1.4单位/毫克蛋白（n = 22，±标准误），而A334T突变产生的酶平均Vmax为0.26±0.02单位/毫克蛋白（n = 6，±标准误），仅为对照Vmax的1.4%。用HhaI进行限制性消化并结合cDNA直接测序，结果显示两名患者为A334T等位基因的纯合子，一名为杂合子，从而确定了家族内的常染色体隐性遗传。尽管A334T酶对ATP的正常Km值为680±226μM（n = 3,±标准误），但在标准测定条件下，（RS）-甲羟戊酸的米氏常数增加了30倍以上，达到4623±1167μM（n = 4,±标准误）。使用对A334T等位基因为纯合子的淋巴母细胞提取物也获得了类似的动力学结果。丙氨酸334在从细菌到人类的MKase中是不变的，并且位于一个推测与ATP结合序列具有同源性的富含甘氨酸的区域。我们的结果表明，用于人类MKase的细菌表达系统将提供一个有用的模型系统，可用于分析遗传突变并确定MKase中与甲羟戊酸结合稳定相关的第一个活性位点残基。
