Cloning and biochemical characterisation of an Aspergillus niger glucokinase - Evidence for the presence of separate glucokinase and hexokinase enzymes
Cloning and biochemical characterisation of an Aspergillus niger glucokinase - Evidence for the presence of separate glucokinase and hexokinase enzymes
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DOI：
10.1111/j.1432-1033.1996.0518h.x
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发表时间：
1996-09-15
期刊：
影响因子：
--
通讯作者：
Visser, J
中科院分区：
文献类型：
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作者：
Panneman, H;Ruijter, GJG;Visser, J
黑曲霉葡萄糖激酶基因 glkA 已使用通过简并寡核苷酸聚合酶链反应产生的探针进行克隆。测定了thr基因的DNA序列，推导的氨基序列显示出与其他真核生物己糖激酶和葡萄糖激酶蛋白，特别是与酿酒酵母葡萄糖激酶蛋白显着相似。从多拷贝glkA 转化体中纯化编码的蛋白质，并进行广泛表征。该蛋白质的分子量为 54536 Da，pI 为 5.2。该酶对葡萄糖具有高亲和力（pH 7.5 时 K-m 0.063 mM），对果糖具有相对较低的亲和力（pH 7.5 时 K-m 120 mM），因此体内葡萄糖激酶对果糖的磷酸化可以忽略不计。底物 C1 和 C4 处的构型似乎对于底物特异性至关重要。黑曲霉葡萄糖激酶表现出ADP对ATP的非竞争性抑制和ADP对葡萄糖的非竞争性抑制。 k(cat)（周转数）在 pH 7.5 以下迅速下降（pH 7.0 时为 56%，pH 6.5 时为 17%），这可能对体内活性调节具有重要意义。此外，还提供了黑曲霉中存在第二种己糖磷酸化酶的证据。这种酶可能是一种己糖激酶，因为与葡萄糖激酶不同，这种活性受到海藻糖 6-磷酸的抑制。
The Aspergillus niger glucokinase gene glkA has been cloned using a probe generated by polymerase chain reaction with degenerate oligonucleotides. The DNA sequence of thr gene was determined, and the deduced amino rid sequence shows significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to the Saccharomyces cerevisiae glucokinase protein. The encoded protein was purified from a multicopy glkA transformant, and extensively characterised. The protein has a molecular mass of 54536 Da and a pI of 5.2. The enzyme has high affinity for glucose (K-m 0.063 mM at pH 7.5) and a relatively low affinity for fructose (K-m 120 mM at pH 7.5), and in vivo fructose phosphorylation by glucokinase is consequently negligible. The configurations at C1 and C4 of the substrate appear to be essential for substrate specificity. The A. niger glucokinase shows non-competitive inhibition by ADP towards ATP and uncompetitive inhibition by ADP towards glucose. The k(cat) (turnover number) decreases rapidly below pH 7.5 (56% at pH 7.0 and 17% at pH 6.5) and this may have important implications for the in vivo regulation of activity. In addition, proof is provided for the presence of a second hexosephosphorylating enzyme in A. niger. This enzyme is probably a hexokinase, since unlike glucokinase, this activity is inhibited by trehalose 6-phosphate.