Reaction mechanism of phosphoglucosamine mutase from Escherichia coli
Reaction mechanism of phosphoglucosamine mutase from Escherichia coli
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DOI：
10.1046/j.1432-1327.1999.00373.x
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发表时间：
1999-05-01
期刊：
影响因子：
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通讯作者：
Mengin-Lecreulx, D
中科院分区：
文献类型：
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作者：
Jolly, L;Ferrari, P;Mengin-Lecreulx, D
分离纯化了6-磷酸氨基葡萄糖和1-磷酸氨基葡萄糖相互转化所需的磷酸葡萄糖变位酶(GlmM)，并对其动力学性质进行了研究。该酶以磷酸化形式存在，并按照经典的乒乓球bi-bi机理催化其反应。高效液相色谱可分离脱磷酸化和磷酸化的GlmM，联用MS显示只有一种磷酸盐共价连接到酶的活性部位。在445个氨基酸组成的多肽中，磷酸化位点明确为Ser102。GlmM也能够催化葡萄糖-L-磷酸和葡萄糖-6-磷酸异构体的相互转化，尽管速率要低得多(1400倍)。有趣的是，Ser100到苏氨酸残基的突变导致GLmM的非特异性磷酸葡萄糖变位酶活性增加了20倍，这表明在己糖磷酸变位酶的共同序列中，这个位置上的丝氨酸或苏氨酸的存在可能是决定这些酶对糖-磷酸或氨基糖-磷酸底物的专一性的因素之一。
The phosphoglucosamine mutase (GlmM) from Escherichia coli, specifically required for the interconversion of glucosamine-6-phosphate and glucosamine-1-phosphate (an essential step in the pathway for cell-wall peptidoglycan and lipopolysaccharide biosyntheses) was purified to homogeneity and its kinetic properties were investigated. The enzyme was active in a phosphorylated form and catalysed its reaction according to a classical ping-pong bi-bi mechanism. The dephosphorylated and phosphorylated forms of GlmM could be separated by HPLC and coupled MS showed that only one phosphate was covalently linked to the active site of the enzyme. The site of phosphorylation was clearly identified as Ser102 in the 445-amino acid polypeptide. GlmM was also capable of catalysing the interconversion of glucose-l-phosphate and glucose-6-phosphate isomers, although at a much lower (1400-fold) rate. Interestingly, the mutational change of the Ser100 to a threonine residue resulted in a 20-fold increase of the nonspecific phosphoglucomutase activity of GLmM, suggesting that the presence of either a serine or a threonine at this position in the consensus sequence of hexosephosphate mutases could be one of the factors that determines the specificity of these enzymes for either sugar-phosphate or amino sugar-phosphate substrates.