Naught Laura E, Regni Catherine, Beamer Lesa J, Tipton Peter A
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.
Biochemistry. 2003 Aug 26;42(33):9946-51. doi: 10.1021/bi034673g.
In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site. An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers. Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type. The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site. In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site. When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108. (31)P NMR spectra of the S108A protein confirm that it is phosphorylated. Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.
在铜绿假单胞菌中，双特异性酶磷酸甘露糖变位酶/磷酸葡萄糖变位酶催化磷酸基团从丝氨酸108转移至底物（6-磷酸甘露糖或6-磷酸葡萄糖）1位的羟基上。然后该酶必须催化底物6位上的磷酸基团再转移回酶上。每次磷酸基团转移预计都需要由酶活性位点的氨基酸残基提供的酸碱催化。通过定点诱变对活性位点残基进行的广泛研究未能鉴定出介导质子转移的单个关键残基。将活性位点残基精氨酸20、赖氨酸118、精氨酸247、组氨酸308和组氨酸329突变为不含可电离基团的残基后，产生的蛋白质其V(max)降至野生型的4%-12%。没有单个残基能更显著地降低催化活性，且几个残基对V(max)有相似影响，这一事实表明活性位点氨基酸整体通过产生正静电势起作用，该正静电势用于降低底物羟基的pK，使其以离子化形式结合在活性位点。通过这种方式，避免了将反应性羟基定位在特定氨基酸残基附近的必要性，这或许可以解释该酶如何能够促进两次磷酸基团转移的催化作用，尽管当底物以两种不同方向结合在活性位点时，1位和6位并不占据完全相同的位置。当丝氨酸108发生突变时，该酶仍保留了惊人数量的活性，这导致有人提出在没有丝氨酸108的情况下，另一个残基会被磷酸化。S108A蛋白的(31)P NMR光谱证实它被磷酸化了。尽管S108A/H329N蛋白没有可检测到的催化活性，但其(31)P NMR光谱与磷酸组氨酸残基不一致。
