Jockel P, Schmid M, Choinowski T, Dimroth P
Institut für Mikrobiologie der Eidgenössischen Technischen Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland.
Biochemistry. 2000 Apr 18;39(15):4320-6. doi: 10.1021/bi992817e.
The membrane-bound beta-subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae catalyzes the decarboxylation of carboxybiotin, which is coupled to Na(+) translocation and consumes a periplasmically derived proton. Upon site-directed mutagenesis of 20 polar and/or conserved residues within putative membrane-integral regions, the specific oxaloacetate decarboxylase activities were reduced to various extents, but only the enzyme with a Y229F mutation was completely inactive. We propose that Y229 is part of the network by which the proton of S382 is delivered to carboxybiotin, where it is consumed upon catalyzing the immediate decarboxylation of this acid-labile compound. Unlike S382 or D203, Y229 appears to be not involved in Na(+) binding, because in the Y229F orY229A mutants, the beta-subunit was protected from tryptic digestion by 50 mM NaCl like in the wild-type enzyme. Oxaloacetate decarboxylase with a betaC291E mutation was unstable in the absence of Na(+) and dissociated into an alpha-gamma subcomplex and the beta-subunit. The enzyme could only be isolated in the presence of 0. 5 M NaCl. These results are consistent with the notion that the beta-subunit changes its conformation upon Na(+) binding.
肺炎克雷伯菌中草酰乙酸脱羧酶的膜结合β亚基催化羧基生物素的脱羧反应，该反应与Na⁺转运偶联，并消耗来自周质的质子。对假定的膜整合区域内的20个极性和/或保守残基进行定点诱变后，草酰乙酸脱羧酶的比活性有不同程度的降低，但只有Y229F突变的酶完全无活性。我们提出，Y229是将S382的质子传递给羧基生物素的网络的一部分，在催化这种酸不稳定化合物的直接脱羧反应时，质子在羧基生物素上被消耗。与S382或D203不同，Y229似乎不参与Na⁺结合，因为在Y229F或Y229A突变体中，β亚基像野生型酶一样受到50 mM NaCl对胰蛋白酶消化的保护。具有βC291E突变的草酰乙酸脱羧酶在没有Na⁺的情况下不稳定，会解离成α-γ亚复合物和β亚基。该酶只能在0.5 M NaCl存在的情况下分离得到。这些结果与β亚基在结合Na⁺时改变其构象的观点一致。
