Shima S, Tziatzios C, Schubert D, Fukada H, Takahashi K, Ermler U, Thauer R K
Max-Planck-Institut für terrestrische Mikrobiologie and Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität, Marburg, Germany.
Eur J Biochem. 1998 Nov 15;258(1):85-92. doi: 10.1046/j.1432-1327.1998.2580085.x.
Formyltransferase from Methanopyrus kandleri is composed of only one type of subunits of molecular mass 32 kDa. The enzyme requires the presence of lyotropic salts for activity and thermostability. We report here that the enzyme is in a monomer/dimer/tetramer association equilibrium, the association constant being affected by lyotropic salts. At 0.01 M K2HPO4/KH2PO4, pH 7.2, the enzyme (0.4 mg/ml) was mainly present in a monomeric form. Upon increase of the phosphate concentration, the concentration of the dimer increased up to a phosphate concentration of 0.6 M, then decrease at the expense of tetramer formation up to a phosphate concentration of 1.0 M. The specific activity at 4 C increased from <0.1 U/mg at 0.01 M, over 1.5 U/mg at 0.6 M to 3.6 U/mg at 1.0 M. Similar results were obtained with ammonium sulfate as lyotropic salt. The findings indicate that both oligomerization and activity increase with increasing salt concentrations, suggesting that there is a causal connection. To determine this, we exploited the observation that oligomer formation was not induced by the weak lyotropic salt NaCl up to a concentration of 1.5 M and that the dissociation of the dimer into the monomer at 4 degrees C proceeded very slowly (50% in approximately 6 h). This allowed us to study the effect of NaCl on the activity of the oligomers at NaCl concentrations not sufficient to induce oligomerization. At 4 degrees C, the activity of the oligomers increased from 0.3 U/mg at 0.25 M NaCl to 3.4 U/mg at 1.0 M NaCl. At these NaCl concentrations, the monomers were inactive. The findings indicate that oligomerization is a prerequisite for enzyme activity in the presence of NaCl. The salt-dependent induction of oligomerization was parallelled by an increase in thermostability; strong lyotropic salts conferred thermostability at much lower concentrations than the weak lyotropic NaCl.
来自坎氏甲烷嗜热菌的甲酰基转移酶仅由一种分子量为32 kDa的亚基组成。该酶的活性和热稳定性需要离液盐的存在。我们在此报告，该酶处于单体/二聚体/四聚体缔合平衡状态，缔合常数受离液盐影响。在0.01 M K2HPO4/KH2PO4，pH 7.2条件下，该酶（0.4 mg/ml）主要以单体形式存在。随着磷酸盐浓度的增加，二聚体的浓度增加，直至磷酸盐浓度达到0.6 M，然后以四聚体形成的形式减少，直至磷酸盐浓度达到1.0 M。4℃时的比活性从0.01 M时的<0.1 U/mg增加到0.6 M时的1.5 U/mg以上，再到1.0 M时的3.6 U/mg。以硫酸铵作为离液盐也得到了类似的结果。这些发现表明，随着盐浓度的增加，寡聚化和活性都增加表明存在因果关系。为了确定这一点，我们利用了以下观察结果：在浓度高达1.5 M的情况下，弱离液盐NaCl不会诱导寡聚体形成，并且在4℃时二聚体解离为单体的过程非常缓慢（约6小时内50%）。这使我们能够研究NaCl在不足以诱导寡聚化的浓度下对寡聚体活性的影响。在4℃时，寡聚体的活性从0.25 M NaCl时的0.3 U/mg增加到1.0 M NaCl时的3.4 U/mg。在这些NaCl浓度下，单体没有活性。这些发现表明，在NaCl存在下，寡聚化是酶活性的先决条件。寡聚化的盐依赖性诱导与热稳定性的增加同时发生；强离液盐在比弱离液盐NaCl低得多的浓度下赋予热稳定性。
