α-Isopropylmalate Synthase from Yeast: Purification, Kinetic Studies, and Effect of Ligands on Stability
α-Isopropylmalate Synthase from Yeast: Purification, Kinetic Studies, and Effect of Ligands on Stability
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来自酵母的 α-异丙基苹果酸合酶：纯化、动力学研究以及配体对稳定性的影响
作者：
E. Ulm;R. Bohme;G. Kohlhaw
α-异丙基苹果酸合酶是亮氨酸生物合成中的第一个特异性酶，从酵母菌（Saccharomyces sp.）（60615株）的提取物中纯化了约100倍，最有效的步骤是用反馈抑制剂亮氨酸从羟基磷灰石柱上特异性洗脱。在纯化的早期步骤中，特别注意保护合酶免受蛋白水解活性的影响。在校准柱上通过凝胶过滤测定的酶的表观分子量在不存在亮氨酸的情况下为137，000，在存在亮氨酸的情况下为121，000。亮氨酸的抑制是特异性的，并且强烈依赖于pH值，随着pH值从7.5增加到8.5，半最大抑制所需的亮氨酸浓度增加约10倍。在此pH范围内，催化活性几乎保持不变。两种底物的表观Km值分别为α-酮异戊酸16 μm和乙酰辅酶A 9 μm。K+是完全活性所必需的，表观Ka值为2 mm。亮氨酸抑制是混合型的，导致两种底物的Vmax降低和表观Km值增加。而没有合作的影响，观察到与任何基板，积极的协同性被视为与亮氨酸在饱和底物浓度的存在下。亮氨酸和（在较小程度上）α-酮异戊酸使纯化的酶稳定，防止热失活。另一方面，乙酰辅酶A的存在加速了失活。在随后的实验中，辅酶A被认为是实际的失活配体，即使在较低的温度和估计在酶浓度范围内的浓度下也有效。
α-Isopropylmalate synthase, the first specific enzyme in leucine biosynthesis, was purified approximately 100-fold from extracts of Saccharomyces sp. (strain 60615), the most effective step being specific elution with the feedback inhibitor leucine from a hydroxyapatite column. In the early steps of purification, special care was taken to protect the synthase against proteolytic activities. The apparent molecular weight of the enzyme as determined from gel filtration on a calibrated column was 137,000 in the absence and 121,000 in the presence of leucine. Inhibition by leucine was specific and strongly pH-dependent, with the leucine concentration necessary for half-maximal inhibition increasing about 10-fold as the pH increased from 7.5 to 8.5. Within this pH range, catalytic activity remained almost unchanged. The apparent Km values for the two substrates were found to be 16 μm for α-ketoisovalerate and 9 μm for acetyl-coenzyme A. K+ was required for full activity, the apparent Ka value being 2 mm. Leucine inhibition was of the mixed type, resulting in decreased Vmax and increased apparent Km values forboth substrates. Whereas no cooperative effects were observed with either substrate, positive cooperativity was seen with leucine in the presence of saturating substrate concentrations. Leucine and, to a lesser extent, α-ketoisovalerate stabilized the purified enzyme against heat-inactivation. The presence of acetyl-coenzyme A, on the other hand, accelerated the inactivation. In subsequent experiments, coenzyme A was recognized as the actual inactivating ligand, being effective even at lower temperatures and in concentrations which were estimated to be in the range of the enzyme concentration.