Merz A, Yee M C, Szadkowski H, Pappenberger G, Crameri A, Stemmer W P, Yanofsky C, Kirschner K
Department of Biophysical Chemistry, Biozentrum, Klingelbergstrasse 70, 4056 Basel, Switzerland.
Biochemistry. 2000 Feb 8;39(5):880-9. doi: 10.1021/bi992333i.
Enzymes from thermophilic organisms often are barely active at low temperatures. To obtain a better understanding of this sluggishness, we used DNA shuffling to mutagenize the trpC gene, which encodes indoleglycerol phosphate synthase, from the hyperthermophile Sulfolobus solfataricus. Mutants producing more active protein variants were selected by genetic complementation of an Escherichia coli mutant bearing a trpC deletion. Single amino acid changes and combinations of these changes improved growth appreciably. Five singly and doubly altered protein variants with changes at the N- and C-termini, or at the phosphate binding site, were purified and characterized with regard to their kinetics of enzymatic catalysis, product binding, cleavage by trypsin, and inactivation by heat. Turnover numbers of the purified variant proteins correlated with the corresponding growth rates, showing that the turnover number was the selected trait. Although the affinities for both the substrate and the product decreased appreciably in most protein variants, these defects were offset by the accumulation of high levels of the enzyme's substrate. Rapid mixing of the product indoleglycerol phosphate with the parental enzyme revealed that the enzyme's turnover number at low temperatures is limited by the dissociation of the enzyme-product complex. In contrast, representative protein variants bind and release the product far more rapidly, shifting the bottleneck to the preceding chemical step. The turnover number of the parental enzyme increases with temperature, suggesting that its structural rigidity is responsible for its poor catalytic activity at low temperatures. In support of this interpretation, the rate of trypsinolysis or of thermal denaturation is accelerated significantly in the activated protein variants.
嗜热生物的酶在低温下通常活性很低。为了更好地理解这种迟缓现象，我们利用DNA改组技术对编码吲哚甘油磷酸合酶的trpC基因进行诱变，该基因来自嗜热栖热菌。通过对携带trpC缺失的大肠杆菌突变体进行遗传互补，筛选出产生活性更高蛋白质变体的突变体。单个氨基酸的变化以及这些变化的组合显著改善了生长情况。对五个在N端和C端或磷酸结合位点发生变化的单突变和双突变蛋白质变体进行了纯化，并对其酶催化动力学、产物结合、胰蛋白酶切割以及热失活进行了表征。纯化的变体蛋白的周转数与相应的生长速率相关，表明周转数是被选择的性状。尽管大多数蛋白质变体对底物和产物的亲和力都明显降低，但这些缺陷被酶底物的高水平积累所抵消。将产物吲哚甘油磷酸与亲本酶快速混合后发现，该酶在低温下的周转数受酶-产物复合物解离的限制。相比之下，代表性的蛋白质变体结合和释放产物的速度要快得多，将瓶颈转移到了前面的化学步骤。亲本酶的周转数随温度升高而增加，这表明其结构刚性是其在低温下催化活性差的原因。支持这一解释的是，在活化的蛋白质变体中，胰蛋白酶消化或热变性的速率显著加快。
