Voordouw G, De Vries P A, Van den Berg W A, De Clerck E P
Eur J Biochem. 1987 Mar 16;163(3):591-8. doi: 10.1111/j.1432-1033.1987.tb10908.x.
Using oligonucleotide-directed mutagenesis of the gene encoding the small subunit (rbcS) from Anacystis nidulans mutant enzymes have been generated with either Trp-54 of the small subunit replaced by a Phe residue, or with Trp-57 replaced by a Phe residue, whereas both Trp-54 and Trp-57 have been replaced by Phe residues in a double mutant. Trp-54 and Trp-57 are conserved in all amino acid sequences or the small subunit (S) that are known at present. The wild-type and mutant forms of Rubisco have all been purified to homogeneity. The wild-type enzyme, purified from Escherichia coli is indistinguishable from enzyme similarly purified from A. nidulans in subunit composition, subunit molecular mass and kinetic parameters (Vmax CO2 = 2.9 U/mg, Km CO2 = 155 microM). The single Trp mutants are indistinguishable from the wild-type enzyme by criteria (a) and (b). However, whereas, Km CO2 is also unchanged, Vmax CO2 is 2.5-fold smaller than the value for the wild-type enzyme for both mutants, demonstrating for the first time that single amino acid replacements in the non-catalytic small subunit influence the catalytic rate of the enzyme. The specificity factor tau, which measures the partitioning of the active site between the carboxylase and oxygenase reactions, was found to be invariant. Since tau is not affected by these mutations we conclude that S is an activating not a regulating subunit.
利用寡核苷酸定向诱变编码来自集胞藻6803的小亚基（rbcS）的基因，已产生了突变酶，其中小亚基的Trp-54被苯丙氨酸残基取代，或者Trp-57被苯丙氨酸残基取代，而在双突变体中Trp-54和Trp-57都被苯丙氨酸残基取代。Trp-54和Trp-57在目前已知的所有小亚基（S）的氨基酸序列中都是保守的。1,5-二磷酸核酮糖羧化酶/加氧酶（Rubisco）的野生型和突变型都已被纯化至均一。从大肠杆菌中纯化的野生型酶在亚基组成、亚基分子量和动力学参数（Vmax CO2 = 2.9 U/mg，Km CO2 = 155 microM）方面与从集胞藻6803中类似纯化的酶没有区别。根据标准（a）和（b），单个色氨酸突变体与野生型酶没有区别。然而，虽然Km CO2也没有变化，但两个突变体的Vmax CO2都比野生型酶的值小2.5倍，首次证明非催化性小亚基中的单个氨基酸替换会影响酶的催化速率。测量活性位点在羧化酶反应和加氧酶反应之间分配的特异性因子tau被发现是不变的。由于tau不受这些突变的影响，我们得出结论，S是一个激活亚基而非调节亚基。
