Lee G J, McFadden B A
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
Biochemistry. 1992 Mar 3;31(8):2304-8. doi: 10.1021/bi00123a014.
Site-directed mutagenesis was used to change Ser376 in the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans to Cys, Thr, or Ala. When expressed in Escherichia coli and purified, the mutant enzymes exhibited carboxylase activities that were reduced by 99% or more with respect to the activity of the wild-type enzyme. The Km values for ribulose bisphosphate at pH 8.0, 30 degrees C, were elevated from 46 microM for wild-type enzyme to 287, 978, and 81 microM for mutants in which Cys, Thr, or Ala, respectively, replaced Ser376. The Cys and Thr variants were almost devoid of oxygenase activity whereas the Ala variant had 16% as much oxygenase as wild-type enzyme, suggesting that this mutation had greatly elevated the oxygenase:carboxylase ratio.
定点诱变被用于将来自蓝细菌集胞藻的1,5-二磷酸核酮糖羧化酶/加氧酶活性位点的丝氨酸376替换为半胱氨酸、苏氨酸或丙氨酸。当在大肠杆菌中表达并纯化后，与野生型酶的活性相比，突变酶的羧化酶活性降低了99%或更多。在pH 8.0、30℃条件下，1,5-二磷酸核酮糖的Km值从野生型酶的46微摩尔升高到分别用半胱氨酸、苏氨酸或丙氨酸取代丝氨酸376的突变体的287、978和81微摩尔。半胱氨酸和苏氨酸变体几乎没有加氧酶活性，而丙氨酸变体的加氧酶活性是野生型酶的16%，这表明该突变大大提高了加氧酶与羧化酶的比例。
