Tobin M B, Cole S C, Miller J R, Baldwin J E, Sutherland J D
Dyson Perrins Laboratory, Oxford, UK.
Gene. 1995 Aug 30;162(1):29-35. doi: 10.1016/0378-1119(95)00369-h.
Site-directed mutagenesis of the penDE gene and expression in Escherichia coli has produced recombinant acylcoenzyme A:isopenicillin N acyltransferase (re-AT) containing amino-acid substitutions in the proenzyme cleavage site (decreases) region (Asp-Gly102 decreases Cys103-Thr-Thr). The effect of these substitutions on proenzyme cleavage and AT activity has been investigated. The re-AT with substitutions at Cys103 (Cys103-->Ser, Cys103-->Ala and Cys103-->Trp) were uncleaved and inactive. Substitutions at Asp101 and Gly102 (Asp101-->Gly, Gly102-->Ala, Gly102-->Val, Gly102-->Met, Gly102-->Val and Asp101Gly102-->GlyPhe) did not prevent proenzyme cleavage or abolish AT activity. Thr105-->Ser and Thr105-->Ala substitutions did not prevent proenzyme cleavage or AT activity; however, AT containing Thr105-->Val resulted in a significant inhibition of proenzyme cleavage.
对penDE基因进行定点诱变并在大肠杆菌中表达，已产生了重组酰基辅酶A：异青霉素N酰基转移酶（re-AT），该酶在酶原切割位点（减少）区域（天冬氨酸-甘氨酸102减少半胱氨酸103-苏氨酸-苏氨酸）含有氨基酸取代。已研究了这些取代对酶原切割和AT活性的影响。在半胱氨酸103处有取代（半胱氨酸103→丝氨酸、半胱氨酸103→丙氨酸和半胱氨酸103→色氨酸）的re-AT未被切割且无活性。在天冬氨酸101和甘氨酸102处的取代（天冬氨酸101→甘氨酸、甘氨酸102→丙氨酸、甘氨酸102→缬氨酸、甘氨酸102→甲硫氨酸、甘氨酸102→缬氨酸和天冬氨酸101甘氨酸102→甘氨酸苯丙氨酸）并未阻止酶原切割或消除AT活性。苏氨酸105→丝氨酸和苏氨酸105→丙氨酸取代并未阻止酶原切割或AT活性；然而，含有苏氨酸105→缬氨酸的AT导致酶原切割受到显著抑制。
