Mäntsälä P, Zalkin H
J Biol Chem. 1984 Nov 25;259(22):14230-6.
Site-directed mutagenesis was employed to replace cysteine 12 with phenylalanine in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase). Glutamine-dependent amidophosphoribosyltransferase activity was abolished as a consequence of the mutation. The mutant enzyme, however, exhibited NH3-dependent activity, contained Fe-S, and was normally regulated by AMP. These results document the role of the active site cysteine in activation of glutamine for amide transfer. NH3-dependent amidophosphoribosyltransferase was utilized for de novo purine nucleotide synthesis. Cells containing the mutant enzyme grew at nearly the wild-type rate in media containing a high concentration of NH4Cl. The Phe-12 mutation was used to study NH2-terminal processing. Whereas the wild-type Cys-12 enzyme is processed correctly in Escherichia coli by removal of 11 amino acid residues from the NH2 terminus, the Phe-12 mutant enzyme was not subject to undecapeptide processing. Neither the mutant nor wild-type enzyme made in vitro was correctly processed. Alternative enzymatic and autocatalytic processing mechanisms were considered. The available evidence favors autocatalytic NH2-terminal undecapeptide processing.
采用定点诱变技术，将枯草芽孢杆菌谷氨酰胺磷酸核糖焦磷酸酰胺转移酶（酰胺磷酸核糖转移酶）中的半胱氨酸12替换为苯丙氨酸。该突变导致谷氨酰胺依赖性酰胺磷酸核糖转移酶活性丧失。然而，突变酶表现出氨依赖性活性，含有铁硫簇，并且通常受AMP调节。这些结果证明了活性位点半胱氨酸在谷氨酰胺酰胺转移激活中的作用。氨依赖性酰胺磷酸核糖转移酶用于从头嘌呤核苷酸合成。含有突变酶的细胞在含有高浓度氯化铵的培养基中以接近野生型的速率生长。苯丙氨酸12突变用于研究氨基末端加工。野生型半胱氨酸12酶在大肠杆菌中通过从氨基末端去除11个氨基酸残基而被正确加工，而苯丙氨酸12突变酶则不进行十一肽加工。体外制备的突变酶和野生型酶均未被正确加工。考虑了替代的酶促和自催化加工机制。现有证据支持自催化氨基末端十一肽加工。
