Jaworski J G, Post-Beittenmiller M A, Ohlrogge J B
Department of Botany and Plant Pathology, Michigan State University.
Eur J Biochem. 1989 Oct 1;184(3):603-9. doi: 10.1111/j.1432-1033.1989.tb15056.x.
Site-directed mutagenesis was used to change the phosphopantetheine attachment site (Ser38) of spinach acyl carrier protein I (ACP-I) from a serine to a threonine or cysteine residue. 1. Although the native ACP-I is fully phosphopantethenylated when expressed in Escherichia coli, the TH-ACP-I and CY-ACP-I mutants were found to be completely devoid of the phosphopantetheine group. Therefore, the E. coli holoACP synthase requires serine for in vivo phosphopantetheine addition to spinach ACP-I. 2. Spinach holoACP synthase was completely inactive in vitro with either the TH-ACP-I or CY-ACP-I mutants. In addition, TH-ACP-I and CY-ACP-I were strong inhibitors of spinach holoACP synthase. 3. The mutant ACPs were weak or ineffective as inhibitors of spinach fatty acid synthesis and spinach oleoyl-ACP hydrolase. 4. Compared to holoACP-I, the mutant apoACP-I analogs had: (a) altered mobility in SDS and native gel electrophoresis, (b) altered binding to anti-(spinach ACP-I) antibodies and (c) altered isoelectric points. The combined physical, immunological and enzyme inhibition data indicate that attachment of the phosphopantheine prosthetic group alters ACP conformation.
定点诱变用于将菠菜酰基载体蛋白I（ACP-I）的磷酸泛酰巯基乙胺附着位点（Ser38）从丝氨酸残基改变为苏氨酸或半胱氨酸残基。1. 尽管天然的ACP-I在大肠杆菌中表达时会完全磷酸泛酰巯基乙胺化，但发现TH-ACP-I和CY-ACP-I突变体完全没有磷酸泛酰巯基乙胺基团。因此，大肠杆菌全ACP合酶在体内将磷酸泛酰巯基乙胺添加到菠菜ACP-I时需要丝氨酸。2. 菠菜全ACP合酶在体外对TH-ACP-I或CY-ACP-I突变体完全无活性。此外，TH-ACP-I和CY-ACP-I是菠菜全ACP合酶的强抑制剂。3. 突变的ACP作为菠菜脂肪酸合成和菠菜油酰-ACP水解酶的抑制剂作用较弱或无效。4. 与全ACP-I相比，突变的脱辅基ACP-I类似物具有：（a）在SDS和天然凝胶电泳中迁移率改变，（b）与抗（菠菜ACP-I）抗体的结合改变，以及（c）等电点改变。综合的物理、免疫和酶抑制数据表明，磷酸泛酰巯基乙胺辅基的附着改变了ACP的构象。
