Beer H D, Wohlfahrt G, Schmid R D, McCarthy J E
Department of Enzymology, National Biotechnology Research Centre (GBF), Braunschweig, Federal Republic of Germany.
Biochem J. 1996 Oct 15;319 ( Pt 2)(Pt 2):351-9. doi: 10.1042/bj3190351.
The fungus Rhizopus oryzae synthesizes an extracellular lipase precursor bearing N-terminal pre- and pro-sequences. Our studies in Escherichia coli and using recombinant lipase in vitro indicate that the prosequence of 97 amino acids has at least two functions. First, it modulates the enzyme activity of the lipase so that this enzyme can initially be synthesized in a non-destructive form. Direct synthesis of the mature form of the lipase in the cell has toxic consequences, at least partly because of phospholipase activity that is suppressed in the proprotein. Secondly, it supports folding of the lipase via a pathway influenced by a single cysteine residue at position - 68. Mutational analysis of the prosequence demonstrates not only the key role of this cysteine residue but also the importance of the neighbouring amino acids. In particular, Arg-69 probably enhances the leaving group character of Cys-68. We propose a model in which Cys-68 acts as an intramolecular thiodisulphide reagent, playing a catalytic role in the folding of the enzyme. The prosequence is capable of performing the described functions both in cis and in trans.
米根霉可合成一种带有N端前导序列和前肽序列的细胞外脂肪酶前体。我们在大肠杆菌中的研究以及体外使用重组脂肪酶的研究表明，由97个氨基酸组成的前肽序列至少具有两种功能。首先，它调节脂肪酶的酶活性，使该酶最初能够以非破坏性形式合成。在细胞中直接合成脂肪酶的成熟形式会产生毒性后果，至少部分原因是前体蛋白中被抑制的磷脂酶活性。其次，它通过受位于 - 68位的单个半胱氨酸残基影响的途径支持脂肪酶的折叠。对前肽序列的突变分析不仅证明了该半胱氨酸残基的关键作用，还证明了相邻氨基酸的重要性。特别是，Arg - 69可能增强了Cys - 68的离去基团特性。我们提出了一个模型，其中Cys - 68作为分子内硫代二硫化物试剂，在酶的折叠中起催化作用。前肽序列能够在顺式和反式中发挥所述功能。
