Hiraiwa N, Nishimura M, Hara-Nishimura I
Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
FEBS Lett. 1999 Mar 26;447(2-3):213-6. doi: 10.1016/s0014-5793(99)00286-0.
A vacuolar processing enzyme (VPE) responsible for maturation of various vacuolar proteins is synthesized as an inactive precursor. To clarify how to convert the VPE precursor into the active enzyme, we expressed point mutated VPE precursors of castor bean in the pep4 strain of Saccharomyces cerevisiae. A VPE with a substitution of the active site Cys with Gly showed no ability to convert itself into the mature form, although a wild VPE had the ability. The mutated VPE was converted by the action of the VPE that had been purified from castor bean. Substitution of the conserved Asp-Asp at the putative cleavage site of the C-terminal propeptide with Gly-Gly abolished both the conversion into the mature form and the activation of the mutated VPE. In vitro assay with synthetic peptides demonstrated that a VPE exhibited activity towards Asp residues and that a VPE cleaved an Asp-Gln bond to remove the N-terminal propeptide. Taken together, the results indicate that the VPE is self-catalytically maturated to be converted into the active enzyme by removal of the C-terminal propeptide and subsequent removal of the N-terminal one.
一种负责多种液泡蛋白成熟的液泡加工酶（VPE）最初以无活性前体的形式合成。为了阐明如何将VPE前体转化为活性酶，我们在酿酒酵母的pep4菌株中表达了蓖麻籽的点突变VPE前体。将活性位点半胱氨酸替换为甘氨酸的VPE没有将自身转化为成熟形式的能力，而野生型VPE则有此能力。突变的VPE通过从蓖麻籽中纯化得到的VPE的作用而被转化。将C端前肽假定切割位点处保守的天冬氨酸-天冬氨酸替换为甘氨酸-甘氨酸，既消除了向成熟形式的转化，也消除了突变VPE的激活。用合成肽进行的体外测定表明，VPE对天冬氨酸残基具有活性，并且VPE切割天冬氨酸-谷氨酰胺键以去除N端前肽。综上所述，结果表明VPE通过去除C端前肽并随后去除N端前肽而自催化成熟，转化为活性酶。
