Sheff D R, Rubenstein P A
Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242.
J Biol Chem. 1992 Feb 5;267(4):2671-8.
Most actins examined to date undergo a unique posttranslational modification termed processing, catalyzed by the actin N-acetylaminopeptidase. Processing is the removal of acetylmethionine from the amino terminus in class I actins with Met-Asp(Glu) amino termini. For class II actins with Met-X-Asp(Glu) amino termini, processing is the removal of the second residue as an N-acetylamino acid. Other cytosolic proteins with these amino termini are not processed suggesting that the reaction may be specific for actins. In actin, X is usually cysteine. However, there are some class II actins in which this residue is other than cysteine, suggesting a broader substrate specificity for actin N-acetylaminopeptidase than acetylmethionine or acetylcysteine. We constructed mutant actins in which this cysteine was replaced with serine, asparagine, glycine, aspartic acid, histidine, phenylalanine, and tyrosine and used these to determine the substrate specificity of rat liver actin N-acetylaminopeptidase in vitro. Amino-terminal acetylmethinonine was cleaved from adjacent aspartic acid, asparagine, or histidine, but not serine, glycine, phenylalanine, or tyrosine. Of the acetylated actin amino termini tested, only acetylmethionine and acetylcysteine were cleaved. Histidine was never N-acetylated and was not cleaved. When phenylalanine and tyrosine were adjacent to the initiator methionine, no initiator methionine was cleaved even though it was acetylated. These results suggest a narrow substrate specificity for the rat liver actin N-acetylaminopeptidase. They also demonstrate that the adjacent residue can effect actin N-acetylaminopeptidase specificity.
迄今为止所检测的大多数肌动蛋白都经历一种独特的翻译后修饰，称为加工过程，该过程由肌动蛋白N - 乙酰氨基肽酶催化。加工过程是指在具有Met - Asp（Glu）氨基末端的I类肌动蛋白中，从氨基末端去除乙酰甲硫氨酸。对于具有Met - X - Asp（Glu）氨基末端的II类肌动蛋白，加工过程是将第二个残基作为N - 乙酰氨基酸去除。其他具有这些氨基末端的胞质蛋白不会被加工，这表明该反应可能对肌动蛋白具有特异性。在肌动蛋白中，X通常是半胱氨酸。然而，有一些II类肌动蛋白中该残基不是半胱氨酸，这表明肌动蛋白N - 乙酰氨基肽酶的底物特异性比乙酰甲硫氨酸或乙酰半胱氨酸更广泛。我们构建了突变型肌动蛋白，其中该半胱氨酸被丝氨酸、天冬酰胺、甘氨酸、天冬氨酸、组氨酸、苯丙氨酸和酪氨酸取代，并利用这些突变型肌动蛋白在体外确定大鼠肝脏肌动蛋白N - 乙酰氨基肽酶的底物特异性。氨基末端的乙酰甲硫氨酸从相邻的天冬氨酸、天冬酰胺或组氨酸处被切割，但不从丝氨酸、甘氨酸、苯丙氨酸或酪氨酸处切割。在所测试的乙酰化肌动蛋白氨基末端中，只有乙酰甲硫氨酸和乙酰半胱氨酸被切割。组氨酸从未被N - 乙酰化，也未被切割。当苯丙氨酸和酪氨酸与起始甲硫氨酸相邻时，即使起始甲硫氨酸被乙酰化，也不会被切割。这些结果表明大鼠肝脏肌动蛋白N - 乙酰氨基肽酶的底物特异性较窄。它们还表明相邻残基可影响肌动蛋白N - 乙酰氨基肽酶的特异性。
