Andres D A, Goldstein J L, Ho Y K, Brown M S
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235-9046.
J Biol Chem. 1993 Jan 15;268(2):1383-90.
Protein farnesyltransferase from rat brain is composed of tightly associated alpha- and beta-subunits of 377 and 437 amino acids that migrate on SDS-polyacrylamide gels with apparent molecular masses of 49 and 46 kDa, respectively. The enzyme attaches farnesyl groups to cysteines in p21ras and other proteins that contain cysteine residues at the fourth position from the COOH terminus. Production of stable enzyme in animal cells requires the simultaneous synthesis of both subunits, and all activity is lost when the subunits are dissociated chemically. The beta-subunit functions in the Zn(2+)-dependent binding of the protein substrate. The role of the alpha-subunit is unknown. In the current studies we used in vitro mutagenesis and transfection of cloned cDNAs to define the parts of the alpha-subunit that are necessary to stabilize the beta-subunit and to support farnesyl transfer. Deletion of 51 amino acids at the NH2 terminus of the alpha-subunit allowed normal stabilization of the beta-subunit and production of normal enzyme activity, but deletion of 106 amino acids abolished both of these properties. A proline-rich region at residues 12-34 of the alpha-subunit is not required for activity, but its presence explains the anomalously slow migration of the polypeptide on SDS-polyacrylamide gels. Deletion of only 5 amino acids at the COOH terminus of the alpha-subunit reduced activity appreciably. Substitution of asparagine for a conserved lysine at position 164 produced an alpha-subunit that stabilized the beta-subunit normally and permitted normal binding of the two substrates, farnesyl pyrophosphate and p21H-ras. Nevertheless, the rate of transfer of the bound farnesyl group to p21H-ras was markedly reduced. The latter finding suggests that the alpha-subunit plays a direct role in the catalytic reaction in addition to its role in the stabilization of the beta-subunit.
大鼠脑内的蛋白质法尼基转移酶由紧密结合的α亚基和β亚基组成，α亚基含377个氨基酸，β亚基含437个氨基酸，它们在SDS-聚丙烯酰胺凝胶上迁移时，表观分子量分别为49 kDa和46 kDa。该酶将法尼基基团连接到p21ras及其他在COOH末端起第4位含有半胱氨酸残基的蛋白质中的半胱氨酸上。在动物细胞中产生稳定的酶需要同时合成两个亚基，当亚基化学解离时所有活性都会丧失。β亚基在蛋白质底物的Zn(2+)依赖性结合中起作用。α亚基的作用尚不清楚。在当前研究中，我们使用体外诱变和克隆cDNA转染来确定α亚基中稳定β亚基和支持法尼基转移所必需的部分。α亚基NH2末端缺失51个氨基酸可使β亚基正常稳定并产生正常的酶活性，但缺失106个氨基酸则会消除这两个特性。α亚基第12 - 34位残基处富含脯氨酸的区域对活性不是必需的，但其存在解释了该多肽在SDS-聚丙烯酰胺凝胶上异常缓慢的迁移。α亚基COOH末端仅缺失5个氨基酸就会明显降低活性。将第164位保守的赖氨酸替换为天冬酰胺产生的α亚基能正常稳定β亚基，并允许两种底物法尼基焦磷酸和p21H-ras正常结合。然而，结合的法尼基基团转移到p21H-ras的速率明显降低。后一发现表明，α亚基除了在稳定β亚基方面起作用外，还在催化反应中起直接作用。
