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首页> 外文期刊>Biochemistry >STRUCTURE AND FUNCTION OF SALMONELLA TYPHIMURIUM OROTATE PHOSPHORIBOSYLTRANSFERASE - PROTEIN COMPLEMENTATION REVEALS SHARED ACTIVE SITES
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STRUCTURE AND FUNCTION OF SALMONELLA TYPHIMURIUM OROTATE PHOSPHORIBOSYLTRANSFERASE - PROTEIN COMPLEMENTATION REVEALS SHARED ACTIVE SITES

机译：鼠伤寒沙门氏菌乳清蛋白磷酸核糖转移酶的结构和功能-蛋白质互补序列揭示了活性位点

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摘要

A solvent-exposed loop, comprising residues 98-119 of S. typhimurium orotate phosphoribosyltransferase (OPRTase), is at the subunit interface of the dimeric enzyme, and its amino acid side chains potentially contact active sites on either subunit. A portion of the loop (103-107) appears to be mobile on the basis of the X-ray structures of enzyme . OMP [Scapin, G., Grubmeyer, C., and Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294] and enzyme . PRPP . orotate complexes [Scapin, G., Ozturk, D. H., Grubmeyer, C., and Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754]. Lys-103, which is essential for activity [Ozturk, D. H., Dorfman, R. H., Scapin, G., Sacchettini, J. C., and Grubmeyer, C. (1995) Biochemistry 34, 10755-10763], may thus be functional in the active site formed by the adjacent subunit. Asp-125 is an essential residue that is in the middle of the active site. Equimolar mixtures of the nearly inactive K103A and D125N mutant ORPTase subunits produced approximately 21-23% of the enzymatic activity of the wild-type OPRTase. Heterodimer formation in the complemented mixtures was evidenced by various physical methods. Thus, the active site of OPRTase requires Asp-125 from one subunit and Lys-103 from the adjacent subunit. As predicted from the three-dimensional structure, increased activity resulting from complementation was also observed with mixtures of the K103A mutant and the poorly active K73A and K73Q mutants but not with mixtures of D125N and either K73A or K73Q mutants. Neither K103A nor D125N mutants exhibited negative complementation with the wild-type enzyme. A K103A/D125N double mutant enzyme was also constructed and was able to inactivate wild-type enzyme. Negative complementation between the wild-type and double mutant enzyme was used to determine the subunit dissociation rate for the wild-type dimer, k(D) = 0.013 min(-1).
机译：暴露于溶剂的环，包括鼠伤寒沙门氏菌磷酸酯核糖基转移酶（OPRTase）的残基98-119，位于二聚酶的亚基界面，其氨基酸侧链可能与任一亚基上的活性位点接触。根据酶的X射线结构，环的一部分（103-107）似乎是可移动的。 OMP [Scapin，G.，Grubmeyer，C。和Sacchettini，J.C。（1994）Biochemistry 33，1287-1294]和酶。 PRPP。乳清酸盐复合物[Scapin，G.，Ozturk，D.H.，Grubmeyer，C。，和Sacchettini，J.C。（1995）Biochemistry 34，10744-10754]。 Lys-103对活性至关重要[Ozturk，DH，Dorfman，RH，Scapin，G.，Sacchettini，JC，and Grubmeyer，C.（1995）Biochemistry 34，10755-10763]，因此可能在活性物质中起作用由相邻亚基形成的位点。 Asp-125是必需的残基，位于活性位点的中间。几乎无活性的K103A和D125N突变ORPTase亚基的等摩尔混合物产生的野生型OPRTase的酶活性约为21-23％。通过各种物理方法证明了在互补混合物中的异二聚体形成。因此，OPRTase的活性位点需要一个亚基的Asp-125和相邻亚基的Lys-103。如从三维结构所预测的，还观察到由K103A突变体与活性较差的K73A和K73Q突变体的混合物互补产生的活性，但与D125N和K73A或K73Q突变体的混合物均未观察到。 K103A和D125N突变体均未显示出与野生型酶的负互补。还构建了K103A / D125N双突变酶，其能够灭活野生型酶。野生型和双突变酶之间的负互补用于确定野生型二聚体的亚基解离速率，k（D）= 0.013 min（-1）。

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