Zhu Hongfa, Shi Jianying, Treber Michelle, Dai Jia, Arvidson Dennis N, Woldegiorgis Gebre
Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health and Science University, 20000 N.W. Walker Road, Beaverton 97006-8921, USA.
Arch Biochem Biophys. 2003 May 1;413(1):67-74. doi: 10.1016/s0003-9861(03)00081-x.
The muscle isoform of carnitine palmitoyltransferase I (M-CPTI) is 30- to 100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). We have previously shown that deletion of the first 28 N-terminal amino acid residues in M-CPTI abolished malonyl CoA inhibition and high-affinity binding [Biochemistry 39 (2000) 712-717]. To determine the role of specific residues within the first 28 N-terminal amino acids of human heart M-CPTI on malonyl CoA sensitivity and binding, we constructed a series of substitution mutations and a mutant M-CPTI composed of deletion 18 combined with substitution mutations V19A, L23A, and S24A. All mutants had CPT activity similar to that of the wild type. A change of Glu3 to Ala resulted in a 60-fold decrease in malonyl CoA sensitivity and loss of high-affinity malonyl CoA binding. A change of His5 to Ala in M-CPTI resulted in only a 2-fold decrease in malonyl CoA sensitivity and a significant loss in the low- but not high-affinity malonyl CoA binding. Deletion of the first 18 N-terminal residues combined with substitution mutations V19A, L23A, and S24A resulted in a mutant M-CPTI with an over 140-fold decrease in malonyl CoA sensitivity and a significant loss in both high- and low-affinity malonyl CoA binding. This was further confirmed by a combined four-residue substitution of Glu3, Val19, Leu23, and Ser24 with alanine. Our site-directed mutagenesis studies demonstrate that Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl CoA inhibition and binding, but not for catalysis.
肉碱棕榈酰转移酶I的肌肉同工型（M-CPTI）对丙二酰辅酶A抑制作用的敏感性比肝脏同工型（L-CPTI）高30至100倍。我们之前已经表明，M-CPTI中N端前28个氨基酸残基的缺失消除了丙二酰辅酶A的抑制作用和高亲和力结合[《生物化学》39(2000)712 - 717]。为了确定人心脏M-CPTI的N端前28个氨基酸内特定残基对丙二酰辅酶A敏感性和结合的作用，我们构建了一系列取代突变体以及一个由缺失18个氨基酸并结合V19A、L23A和S24A取代突变组成的突变型M-CPTI。所有突变体的CPT活性与野生型相似。将Glu3突变为Ala导致丙二酰辅酶A敏感性降低60倍，并丧失高亲和力丙二酰辅酶A结合能力。M-CPTI中His5突变为Ala仅导致丙二酰辅酶A敏感性降低2倍，并且低亲和力而非高亲和力丙二酰辅酶A结合能力显著丧失。N端前18个残基的缺失与V19A、L23A和S24A取代突变相结合，产生了一个突变型M-CPTI，其丙二酰辅酶A敏感性降低超过140倍，高亲和力和低亲和力丙二酰辅酶A结合能力均显著丧失。用丙氨酸对Glu3、Val19、Leu23和Ser24进行四个残基的联合取代进一步证实了这一点。我们的定点诱变研究表明，M-CPTI中的Glu3、Val19、Leu23和Ser24对丙二酰辅酶A的抑制作用和结合很重要，但对催化作用不重要。
