Kendall R L, Bradshaw R A
Department of Biological Chemistry, College of Medicine, University of California, Irvine 92717-1700.
J Biol Chem. 1992 Oct 15;267(29):20667-73.
A methionine aminopeptidase that specifically removes methionine residues from peptides with amino-terminal sequences of Met-Ala-, Met-Val-, Met-Ser-, Met-Gly-, and Met-Pro- but not Met-Leu- or Met-Lys- has been isolated to homogeneity from porcine liver by a procedure involving five chromatographic steps. The enzyme, whose specificity matches that predicted for the entity responsible for the co-translational amino-terminal processing of nascent polypeptide chains, has a measured molecular mass of 70,000 Da by SDS-polyacrylamide electrophoresis and 67,000 Da by gel chromatography (under nondenaturing conditions), suggesting the native molecule is a monomer. It is activated by Co2+ and inhibited by beta-mercaptoethanol and EDTA. With octapeptide substrates related to the amino-terminal portion of the beta-chain of human hemoglobin (with a histidine in position 3), the enzyme had a pH optimum of 6.0. With a synthetic peptide devoid of histidine, it showed no pH dependence from 6.0 to 8.0. This sensitivity may be due to the propensity of peptides with histidine in the third position to bind divalent cations such as Co2+. The measured Km and kappa cat values were affected by residues in the second position. The peptide corresponding to the natural sequence (Met-Val-His-) gave a kappa cat/Km value of 260 mM-1 s-1; substitution of alanine in the second position raised the kappa cat/Km to 1523 mM-1 s-1, but substitution of proline lowered the value to 130. The effects are primarily on the kappa cat. The substitution of proline (for histidine) in the third position, the mutation found in hemoglobin Long Island, prevents the removal of the methionine residue, as occurs with the mutant protein. The porcine liver enzyme is similar to methionine aminopeptidases isolated from Escherichia coli, Salmonella typhimurium, and yeast in that it also is stimulated by Co2+. However, it is much larger than these enzymes and differs somewhat in specificity, particularly with the yeast enzyme.
通过包含五个色谱步骤的程序，已从猪肝中分离出一种甲硫氨酸氨肽酶，该酶能特异性地从具有Met-Ala-、Met-Val-、Met-Ser-、Met-Gly-和Met-Pro-氨基末端序列的肽中去除甲硫氨酸残基，但不能从Met-Leu-或Met-Lys-序列的肽中去除。该酶的特异性与预测的负责新生多肽链共翻译氨基末端加工的实体的特异性相匹配，通过SDS-聚丙烯酰胺电泳测得的分子量为70,000 Da，通过凝胶色谱法（在非变性条件下）测得的分子量为67,000 Da，表明天然分子是单体。它被Co2+激活，被β-巯基乙醇和EDTA抑制。对于与人血红蛋白β链氨基末端部分相关的八肽底物（第3位为组氨酸），该酶的最适pH为6.0。对于不含组氨酸的合成肽，在6.0至8.0范围内它没有pH依赖性。这种敏感性可能是由于第3位带有组氨酸的肽倾向于结合二价阳离子如Co2+。测得的Km和κcat值受第2位残基的影响。对应于天然序列（Met-Val-His-）的肽的κcat/Km值为260 mM-1 s-1；第2位的丙氨酸取代将κcat/Km提高到1523 mM-1 s-1，但脯氨酸取代将该值降低到130。这些影响主要作用于κcat。第3位脯氨酸（取代组氨酸）的取代，即血红蛋白长岛型中发现的突变，阻止了甲硫氨酸残基的去除，就像突变蛋白那样。猪肝酶与从大肠杆菌、鼠伤寒沙门氏菌和酵母中分离出的甲硫氨酸氨肽酶相似，因为它也受Co2+刺激。然而，它比这些酶大得多，并且在特异性上有所不同，特别是与酵母酶不同。
