Markovic O, Jörnvall H
Eur J Biochem. 1986 Aug 1;158(3):455-62. doi: 10.1111/j.1432-1033.1986.tb09775.x.
The first primary structure of a pectinesterase has been determined by analysis of the main form of this plant enzyme from tomatoes. The analysis was untraditional in the sense that few data were obtained by frequently used methods. Thus, digestion with trypsin, Glu-specific protease and several other enzymes gave limited cleavages and highly insoluble products. Instead, the determination was to a large extent based on peptides derived from chemical cleavages with CNBr at Met, N-chlorosuccinimide at Trp and hydroxylamine at Asn-Gly, plus an enzymatic cleavage at modified cysteine residues after chemical derivatizations. The structure shows the protein chain to be 305 residues long, with four half-cystine residues and five tryptophan residues. The N-terminus has a free alpha-amino group (from isoleucine). Two types of residue, tyrosine and histidine, have been functionally implied in the catalytic activity of the enzyme. Tyrosine is common in the protein (at 18 positions). However, only two histidine residues are found, and both are close to tyrosine residues in the primary structure (His-128 separated by one intervening residue from Tyr-126, and His-269 adjacent to Tyr-268), defining segment(s) of possible interest in relation to the active site.
通过对番茄中这种植物酶的主要形式进行分析，确定了果胶酯酶的首个一级结构。该分析方法非传统，因为通过常用方法获得的数据很少。因此，用胰蛋白酶、谷氨酸特异性蛋白酶和其他几种酶进行消化，得到的裂解产物有限且高度不溶。相反，该结构的确定在很大程度上基于用溴化氰在甲硫氨酸处、N-氯代琥珀酰亚胺在色氨酸处以及羟胺在天冬酰胺-甘氨酸处进行化学裂解产生的肽段，再加上化学衍生后在修饰的半胱氨酸残基处进行的酶促裂解。该结构表明蛋白质链长305个残基，有四个半胱氨酸残基和五个色氨酸残基。N端有一个游离的α-氨基（来自异亮氨酸）。两种残基，酪氨酸和组氨酸，在酶的催化活性中具有功能意义。酪氨酸在蛋白质中很常见（有18个位置）。然而，仅发现两个组氨酸残基，且在一级结构中两者都靠近酪氨酸残基（His-128与Tyr-126之间相隔一个中间残基，His-269与Tyr-268相邻），这确定了与活性位点相关的可能感兴趣的片段。
