Milović Nebojsa M, Kostić Nenad M
Department of Chemistry, Gilman Hall, Iowa State University, Ames 50011-3111, USA.
J Am Chem Soc. 2003 Jan 22;125(3):781-8. doi: 10.1021/ja027408b.
The X-Pro peptide bond (in which X represents any amino acid residue) in peptides and proteins is resistant to cleavage by most proteolytic enzymes. We show that Pd(H(2)O)(4) ion can selectively hydrolyze this tertiary peptide bond within the X-Pro-Met and X-Pro-His sequence segments. The hydrolysis requires an equimolar amount of the Pd(II) reagent and occurs under mild conditions-at temperature as low as 20 degrees C (with half-life of 1.0 h at pH 2.0) and at pH as high as 7.0 (with half-life of 4.2 h at pH 7.0 and 40 degrees C). The secondary peptide bond, exemplified by X-Gly in the X-Gly-Met and X-Gly-His sequence segments, however, is cleaved only in weakly acidic solution (pH < 4.0) and more slowly (half-life is 4.2 h at pH 2.0 and 60 degrees C). We explain the sequence-specificity of X-Pro cleavage by NMR spectroscopic analysis of the coordination of the X-Pro-Met segment to the Pd(II) ion. We give indirect evidence for the mechanism of cleavage by analyzing the conformation of the scissile X-Pro peptide bond, and by comparing the rate constants for the cleavage of the tertiary X-Pro peptide bond, the tertiary X-Sar peptide bond (Sar is N-methyl glycine), and the typical secondary X-Gly peptide bond in a set of analogous oligopeptides. Methionine and histidine side chains provide the recognition by selectively binding (anchoring) the Pd(II) ion. The proline residue provides the enhanced activity because its tertiary X-Pro peptide bond favors the cleavage-enhancing binding of the Pd(II) ion to the peptide oxygen atom and prevents the cleavage-inhibiting binding of the Pd(II) ion upstream of the anchoring (histidine or methionine) residue. Cleavage can be switched from the residue-selective to the sequence-specific mode by simply adjusting the pH of the aqueous solution. In acidic solutions, any X-Y bond in X-Y-Met and X-Y-His segments is cleaved because the cleavage is directed by anchoring methionine and histidine residues. In mildly acidic and neutral solutions, only the X-Pro bond in X-Pro-Met and X-Pro-His sequences is cleaved because of an interplay between the anchoring residue and the proline residue preceding it. Because Pro-Met and Pro-His sequences are rare in proteins, this sequence-specific cleavage is potentially useful for the removal of the fusion tags from the bioengineered fusion proteins.
肽和蛋白质中的X-脯氨酸肽键（其中X代表任何氨基酸残基）对大多数蛋白水解酶的切割具有抗性。我们发现，[Pd(H₂O)₄]²⁺离子可以选择性地水解X-脯氨酸-甲硫氨酸和X-脯氨酸-组氨酸序列段内的这种三级肽键。水解反应需要等摩尔量的Pd(II)试剂，并且在温和条件下发生——温度低至20℃（在pH 2.0时半衰期为1.0小时）以及pH高达7.0（在pH 7.0和40℃时半衰期为4.2小时）。然而，以X-甘氨酸-甲硫氨酸和X-甘氨酸-组氨酸序列段中的X-甘氨酸为例的二级肽键，仅在弱酸性溶液（pH < 4.0）中被切割，且速度更慢（在pH 2.0和60℃时半衰期为4.2小时）。我们通过对X-脯氨酸-甲硫氨酸片段与Pd(II)离子配位的NMR光谱分析来解释X-脯氨酸切割的序列特异性。我们通过分析可裂解的X-脯氨酸肽键的构象，并比较一组类似寡肽中三级X-脯氨酸肽键、三级X-肌氨酸肽键（肌氨酸是N-甲基甘氨酸）和典型二级X-甘氨酸肽键的切割速率常数，给出了切割机制的间接证据。甲硫氨酸和组氨酸侧链通过选择性结合（锚定）Pd(II)离子来实现识别。脯氨酸残基提供了增强的活性，因为其三级X-脯氨酸肽键有利于Pd(II)离子与肽氧原子的结合从而增强切割，并且阻止了Pd(II)离子在锚定（组氨酸或甲硫氨酸）残基上游的抑制切割的结合。通过简单地调节水溶液的pH，切割可以从残基选择性模式切换到序列特异性模式。在酸性溶液中，X-甲硫氨酸和X-组氨酸片段中的任何X-Y键都会被切割，因为切割是由锚定的甲硫氨酸和组氨酸残基引导的。在弱酸性和中性溶液中，只有X-脯氨酸-甲硫氨酸和X-脯氨酸-组氨酸序列中的X-脯氨酸键会被切割，这是由于锚定残基与其前面的脯氨酸残基之间的相互作用。由于脯氨酸-甲硫氨酸和脯氨酸-组氨酸序列在蛋白质中很少见，这种序列特异性切割对于从生物工程融合蛋白中去除融合标签可能是有用的。
