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首页> 外文期刊>Chemistry: A European journal >Setting the Stage for New Catalytic Functions in Designed Proteins - Exploring the Imine Pathway in the Efficient Decarboxylation of Oxaloacetate by an Arg-Lys Site in a Four-Helix Bundle Protein Scaffold
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Setting the Stage for New Catalytic Functions in Designed Proteins - Exploring the Imine Pathway in the Efficient Decarboxylation of Oxaloacetate by an Arg-Lys Site in a Four-Helix Bundle Protein Scaffold

机译：在设计的蛋白质中为新的催化功能设定阶段-探索四螺旋束蛋白质支架中Arg-Lys位点有效催化草酰乙酸酯化的亚胺途径

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摘要

Fourteen 42-residue polypeptides have been designed to identify reactive sites for the catalysis of the decaroxylation of oxaloacetate, a chemial transformation that proceeds through the formation of an imine intermediate. The sequences fold into helix-loop-helix motifs and dimerise to four-helix bundles. The catalytically active lysine residues were incorporated in several surface exposed positions, but also in positions characterised by hyrophobic properties to reduce their pK_a values. The molecular environments of the Lys residues were systematically varied, to find which residues were able to stabilse and bind the imine intermediate in the decarboxylation reaction. A two-residue Arg-Lys site formed the main component of the reactive site of the helix-loop-helix dimer Decarb-K34_R33, which obeyed saturation kinetics in catalysing the reaction with a k_cat/K_M of 0.59 M~(-1) s~(-1). The rate constant measured was nearly three orders of magnitude was nearly three orders of magnitude larger than the second-order rate constant of the butylamine-catalysed reaction (0.01 1 M~(-1)s~(-1)), and four orders of magnitude larger than the pseudo first-order rate constant of the uncatalysed reaction (1.3 X 10~(-5) s(~-1)). The sequence of Decarb-K34_R33 contained only a single lysine residue. It was flanked by an arginine in the preceding position in the sequence. A flanking Arg residue provide more efficient catalysis than a flanking Lys or Gln residue. Arginines in flaking positions in the helix, in positions four residues before or after the Lys in the sequence, are not as important in cat alysis as the Arg of the Arg-Lys pair. The effect of pK_a on the catalytic efficiency of the Lys residue in the decarboxylation reaction is well known. The identification of the role of the flanking Arg residue in catalysing decarboxylation, its optimal position, and the importance of conformational stability reported here sets the stage for developing a number of catalytic systems that depend on the formation of imine intermediates, but that lead to different reaction products.
机译：已经设计了十四种具有42个残基的多肽，以识别用于催化草酰乙酸脱羧反应的反应性位点，草酸乙酸是通过形成亚胺中间体而进行的化学转化。序列折叠成螺旋-环-螺旋基序，二聚化为四螺旋束。将催化活性的赖氨酸残基掺入几个表面暴露的位置，但也掺入以疏水性质为特征的位置，以降低其pK_a值。 Lys残基的分子环境被系统地改变，以发现哪些残基能够在脱羧反应中稳定并结合亚胺中间体。螺旋-环-螺旋二聚体Decarb-K34_R33的两个残基Arg-Lys位点构成了反应位点的主要成分，在0.59 M〜（-1）s的k_cat / K_M催化反应时，其遵循饱和动力学。 〜（-1）。测得的速率常数比丁胺催化的反应的二阶速率常数（0.01 1 M〜（-1）s〜（-1））高出近三个数量级，比二阶速率常数大三倍。其大小大于未催化反应的拟一级反应速率常数（1.3 X 10〜（-5）s（〜-1））。 Decarb-K34_R33的序列仅包含一个赖氨酸残基。在序列的前面位置，它的两侧是一个精氨酸。侧翼的Arg残基提供比侧翼的Lys或Gln残基更有效的催化作用。在螺旋分析中，位于序列中Lys之前或之后四个残基的薄片位置中的精氨酸，在催化分析中不如Arg-Lys对的Arg重要。 pK_a对脱羧反应中Lys残基的催化效率的影响是众所周知的。侧链精氨酸残基在催化脱羧反应中的作用，其最佳位置以及构象稳定性的重要性的鉴定，为本文开发的许多催化系统奠定了基础，这些催化系统取决于亚胺中间体的形成，但会导致反应产物。

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