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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
Author(s) -
Allert Malin,
Baltzer Lars
Publication year - 2002
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/1521-3765(20020603)8:11<2549::aid-chem2549>3.0.co;2-x
Subject(s) - decarboxylation , chemistry , imine , stereochemistry , residue (chemistry) , reaction rate constant , helix (gastropod) , asparagine , enzyme kinetics , active site , dimer , lysine , catalysis , amino acid , kinetics , biochemistry , organic chemistry , biology , ecology , physics , quantum mechanics , snail
Fourteen 42‐residue polypeptides have been designed to identify reactive sites for the catalysis of the decarboxylation of oxaloacetate, a chemical 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 hydrophobic properties to reduce their p K a values. The molecular environments of the Lys residues were systematically varied, to find which residues were able to stabilise 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 larger than the second‐order rate constant of the butylamine‐catalysed reaction (0.0011 M −1 s −1 ), and four orders of magnitude larger than the pseudo first‐order rate constant of the uncatalysed reaction (1.3×10 −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 provided more efficient catalysis than a flanking Lys or Gln residue. Arginines in flanking positions in the helix, in positions four residues before or after the Lys in the sequence, are not as important in catalysis as the Arg of the Arg–Lys pair. The effect of p K 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.