Noncovalent binding of a reaction intermediate by a designed helix-loop-helix motif-implications for catalyst design.
In our search for a catalyst for the transamination reaction of aspartic acid to form oxaloacetate, twenty-five forty-two-residue sequences were designed to fold into helix-loop-helix dimers and form binding sites for the key intermediate along the reaction pathway, the aldimine. This intermediate is formed from aspartic acid and the cofactor pyridoxal phosphate. The design of the binding sites followed a strategy in which exclusively noncovalent forces were used for binding the aldimine. Histidine residues were incorporated to catalyse the rate-limiting 1,3 proton transfer reaction that converts the aldimine into the ketimine, an intermediate that is subsequently hydrolysed to form oxaloacetate and pyridoxamine phosphate. The two most efficient catalysts, T-4 and T-16, selected from the pool of sequences by a simple screening procedure, were shown by CD and NMR spectroscopies to bind the aldimine intermediate with dissociation constants in the millimolar range. The mean residue ellipticity of T-4 in aqueous solution at pH 7.4 and a concentration of 0.75 mM was -18500 deg x cm(2) dmol(-1). Upon addition of 6 mm l-aspartic acid and 1.5 mM pyridoxal phosphate to form the aldimine, the mean residue ellipticity changed to -19900 deg x cm(2) dmol(-1). The corresponding mean residue ellipticities of T-16 were -21200 deg x cm(2) dmol(-1) and -24000 deg x cm(2) dmol(-1). These results show that the helical content increased in the presence of the aldimine, and that the folded polypeptides bound the aldimine. The (1)H NMR relaxation time of the imine CH proton of the aldimine was affected by the presence of T-4 as was the (31)P NMR resonance linewidth. The catalytic efficiencies of T-4 and T-16 were compared to that of imidazole and found to be more than three orders of magnitude larger. The designed binding sites were thus shown to be capable of binding the aldimine in close proximity to His residues, by noncovalent forces, into conformations that proved to be catalytically active. The results show for the first time the design of well-defined catalytic sites that bind a reaction intermediate with enzyme-like affinities under equilibrium conditions and represent an important advance in de novo catalyst design.
Allert
Malin
M
Department of Chemistry, Organic Chemistry, Göteborg University 41296 Göteborg, Sweden.
Baltzer
Lars
L
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D004798:Enzymes; D018257:Helix-Loop-Helix Motifs; D007097:Imines / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D062907:Oxaloacetic Acid / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet
D004798:Enzymes; D007097:Imines; D010455:Peptides; D062907:Oxaloacetic Acid; D001224:Aspartic Acid; D011732:Pyridoxal Phosphate