Two-dimensional 1H-NMR studies of horseradish peroxidase C and its interaction with indole-3-propionic acid.
The binding of aromatic donor molecules to plant peroxidases has been investigated by examining the complex formed between horseradish peroxidase isoenzyme C and indole-3-propionic acid using two-dimensional 1H-NMR spectroscopy. Despite the relatively high molecular mass and paramagnetism of the protein, this technique can be successfully applied to provide new information on the structure of the complex. A number of relatively well-resolved resonances in certain regions of the one-dimensional spectrum are assigned to amino acid type on the basis of the two-dimensional experiments. Two phenylalanine side chains are found to interact at positions close to the haem group as shown by nuclear Overhauser effect spectroscopy (NOESY). Furthermore, the NOESY spectrum of the complex reveals distinct interactions between these phenylalanine residues and the indole ring of the donor molecule. The binding site is found to comprise of these phenylalanine side chains and also the methyl group of a leucine or valine residue. On the basis of the model structure of horseradish peroxidase isoenzyme C proposed by Welinder and Nørskov-Lauritsen and information from previous studies of the related turnip peroxidases, possible locations for this binding site are discussed. The NMR methods adopted here may be generally applicable to the study of peroxidase--aromatic-donor interactions.
Veitch
N C
NC
Inorganic Chemistry Laboratory, University of Oxford, England.
Williams
R J
RJ
D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism; D006639:Histidine; D006735:Horseradish Peroxidase / Q000235:genetics / Q000378:metabolism*; D006859:Hydrogen; D007211:Indoles / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid
D001386:Azides; D007211:Indoles; D007527:Isoenzymes; D006639:Histidine; D006859:Hydrogen; C015292:indolepropionic acid; D006735:Horseradish Peroxidase; D010544:Peroxidases