Spectroscopic analysis of recombinant rat histidine decarboxylase.
Mammalian histidine decarboxylases have not been characterized well owing to their low amounts in tissues and instability. We describe here the first spectroscopic characterization of a mammalian histidine decarboxylase, i.e. a recombinant version of the rat enzyme purified from transformed Escherichia coli cultures, with similar kinetic constants to those reported for mammalian histidine decarboxylases purified from native sources. We analyzed the absorption, fluorescence and circular dichroism spectra of the enzyme and its complexes with the substrate and substrate analogues. The pyridoxal-5'-phosphate-enzyme internal Schiff base is mainly in an enolimine tautomeric form, suggesting an apolar environment around the coenzyme. Michaelis complex formation leads to a polarized, ketoenamine form of the Schiff base. After transaldimination, the coenzyme-substrate Schiff base exists mainly as an unprotonated aldimine, like that observed for dopa decarboxylase. However, the coenzyme-substrate Schiff base suffers greater torsion than that observed in other L-amino acid decarboxylases, which may explain the relatively low catalytic efficiency of this enzyme. The active center is more resistant to the formation of substituted aldamines than the prokaryotic homologous enzyme and other L-amino acid decarboxylases. Characterization of the similarities and differences of mammalian histidine decarboxylase with respect to other homologous enzymes would open new perspectives for the development of new and more specific inhibitors with pharmacological potential.
Department of Molecular Biology and Biochemistry, Faculty of Sciences, University of Málaga, 29071 Málaga, Spain.
María Teresa
MT
Olmo
Francisca
F
Sánchez-Jiménez
Miguel Angel
MA
Medina
Hideyuki
H
Hayashi
D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006640:Histidine Decarboxylase / Q000737:chemistry*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D011994:Recombinant Proteins; D006640:Histidine Decarboxylase