tailieunhanh - Báo cáo khoa học: Local changes in the catalytic site of mammalian histidine decarboxylase can affect its global conformation and stability

Mature, active mammalian histidine decarboxylase is a di-meric enzyme of carboxy-truncatedmonomers ( 53 kDa). By using a biocomputational approach, we have generateda three-dimensional model of a recombinant 1/512 fragment of the rat enzyme, which shows kinetic constants similar to those of the mature enzyme purified from rodent tissues. This model, together with previous spectroscopic data, al-lowed us to postulate that the occupation of the catalytic center by the natural substrate, or by substrate-analogs, would induce remarkable changes in the conformation of the intact holoenzyme | Eur. J. Biochem. 270 4376-4387 2003 FEBS 2003 doi Local changes in the catalytic site of mammalian histidine decarboxylase can affect its global conformation and stability Carlos Rodriguez-Caso1 Daniel Rodriguez-Agudo1 Aurelio A. Moya-Garcia1 Ignacio Fajardo1 Miguel Angel Medina1 Vinod Subramaniam2 and Francisca Sanchez-Jimenez1 1 Department of Molecular Biology and Biochemistry Faculty of Sciences Malaga Spain 2Max Planck Institute for Biophysical Chemistry Goettingen Germany Mature active mammalian histidine decarboxylase is a dimeric enzyme of carboxy-truncated monomers w 53 kDa . By using a biocomputational approach we have generated a three-dimensional model of a recombinant 1 512 fragment of the rat enzyme which shows kinetic constants similar to those of the mature enzyme purified from rodent tissues. This model together with previous spectroscopic data allowed us to postulate that the occupation of the catalytic center by the natural substrate or by substrate-analogs would induce remarkable changes in the conformation of the intact holoenzyme. To investigate the proposal conformational changes during catalysis we have carried out electrophoretic chromatographic and spectroscopic analyses of purified recombinant rat 1 512 histidine decarboxylase in the presence of the natural substrate or substrate-analogs. Our results suggest that local changes in the catalytic site indeed affect the global conformation and stability of the dimeric protein. Tlrese reuults icovi íná gl ts l ir new alternatives to inhibit histamine production efficiently in vivo. Keywords histidine decarboxylase histamine a-fluoro-methylhistidine L-histidine methyl ester pyridoxal phosphate-dependent enzymes. Mammalian histidine decarboxylase HDC the enzyme responsible for the biosynthesis of histamine is a pyridoxal 5 -phosphate PLP -dependent enzyme that belongs to the evolutionary group II of L-amino acid decarboxylases 1-3 . Histamine is involved in .