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Subsequent to the demonstration in the late 1950s of the catalytic power of the C2 anion⁄ylid of thiamin diphosphate, further convincing evidence was provided demonstrating that the 4¢-aminopyrimidine group plays a vital role in activation of this cofactor. | ỊFEBS Journal MINIREVIEW Reaction mechanisms of thiamin diphosphate enzymes new insights into the role of a conserved glutamate residue Boaz Shaanan and David M. Chipman Department of Life Sciences Ben-Gurion University Beer-Sheva Israel Keywords aminopyrimidine-iminopyrimidine tautomerization carboligation conserved glutamate 3D structure effective dielectric constant glyoxylate carboligase thiamin thiamin diphosphate-dependent enzyme Correspondence B. Shaanan Department of Life Sciences Ben-Gurion University Beer-Sheva 84105 Israel Fax 972 8 646 1710 Tel 972 8 647 2220 E-mail bshaanan@bgu.ac.il D. Chipman Department of Life Sciences Ben-Gurion University Beer-Sheva 84105 Israel Fax 972 8 646 1710 Tel 972 8 647 2646 E-mail chipman@bgu.ac.il Received 16 December 2008 revised 4 February 2009 accepted 9 February 2009 doi 10.1111 j.1742-4658.2009.06965.x Subsequent to the demonstration in the late 1950s of the catalytic power of the C2 anion ylid of thiamin diphosphate further convincing evidence was provided demonstrating that the 4 -aminopyrimidine group plays a vital role in activation of this cofactor. Structural evidence from several crystal structures of thiamin diphosphate-dependent enzymes emphasized the presence of a glutamate residue in hydrogen-bonding distance from N1 as a conserved element in these enzymes. The important role of this conserved glutamate in promoting C2-H ionization and activation of thiamin diphosphate was emphasized by site-directed mutagenesis studies. This role was further elaborated by spectroscopic studies of the 4 -aminopyrimidine-imi-nopyrimidine tautomerization. The low polarity of the environment of the protein-bound thiazolium is an additional factor in the stabilization of the C2 anion ylid. The recently determined crystal structure and mutagenesis studies of glyoxylate carboligase in which the position of the conserved glutamate is occupied by valine now show that for the multi-step reaction catalyzed by this enzyme the .
