The mutant N143P reveals how Na+ activates thrombin Export

@article{citeulike:5986069, abstract = {10.1074/jbc.M109.069500 The molecular mechanism of thrombin activation by Na+ remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E and E:Na+ forms. The extended scheme establishes that analysis of kcat unequivocally identifies allosteric transduction of Na+ binding into enhanced catalytic activity. The thrombin mutant N143P features no Na+-dependent enhancement of kcat, yet binds Na+ with an affinity comparable to that of wild-type. Crystal structures of the mutant in the presence and absence of Na+ confirm that Pro143 abrogates the important H-bond between the backbone N atom of residue 143 and the carbonyl O atom of Glu192, which in turn controls the orientation of the Glu192-Gly193 peptide bond and the correct architecture of the oxyanion hole. We conclude that Na+ activates thrombin by securing the correct orientation of the Glu192-Gly193 peptide bond, which is likely flipped in the absence of cation. Absolute conservation of the 143-192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in protease function suggest that this mechanism of Na+ activation is present in all Na+-activated trypsin-like proteases.}, author = {Niu, Weiling and Chen, Zhiwei and Bush-Pelc, Leslie A. and Bah, Alaji and Gandhi, Prafull S. and Di Cera, Enrico}, citeulike-article-id = {5986069}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M109.069500}, citeulike-linkout-1 = {http://www.jbc.org/content/early/2009/10/21/jbc.M109.069500.abstract}, citeulike-linkout-2 = {http://www.jbc.org/content/early/2009/10/21/jbc.M109.069500.full.pdf}, citeulike-linkout-3 = {http://view.ncbi.nlm.nih.gov/pubmed/19846563}, citeulike-linkout-4 = {http://www.hubmed.org/display.cgi?uids=19846563}, day = {21}, doi = {10.1074/jbc.M109.069500}, journal = {Journal of Biological Chemistry}, keywords = {glu, glutamate, gly, glycine, pro, proline}, month = {October}, posted-at = {2009-10-22 18:00:00}, priority = {2}, title = {The mutant N143P reveals how Na+ activates thrombin}, url = {http://dx.doi.org/10.1074/jbc.M109.069500}, year = {2009} }

TY - JOUR ID - citeulike:5986069 L3 - citeulike-article-id:5986069 N2 - 10.1074/jbc.M109.069500 The molecular mechanism of thrombin activation by Na+ remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E and E:Na+ forms. The extended scheme establishes that analysis of kcat unequivocally identifies allosteric transduction of Na+ binding into enhanced catalytic activity. The thrombin mutant N143P features no Na+-dependent enhancement of kcat, yet binds Na+ with an affinity comparable to that of wild-type. Crystal structures of the mutant in the presence and absence of Na+ confirm that Pro143 abrogates the important H-bond between the backbone N atom of residue 143 and the carbonyl O atom of Glu192, which in turn controls the orientation of the Glu192-Gly193 peptide bond and the correct architecture of the oxyanion hole. We conclude that Na+ activates thrombin by securing the correct orientation of the Glu192-Gly193 peptide bond, which is likely flipped in the absence of cation. Absolute conservation of the 143-192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in protease function suggest that this mechanism of Na+ activation is present in all Na+-activated trypsin-like proteases. JF - Journal of Biological Chemistry TI - The mutant N143P reveals how Na+ activates thrombin KW - glu KW - glutamate KW - gly KW - glycine KW - pro KW - proline AU - Niu, Weiling AU - Chen, Zhiwei AU - Bush-Pelc, Leslie AU - Bah, Alaji AU - Gandhi, Prafull AU - Di Cera, Enrico PY - 2009/10/21/ UR - http://dx.doi.org/10.1074/jbc.M109.069500 ER -