Mutant N143P reveals how Na+ activates thrombin.
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 k(cat) unequivocally identifies allosteric transduction of Na(+) binding into enhanced catalytic activity. The thrombin mutant N143P features no Na(+)-dependent enhancement of k(cat) 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 Pro(143) abrogates the important H-bond between the backbone N atom of residue 143 and the carbonyl O atom of Glu(192), which in turn controls the orientation of the Glu(192)-Gly(193) peptide bond and the correct architecture of the oxyanion hole. We conclude that Na(+) activates thrombin by securing the correct orientation of the Glu(192)-Gly(193) 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.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Weiling
W
Niu
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Zhiwei
Z
Chen
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Leslie A
LA
Bush-Pelc
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Alaji
A
Bah
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Prafull S
PS
Gandhi
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110. Electronic address: enrico@wustl.edu.
Enrico
E
Di Cera
D000494:Allosteric Regulation / Q000235:genetics; D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D020125:Mutation, Missense; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D012964:Sodium; D013917:Thrombin