Mutant N143P Reveals How Na+ Activates Thrombin

• Published in: The Journal of biological chemistry Vol. 284; no. 52; pp. 36175 - 36185
• Main Authors: Niu, Weiling, Chen, Zhiwei, Bush-Pelc, Leslie A., Bah, Alaji, Gandhi, Prafull S., Di Cera, Enrico
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.12.2009; American Society for Biochemistry and Molecular Biology
• Subjects: AFFINITY; Allosteric Regulation - genetics; Amino Acid Substitution; ARCHITECTURE; ATOMS; CARBONYLS; CRYSTAL STRUCTURE; Crystallography, X-Ray; Enzyme Activation - genetics; Enzyme Catalysis and Regulation; ENZYMES; Humans; Hydrogen Bonding; KINETICS; MATERIALS SCIENCE; MUTANTS; Mutation, Missense; ORIENTATION; PEPTIDES; Protein Binding - genetics; Protein Structure, Tertiary - physiology; RESIDUES; Sodium - chemistry; Sodium - metabolism; Structure-Activity Relationship; THROMBIN; Thrombin - chemistry; Thrombin - genetics; Thrombin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 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.