Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin

详细信息    查看全文

• 作者：Nicola Pozzi ; Mirco Zerbetto ; Laura Acquasaliente ; Simone Tescari ; Diego Frezzato ; Antonino Polimeno ; David W. Gohara ; Enrico Di Cera ; Vincenzo De Filippis
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：July 19, 2016
• 年：2016
• 卷：55
• 期：28
• 页码：3984-3994
• 全文大小：728K
• 年卷期：0
• ISSN：1520-4995

文摘

Thrombin exists as an ensemble of active (E) and inactive (E*) conformations that differ in their accessibility to the active site. Here we show that redistribution of the E*–E equilibrium can be achieved by perturbing the electrostatic properties of the enzyme. Removal of the negative charge of the catalytic Asp102 or Asp189 in the primary specificity site destabilizes the E form and causes a shift in the 215–217 segment that compromises substrate entrance. Solution studies and existing structures of D102N document stabilization of the E* form. A new high-resolution structure of D189A also reveals the mutant in the collapsed E* form. These findings establish a new paradigm for the control of the E*–E equilibrium in the trypsin fold.