Probing Conformational Plasticity of the Activation Domain of Trypsin: The Role of Glycine Hinges

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• 作者：Linda Gombos ; Jó ; zsef Kardos ; Andr&aacute ; s Patthy ; Pé ; ter Medveczky ; L&aacute ; szló ; Szil&aacute ; gyi ; Andr&aacute ; s M&aacute ; ln&aacute ; si-Csizmadia ; L&aacute ; szló ; Gr&aacute ; f
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：February 12, 2008
• 年：2008
• 卷：47
• 期：6
• 页码：1675 - 1684
• 全文大小：283K
• 年卷期：v.47,no.6(February 12, 2008)
• ISSN：1520-4995

文摘

Trypsin-like serine proteases play essential roles in diverse physiological processes such ashemostasis, apoptosis, signal transduction, reproduction, immune response, matrix remodeling, development,and differentiation. All of these proteases share an intriguing activation mechanism that involves thetransition of an unfolded domain (activation domain) of the zymogen to a folded one in the active enzyme.During this conformational change, activation domain segments move around highly conserved glycinehinges. In the present study, hinge glycines were replaced by alanine residues via site directed mutagenesis.The effects of these mutations on the interconversion of the zymogen-like and active conformations aswell as on catalytic activity were studied. Mutant trypsins showed zymogen-like structures to varyingextents characterized by increased flexibility of some activation domain segments, a more accessibleN-terminus and a deformed substrate binding site. Our results suggest that the trypsinogen to trypsintransition is hindered by the mutations, which results in a shift of the equilibrium between the inactivezymogen-like and active enzyme conformations toward the inactive state. Our data also showed, however,that the inactive conformations of the various mutants differ from each other. Binding of substrate analoguesshifted the conformational equilibrium toward the active enzyme since inhibited forms of the trypsinmutants showed similar structural features as the wild-type enzyme. The catalytic activity of the mutantscorrelated with the proper conformation of the active site, which could be supported by varyingconformations of the N-terminus and the autolysis loop. Transient kinetic measurements confirmed theexistence of an inactive to active conformational transition occurring prior to substrate binding.