Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Biochemistry

Language

English (en)

Date of Award

January 2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Enrico Di Cera

Abstract

Serine proteases of the chymotrypsin family play important roles in the regulation and function of numerous biological processes including digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis and immunity. For many of these proteases, activity unfolds when a zymogen is activated by limited proteolysis and the associated conformational changes result in the formation of a proper active site and oxyanion hole, both of which are required for efficient hydrolysis of peptide bonds. The transition from zymogen to active enzyme, E, thus provides critical temporal and spatial regulatory mechanism of protease function. Catalytic activity of serine proteases belonging to Vitamin K-dependent clotting factors is significantly affected by Na+ through an allosteric mechanism. Over the past 30 years, structural and biochemical studies revealed that Na+ enhances the enzymatic properties of these proteases from a low activity, E, to a high activity: E:Na+) conformation. However, investigation of the effects of Na+ on these proteases has mainly focused on the thermodynamics of interaction and the resulting catalytic enhancement, with little emphasis on characterizing the kinetics of Na+ binding. In deed, the kinetic mechanism of Na+ binding to many Na+-activated enzymes remain for the most part unexplored due to lack of convenient probes to monitor the interaction or the difficulty of resolving rate constants for reactions that likely occur on a very fast time scale. My thesis project aims to fill this gap in the investigation of Na+-activated proteases by elucidating the kinetic mechanism of Na+ binding to vitamin K-dependent clotting factors. While studying the kinetics of Na+ binding to human α-thrombin, we observed a biphasic mechanism of binding whose analysis led to the discovery that in the absence of Na+, the enzyme exists in dynamic equilibrium between two conformations, E* and E. Structural and kinetic studies indicate that E is the active form of the enzyme responsible for its catalytic properties while E* is an inactive conformation that features a collapsed active site cleft, a disrupted oxyanion hole and an abrogated Na+ binding site. E* is not unique to α-thrombin, however, as we have observed a similar E* to E transition in meizo-thrombin-des F1, factor IXa, factor Xa and activated protein C. Discovery of E* to E transition embedded in these trypsin-like enzymes is novel, and the observation of E*-like features in structures of other serine proteases reveal a level of unprecedented conformational plasticity present in the chymotrypsin fold. The inter-conversion between E* and E has mechanistic significance on how these proteases function in vivo. Based on the physiological role of each protease, catalytic activity can be regulated by properly setting the E*-E equilibrium, favoring E* or E depending on whether that protease requires low or high catalytic activity for its in vivo function. More importantly, stabilization of E* through mutagenesis can provide a low activity enzyme incapable of interacting with substrate or binding inhibitor until an appropriate cofactor binds and unleashes its full catalytic activity. Using α &ndash thrombin, a key enzyme of blood coagulation as a model system, we demonstrated how each conformation could be stabilized through rational protein engineering using site-directed mutagenesis. Stabilization of its E* form will turn α-thrombin into an effective anticoagulant agent that can be utilized for in vivo therapeutic purposes. In fact, α-thrombin mutants, E217K and W215A/E217A that show anticoagulant and antithrombotic effects in non-human primates both exhibit some structural features of E* like partial collapse of the 215-217 β-strand and disruption of the oxyanion hole. Thus stabilization of E* through mutagenesis or binding of a small molecule can provide an elegant regulatory control that can fine tune specificity along a particular pathway. In addition, discovery of E* in Na+-activated clotting proteases expands our understanding of allostery in monomeric enzymes in general and in particular explains why the activity of some thrombin mutants is orders of magnitude lower than the activity of the wild-type in the absence of Na+. Findings from this thesis project reveal a fundamental property of structure-function regulation in the vitamin K dependent clotting enzymes and thus set the stage for further investigation of inactive conformations in other serine proteases of the chymotrypsin family. Whether the presence of E* is a universal property of all serine proteases will await future studies.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7MC8X18

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