Murine Thrombin Lacks Na+ Activation but Retains High Catalytic Activity

Leslie A Bush,Ryan W Nelson,Enrico Di Cera

doi:10.1074/jbc.m512082200

Leslie A Bush, Ryan W Nelson + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m512082200

Copy DOI

Abstract

Human thrombin utilizes Na+ as a driving force for the cleavage of substrates mediating its procoagulant, prothrombotic, and signaling functions. Murine thrombin has Asp-222 in the Na+ binding site of the human enzyme replaced by Lys. The charge reversal substitution abrogates Na+ activation, which is partially restored with the K222D mutation, and ensures high activity even in the absence of Na+. This property makes the murine enzyme more resistant to the effect of mutations that destabilize Na+ binding and shift thrombin to its anticoagulant slow form. Compared with the human enzyme, murine thrombin cleaves fibrinogen and protein C with similar k(cat)/K(m) values but activates PAR1 and PAR4 with k(cat)/K(m) values 4- and 26-fold higher, respectively. The significantly higher specificity constant toward PAR4 accounts for the dominant role of this receptor in platelet activation in the mouse. Murine thrombin can also cleave substrates carrying Phe at P1, which potentially broadens the repertoire of molecular targets available to the enzyme in vivo.

Highlights

Details of the molecular mechanism of Naϩ activation in thrombin have emerged recently from mutagenesis and structural analysis [9]
Does the presence of Lys-222 in murine thrombin preclude Naϩ binding? If so, what compensates for the potential loss of catalytic activity? The D221A/D222K mutant of human thrombin is devoid of Naϩ activation [15] due to complete disruption of the Naϩ binding site [16]
The mutant has functional properties intermediate between those of the Naϩ-free and Naϩ-bound forms of the wild type [15], which suggests that murine thrombin can retain significant catalytic activity even in the absence of Naϩ activation

Summary

Introduction

Details of the molecular mechanism of Naϩ activation in thrombin have emerged recently from mutagenesis and structural analysis [9]. The side chain of Tyr-225 secures the integrity of the water channel embedding the primary specificity pocket [12], and the backbone around Tyr-225 is oriented like the selectivity filter of the KcsA Kϩ channel [3, 13] Of these four residues, Glu-217 and Tyr-225 are conserved in thrombin from all species sequenced to date, from hagfish to human [14]. Asp-189 is a Ser in the sturgeon, and the D189S mutant of human thrombin has impaired Naϩ binding and substrate recognition [10]. The alternative possibility exists that the D222K substitution in murine thrombin is inconsequential on Naϩ binding. The results presented here fill an important gap in our understanding of an enzyme that has been the subject of numerous transgenic studies in recent years (18 –21) but for which no biochemical characterization has been reported

Methods

Results

Conclusion