Replacement of thrombin residue G184 with Lys or Arg fails to mimic Na+ binding

Citation
Db. Roy et al., Replacement of thrombin residue G184 with Lys or Arg fails to mimic Na+ binding, PROTEINS, 43(3), 2001, pp. 315-318
Citations number
10
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEINS-STRUCTURE FUNCTION AND GENETICS
ISSN journal
0887-3585 → ACNP
Volume
43
Issue
3
Year of publication
2001
Pages
315 - 318
Database
ISI
SICI code
0887-3585(20010515)43:3<315:ROTRGW>2.0.ZU;2-R
Abstract
Na+ binding to thrombin enhances the catalytic activity toward numerous syn thetic and natural substrates, The bound Na+ is located in a solvent channe l 16 Angstrom away from the catalytic triad, and connects with D189 in the S1 site through an intervening water molecule, Molecular modeling indicates that the G184K substitution in thrombin positions the protonated E-amino g roup of the Lys side-chain to replace the bound Nat. Likewise, the G184R su bstitution positions the guanidinium group of the longer Arg side-chain to replace both the bound Na+ and the connecting water molecule to D189, We ex plored whether the G184K or G184R substitution would replace the bound Naand yield a thrombin derivative stabilized in the highly active fast form. Both the G184K and G184R mutants lost sensitivity to monovalent cations, as expected, but their activity toward a chromogenic substrate was compromise d up to 200-fold as a result of impaired diffusion into the S1 site and dec reased deacylation rate. Interestingly, both G184K and G184R substitutions compromised cleavage of procoagulant substrates fibrinogen and PAR1 more th an that of the anticoagulant substrate protein C. These findings demonstrat e that Na+ binding to thrombin is difficult to mimic functionally with resi due side-chains, in analogy with results from other systems. (C) 2001 Wiley -Liss, Inc.