J. Biol. Chem., Vol. 263, Issue 8, 3729-3734, 03, 1988
The reaction of bovine alpha-thrombin with tetranitromethane. Characterization of the modified protein
RL Lundblad, CM Noyes, GL Featherstone, JH Harrison and JW Jenzano
Dental Research Center, School of Dentistry, University of North Carolina at Chapel Hill 27514.
Previous studies from several laboratories have shown that thrombin is
inactivated by tetranitromethane with the formation of nitrotyrosine. The
inactivation is characterized by an apparently greater loss of
fibrinogen-clotting activity than activity toward synthetic ester
substrates, suggesting that the residues modified by tetranitromethane are
involved in the interaction of thrombin with fibrinogen. This study was
designed 1) to determine the effect of solvent conditions on the rate of
modification and the stoichiometry of the reaction of tetranitromethane
with bovine alpha-thrombin; 2) to identify the residue(s) modified; and 3)
to characterize the modified enzyme with respect to its interaction with
peptide nitroanilide substrates and fibrinogen. The inactivation of
thrombin by tetranitromethane proceeded more rapidly in 50 mM Tris, pH 8.0,
than in 50 mM sodium phosphate, 100 mM NaCl, pH 8.0. Approximately 10%
fibrinogen-clotting activity remained at maximal inactivation. A study of
the effect of tetranitromethane concentration on the rate of inactivation
suggested that the loss of activity was the result of the modification of 1
mol of tyrosine/mol of thrombin. A similar result was obtained from the
analysis of the extent of inactivation as a function of the extent of
protein modification. Structural analysis of the modified protein showed
substantial modification at both Tyr71 and Tyr85. Enzyme kinetic studies
were performed with the modified protein and a control thrombin with
N2-tosylglycylprolylarginine p-nitroanilide. H-D-
phenylalanylpipecolylarginine p-nitronailide, and purified bovine
fibrinogen. With all three substrates, a substantial decrease in kcat was
observed, whereas there was essentially no change in Km. These results
suggest that, contrary to previous suggestions, the modification of Tyr71
and Tyr85 in thrombin does not influence the binding of substrates, but
rather influences active site reactivity.
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