Oxidation of methionine residues in antithrombin. Effects on biological activity and heparin binding.
Commercially available human plasma-derived preparations of the serine protease inhibitor antithrombin (AT) were shown to contain low levels of oxidation, and we sought to determine whether oxidation might be a means of regulating the protein's inhibitory activity. A recombinant form of AT, with similarly low levels of oxidation as purified, was treated with hydrogen peroxide in order to study the effect of oxidation, specifically methionine oxidation, on the biochemical properties of this protein. AT contains two adjacent methionine residues near the reactive site loop cleaved by thrombin (Met314 and Met315) and two exposed methionines that border on the heparin binding region of AT (Met17 and Met20). In forced oxidations with hydrogen peroxide, the methionines at 314 and 315 were found to be the most susceptible to oxidation, but their oxidation did not affect either thrombin-inhibitory activity or heparin binding. Methionines at positions 17 and 20 were significantly oxidized only at higher concentrations of peroxide, at which point heparin affinity was decreased. However at saturating heparin concentrations, activity was only marginally decreased for these highly oxidized samples of AT. Structural studies indicate that highly oxidized AT is less able to undergo the complete conformational change induced by heparin, most probably due to oxidation of Met17. Since this does not occur in less oxidized, and presumably more physiologically relevant, forms of AT such as those found in plasma preparations, oxidation does not appear to be a means of controlling AT activity.
Van Patten
S M
SM
Structural Protein Chemistry Group, Genzyme Corp., Framingham, Massachusetts 01701-9322, USA. svanpatten@genzyme.com
Hanson
E
E
Bernasconi
R
R
Zhang
K
K
Manavalan
P
P
Cole
E S
ES
McPherson
J M
JM
Edmunds
T
T
D000595:Amino Acid Sequence; D000990:Antithrombin III / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008715:Methionine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D000990:Antithrombin III; D006493:Heparin; D008715:Methionine; D006861:Hydrogen Peroxide