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Thermodynamic linkage between the S1 site, the Na+ site, and the Ca2+ site in the protease domain of human activated protein C (APC) - Sodium ion in the APC crystal structure is coordinated to four carbonyl groups from two separate loops

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http://pubman.mpdl.mpg.de/cone/persons/resource/persons77772

Bode,  W.
Fässler, Reinhard / Molecular Medicine, Max Planck Institute of Biochemistry, Max Planck Society;
Huber, Robert / Structure Research, Max Planck Institute of Biochemistry, Max Planck Society;
Conti, Elena / Structural Cell Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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Schmidt, A. E., Padmanabhan, K., Underwood, M. C., Bode, W., Mather, T., & Bajaj, S. P. (2002). Thermodynamic linkage between the S1 site, the Na+ site, and the Ca2+ site in the protease domain of human activated protein C (APC) - Sodium ion in the APC crystal structure is coordinated to four carbonyl groups from two separate loops. Journal of Biological Chemistry, 277(32), 28987-28995.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0010-6E8A-9
Abstract
The serine protease domain of activated protein C (APC) contains a Na+ and a Ca2+ site. However, the number and identity of the APC residues that coordinate to Na+ is not precisely known. Further, the functional link between the Na+ and the Ca2+ site is insufficiently defined, and their linkage to the substrate S1 site has not been studied. Here, we systematically investigate the functional significance of these two cation sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na+ binds to the substrate-occupied APC with K-d values of similar to24 mM in the absence and similar to6 mM in the presence of Ca2+. Sodium-occupied APC has similar to100-fold increased catalytic efficiency (similar to4- fold decrease in K-m and similar to25-fold increase in k(cat)) in hydrolyzing S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) and Ca2+ further increases this k(cat) slightly (similar to1.2-fold). Ca2+ binds to the protease domain of APC with K-d values of similar to438 muM in the absence and similar to105 muM in the presence of Na+. Ca2+ binding to the protease domain of APC does not affect K-m but increases the k(cat) similar to10-fold, and Na+ further increases this k(cat) similar to3-fold and decreases the K-m value similar to3.7-fold. In agreement with the K-m data, sodium-occupied APC has similar to4-fold increased affinity in binding to p-aminobenzamidine (S1 probe). Crystallographically, the Ca2+ site in APC is similar to that in trypsin, and the Na+ site is similar to that in factor Xa but not thrombin. Collectively, the Na+ site is thermodynamically linked to the S1 site as well as to the protease domain Ca2+ site, whereas the Ca2+ site is only linked to the Na+ site. The significance of these findings is that under physiologic conditions, most of the APC will exist in Na2+-APC-Ca2+ form, which has 110-fold increased proteolytic activity.