Experimental and theoretical evidence supporting the role of Gly³⁶³ in blood coagulation factor IXa (Gly¹⁹³ in Chymotrypsin) for proper activation of the proenzyme

Factor IX is the zymogen of the serine protease factor IXa involved in blood coagulation. In addition to a catalytic domain homologous to the chymotrypsin family, it has Ca²⁺, phospholipid, and factor VIIIa binding regions needed for full biologic activity. We isolated a nonfunctional factor IX protein designated factor IX_{Eagle Rock} (IX_ER) from a patient with hemophilia B. The variant protein is indistinguishable from normal factor IX (IX_N) in its migration on sodium dodecyl sulfate-gel electrophoresis, isoelectric point in urea, carbohydrate content and distribution, number of γ-carboxyglutamic acid residues, and β-OH aspartic acid content, and in its binding to an anti-IX_N monoclonal antibody which has been shown previously to inhibit the interaction of factor VIIIa with factor IXa_N. Further, IX_ER is cleaved to yield a factor IXa-like molecule by factor XIa/Ca²⁺ at a rate similar to that observed for IX_N. However, in contrast to IXa_N, IXa_ER does not bind to antithrombin-III (specific inhibitor of IXa_N) and does not catalyze the activation of factor X (substrate) to factor Xa. To identify the mutation in IX_ER, all eight exons of IX_N and IX_ER gene were amplified by the polymerase chain reaction technique and cloned. A single point mutation (G → T) which results in the replacement of Val for Gly³⁶³ in the catalytic domain of IX_ER was identified. Gly³⁶³ in factor IXa corresponds to the universally conserved Gly¹⁹³ in the active site sequence of the chymotrypsin serine protease family. X-ray crystallographic data in the literature demonstrate a critical role of this Gly in stabilizing the active conformation of chymotrypsin/trypsin in two major ways: 1) in the formation of the substrate binding site; and 2) in the development of the oxyanion hole. Our computer structural data support a concept that the Gly³⁶³ → Val change prevents the development of the active site conformation in factor IXa such that the substrate binding site and the oxyanion hole are not formed in the mutated enzyme.