Abstract
Two loop segments (183-189 and 221-225) in the protease domain of factor Xa contribute to the formation of a Na(+)-binding site. Studies with factor Xa indicate that binding of a single Na(+) ion to this site influences its activity by altering the S1 specificity site, and substitution of Tyr(225) with Pro diminishes sensitivity to Na(+). Using full-length factor Xa(Y225P), the allosteric relationship between the Na(+) site and other structural determinants in factor Xa and prothrombinase was investigated. Direct binding and kinetic measurements with probes that target the S1 specificity pocket indicate that assembly of the mutant in prothrombinase corrected the impaired binding of these probes observed with free factor Xa(Y225P). This appears to result from the apparent allosteric linkage between the factor Va, S1, and Na(+)-binding sites, since binding of the cofactor to membrane-bound factor Xa(Y225P) enhances binding at the S1 site and vice versa. Additional studies revealed that the internal salt bridge (Ile(16)-Asp(194)) of factor Xa(Y225P) is partially destabilized, a process that is reversible upon occupation of the S1 site. The data establish that alterations at the factor Xa Na(+)-binding site shift the zymogen-protease equilibrium to a more zymogen-like state, and as a consequence binding of S1-directed probes and factor Va are adversely affected. Therefore, the zymogen-like characteristics of factor Xa(Y225P) have allowed for the apparent allosteric linkage between the S1, factor Va, and Na(+) sites to become evident and has provided insight into the structural transitions which accompany the conversion of factor X to factor Xa.
Highlights
Factor X (FX)1 is a vitamin K-dependent two-chain glycoprotein that plays a central role in blood coagulation
Studies with factor Xa indicate that binding of a single Na؉ ion to this site influences its activity by altering the S1 specificity site, and substitution of Tyr225 with Pro diminishes sensitivity to Na؉
These findings suggest that FXaY225P has a reduced ability to bind benzamidine at the S1 specificity pocket
Summary
Materials—Hepes, sodium chloride, sodium cyanate, and polyethylene glycol (average Mr ϭ 8000) were from J. At selected time intervals (5–300 min), 5 l of the reaction mixture was placed in 95 l of assay buffer, and the residual enzymatic activity was determined from initial steady state rates of SpecXa hydrolysis, or aliquots were mixed with 1.5 M hydroxylamine, pH 8.0, and frozen at Ϫ80 °C for N-terminal sequence analysis. Inhibition Studies—Initial velocity measurements of SpecXa hydrolysis by FXa or prothrombinase using increasing concentrations of substrate at different fixed concentrations of PAB were analyzed according to the rate expression for linear competitive inhibition [50] to yield the fitted values for Km, Vmax, and Ki. Initial velocity data obtained following incubation of increasing concentrations of rTAP with two fixed concentrations of FXa or prothrombinase were analyzed as described [45]. Carbamylation of FXa in the Presence of PAB: Global Analysis of First Order Inactivation Rate Constants—The first order rate inactivation constant for FXa by NaNCO in the absence of PAB (k0) and in the presence of saturating concentrations of PAB (k1) as well as the equilibrium dissociation constant (Kd) for PAB binding to FXa were calculated by globally fitting activity data as a function of time to the ordinary differential equations describing the reaction mechanism shown in Scheme II using the program Dynafit [53] to extract Kd, k0, and k1
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