The critical role of the 185-189-loop in the factor Xa interaction with Na+ and factor Va in the prothrombinase complex.
The S1 site (Asp(189)) of factor Xa (fXa) is located on a loop (residues 185-189) that contains three solvent-exposed charged residues (Asp(185), Lys(186), and Glu(188)) below the active-site pocket of the protease. To investigate the role of these residues in the catalytic function of fXa, we expressed three mutants of the protease in which the charges of these residues were neutralized by their substitutions with Ala (D185A, K186A, and E188A). Kinetic studies revealed that E188A has a normal catalytic activity toward small synthetic and natural substrates and inhibitors of fXa; however, the same activities were slightly ( approximately 2-fold) and dramatically ( approximately 20-50-fold) impaired for the D185A and K186A mutants, respectively. Further studies revealed that the affinity of D185A and K186A for interaction with Na(+) has also been altered, with a modest impairment ( approximately 2-fold) for the former and a dramatic impairment for the latter mutant. Both prothrombinase and direct binding studies indicated that K186A also has an approximately 6-fold impaired affinity for factor Va. Interestingly, a saturating concentration of factor Va restored the catalytic defect of K186A in reactions with prothrombin and the recombinant tick anticoagulant peptide that is known to interact with the Na(+) loop of fXa, but not with other substrates. These results suggest that factor Va interacts with 185-189-loop for fXa, which is energetically linked to the Na(+)-binding site of the protease.
Rezaie
Alireza R
AR
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA. rezaiear@slu.edu
Kittur
Farooqahmed S
FS
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000378:metabolism; D060829:Arthropod Proteins; D001665:Binding Sites; D002460:Cell Line; D005165:Factor V / Q000235:genetics / Q000378:metabolism*; D015943:Factor Va / Q000235:genetics / Q000378:metabolism*; D015951:Factor Xa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008074:Lipoproteins / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012964:Sodium / Q000378:metabolism*
D000991:Antithrombins; D060829:Arthropod Proteins; D065427:Factor Xa Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D008074:Lipoproteins; D010455:Peptides; C051928:lipoprotein-associated coagulation inhibitor; C022475:prothrombinase complex; C063967:tick anticoagulant peptide; D015943:Factor Va; D005165:Factor V; D012964:Sodium; D015951:Factor Xa; D000409:Alanine