Protonation state of a single histidine residue contributes significantly to the kinetics of the reaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator.
Stopped-flow fluorometry was used to study the kinetics of the reactive center loop insertion occurring during the reaction of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 plasminogen activator inhibitor-1 (PAI-1) with tissue-(tPA) and urokinase (uPA)-type plasminogen activators and human pancreatic elastase at pH 5.5-8.5. The limiting rate constants of reactive center loop insertion (k(lim)) and concentrations of proteinase at half-saturation (K(0.5)) for tPA and uPA and the specificity constants (k(lim)/K(0.5)) for elastase were determined. The pH dependences of k(lim)/K(0.5) reflected inactivation of each enzyme due to protonation of His57 of the catalytic triad. However, the specificity of the inhibitory reaction with tPA and uPA was notably higher than that for the substrate reaction catalyzed by elastase. pH dependences of k(lim) and K(0.5) obtained for tPA revealed an additional ionizable group (pKa, 6.0-6.2) affecting the reaction. Protonation of this group resulted in a significant increase in both k(lim) and K(0.5) and a 4.6-fold decrease in the specificity of the reaction of tPA with NBD P9 PAI-1. Binding of monoclonal antibody MA-55F4C12 to PAI-1 induced a decrease in k(lim) and K(0.5) at any pH but did not affect either the pKa of the group or an observed decrease in k(lim)/K(0.5) due to protonation of the group. In contrast to tPA, the k(lim) and K(0.5) for the reactions of uPA with NBD P9 PAI-1 or its complex with the monoclonal antibody were independent of pH in the 6.5-8.5 range. Since slightly acidic pH is a feature of a number of malignant tumors, alterations in PAI-1/tPA kinetics could play a role in the cancerogenesis. Changes in the protonation state of His(188), which is placed closely to the S1 site and is unique for tPA, has been proposed to contribute to the observed pH dependences of k(lim) and K(0.5).
Komissarov
Andrey A
AA
Division of Biochemical Research, Department of Pathology, Henry Ford Health System, Detroit, Michigan 48202, USA. akomiss1@hfhs.org
Declerck
Paul J
PJ
Shore
Joseph D
JD
D001665:Binding Sites; D002384:Catalysis; D006639:Histidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*
D017395:Plasminogen Activator Inhibitor 1; D006639:Histidine; D010959:Tissue Plasminogen Activator