COMPLEMENTARY-DNA CLONING AND KINETIC CHARACTERIZATION OF A NOVEL INTRACELLULAR SERINE PROTEINASE-INHIBITOR - MECHANISM OF ACTION WITH TRYPSIN AND FACTOR XA AS MODEL PROTEINASES

The full-length cDNA encoding a novel human intracellular serine prote inase inhibitor has been sequenced and found to encode a 376 amino aci d protein (M(r) approximate to 42.5K) that we designate as cytoplasmic antiproteinase. Analysis of the primary structure revealed that the c ytoplasmic antiproteinase has the majority of structural motifs conser ved among the greater superfamily of serine proteinase inhibitors, or serpins. On the basis of several criteria such as amino acid identity and the absence of a classical N-terminal signal peptide, the cytoplas mic antiproteinase represents a new member of the intracellular serpin family. Further inspection of the cytoplasmic antiproteinase amino ac id sequence identified three potential N-glycosylation sites and Arg(3 41)-Cys(342) as the reactive site P-1-P-1' residues, respectively. We have also employed the slow binding kinetic approach to detail the mec hanism of bovine trypsin and human factor Xa inhibition by the novel c ytoplasmic antiproteinase. Inhibition of trypsin by the cytoplasmic an tiproteinase was preceded by a two-step mechanism corresponding to the formation of an initial loose complex, followed by an isomerization s tep to a more stable, tight complex. The binding of the cytoplasmic an tiproteinase to trypsin occurred with a second-order association rate constant of 2.8 x 10(6) M(-1) s(-1) and an overall equilibrium constan t of 22.5 pM, demonstrating that the factor is a potent inhibitor of t his proteinase. Under the appropriate conditions, the tight complex be tween trypsin and the cytoplasmic inhibitor was reversible, indicated by an exponential regeneration of proteinase amidolytic activity from the preformed complex. Therefore, the tight complex appears to be stab ilized predominantly by reversible bonds that form between trypsin and the cytoplasmic inhibitor. In contrast to the inhibition of trypsin, the inhibition of factor Xa amidolytic activity by the cytoplasmic ant iproteinase followed a single-step binding mechanism. The apparent fir st-order rate constant for factor Xa inhibition was found to increase as a linear function of the inhibitor concentration range studied. For mation of the inhibitory complex between factor Xa and the cytoplasmic antiproteinase occurred with a second-order association rate constant of approximately 1.3 x 10(5) M(-1) s(-1) and a equilibrium constant o f 3.7 nM. These findings suggests that the cytoplasmic inhibitor may i nitially encounter significant energy barriers for proper alignment wi th the substrate binding cleft of factor Xa. However, once aligned, th e reaction proceeds rapidly to a tight factor Xa.inhibitor complex tha t dissociates at a slow rate.