Mature cathepsin L is substantially active in the ionic milieu of the extracellular medium

The activity of cathepsin L is affected by ionic strength, resulting in the measured pH optimum being higher in acetate-4-morpholineethane sulfonic acid (MES)-Tris buffers of constant ionic strength than in phosphate buffers of constant molarity (and hence varying ionic strength). In acetate-MES-Tris and phosphate buffers of constant ionic strength across the pH range, the catalytic constant, k_cat, generally peaked at ca. pH 6.5 and essentially independently of ionic strength. K_m values, of ca. 5 μM, manifested a slight rising trend with increasing ionic strength, with a sharp increase to 20-25 μM, specifically at pH 6.5 and I = 0.4. At physiological ionic strengths, the specific buffer ions present affected the activity of mature cathepsin L, k_cat/K_m declining above pH 6.5 in phosphate buffer, but only above pH 7 in acetate-MES-Tris buffer. In Hanks' balanced salt solution, a model of the extracellular fluid, measured values at pH 7.2 were k_cat, 18.9 s^-1; K_m, 13.5 μM; and k_cat/K_m, 1.4 × 10⁶ M^-1 s^-1. The stability of cathepsin L in the physiological pH range was also differentially affected by the specific buffer ions, generally in parallel with the enzyme activity. In Hanks' balanced salt solution, mature cathepsin L was substantially active and stable, having a half-life of 179 s at pH 7.2 and 657 s at pH 6.8 (the peritumor pH).