The use of proteolysis to study the structure of nardilysin

Treatment of a 128 kDa mouse nardilysin with trypsin initially produced an active 105 kDa N-terminally cleaved form. Continued trypsin digestion occurred at the C-terminus, producing inactive core species of ∼92, 76.5, and 62 kDa. Protease V8 digestion generated a stable ∼105 kDa form, nardilysin^V8, that was cleaved near the N-terminal trypsin site. The ∼105 kDa nardilysin^V8 exhibited the same K_m as did the uncleaved enzyme for substrates of the type Abz-GGFX₁X₂X₃VGQ-EDDnp, where X residues were varied. However, k_cat for nardilysin^V8 was 5-6 times greater. Both uncleaved nardilysin and nardilysin^V8 are inhibited by NaCl; however, nardilysin^V8 exhibits an IC₅₀ of ∼2 mM compared to an IC₅₀ of ∼50 mM for uncleaved nardilysin. Nardilysin^V8 is more sensitive to inhibition by phosphate buffer. Treatment of nardilysin^V8 with trypsin generated primarily the 92 kDa form which was inactive. Attempts to express nardilysin as a 105 kDa truncated N-terminal form or as a C-terminally truncated form led to inactive proteins.