N-α-Benzoyl-L-leucine

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respectively) to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21 degrees C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from approximately 6.9, in the free Leu-proteinase, to approximately 5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c-Ka = 2.2 x 10(11) M-1, delta G degree = -64 kJ/mol, delta H degree = +5.9 kJ/mol, and delta S degree = +240 kJ/molK; Leu-proteinase:BBI-Ka = 3.2 x 10(10) M-1, delta G degree = -59 kJ/mol, delta H degree = +8.8 kJ/mol, and delta S degree = +230 J/molK; and Leu-proteinase:F-C-Ka = 1.1 x 10(6) M-1, delta G degree = -34 kJ/mol, delta H degree = +18 J/mol, and delta S degree = +180 J/molK (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature-independent over the range explored, i.e. between 10.0 degrees C and 40.0 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Steady-state and pre-steady-state kinetics for the hydrolysis of p-nitrophenyl esters of N-alpha-carbobenzoxy(-l-)amino acids catalyzed by leucine-proteinase were determined between pH 5 and 10 (I = 0.1 molar) at 23 +/- 0.5 degrees C. For the substrates considered: (a) the acylation step is rate-limiting in catalysis; (b) the pH profiles of k(cat) and k(cat)/K(m) reflect the ionization of two groups with pK(a) values ranging between 6.5 and 6.9, and 8.1 and 8.3 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); and (c) values of K(m) are pH independent. Among the substrates examined, N-alpha-carbobenzoxy-l-leucine-p-nitrophenyl ester shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 5 x 10(-10) molar at the optimum pH value (approximately 7.5).

A proteinase of Mr 27,000 with a possible role in the metabolism and invasion of host tissues was purified from the conditioned medium of Trichophyton rubrum by concanavalin A and anion-exchange chromatography. Peaks of proteolytic activity were analyzed by substrate gel electrophoresis. The 27,000-Mr proteinase had a pH optimum of 8.0, a calcium dependence of 2 mM, and was inhibited by serine proteinase inhibitors, especially phenylmethylsulfonyl fluoride and Phe-Gly-Ala-Leu-chloromethyl ketone. By polyacrylamide gel electrophoresis, the 27,000-Mr proteinase had a reduced molecular weight of 44,000 and reacted with [3H]diisopropyl fluorophosphate. The proteinase degraded azocoll, type III collagen, type IV procollagen, laminin, fibronectin, and the peptide substrates succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (1,573 M-1 s-1) and t-butyloxy carbonyl-Ala-Ala-Leu-p-nitroanilide (1,614 M-1 s-1).