Esterolytic Properties of Leucine-Proteinase, the Leucine-Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves 1: A Steady-State and Pre-Steady-State Study

AUTOR(ES)

Aducci, Patrizia

RESUMO

Steady-state and pre-steady-state kinetics for the hydrolysis of p-nitrophenyl esters of N-α-carbobenzoxy(-l-)amino acids catalyzed by leucine-proteinase were determined between pH 5 and 10 (I = 0.1 molar) at 23 ± 0.5°C. For the substrates considered: (a) the acylation step is rate-limiting in catalysis; (b) the pH profiles of kcat and kcat/Km reflect the ionization of two groups with pKa 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 Km are pH independent. Among the substrates examined, N-α-carbobenzoxy-l-leucine-p-nitrophenyl ester shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 5 × 10−10 molar at the optimum pH value (approximately 7.5).

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