Esterolytic Properties of Leucine-Proteinase, the Leucine-Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves

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Abstract
Steady-state and pre-steady-state kinetics for the hydrolysis for 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.degree. 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-.alpha.-carbobenzoxyl-L-leucine-p-nitrophenyl ester shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 5 .times. 10-10 molar at the optimum pH value (approximately 7.5).
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