Selective, Reversible Loss of Elastolytic Activity of Elastase and Subtilisin Resulting from Electrostatic Changes Due to Maleylation

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Abstract
Maleylation of ɛ‐amino groups of elastase and subtilisin causes a complete loss of their elastolytic activites while their esterolytic activities are almost unaffected. This results from a change in their electrostatic properties and is expressed in a complete loss of their ability to be adsorbed on elastin. The proteolytic activity of maleylated subtilisin toward casein and hemoglobin and V to ward protamine sulphate did not change, but the Km value for the last substrate was significantly incrased. The proteolytic activity of maleylated elastase toward casein and hemoglobin was reduced respectively to 30 and 70%, while the activity toward protamine sulphate was increased as a result of a ten‐fold decrease in Km value. These results indicate that ɛ‐amino groups of elastase serve as secondary binding sites. Elastolytic activity of 2,3‐dimethyl‐maleylated elastase and subtilisin could be recovered by hydrolysis of the 2,3‐dimethyl‐maleylamino bonds, at room temperature, pH 6.9. Acetyl‐L‐alanyl‐L‐alanine methyl ester was found to be an excellent esteratic substrate for subtilisin. The apparent Km and kcat values at 30°C, pH 8.0 are 1.7mM, and 1190 sec−1, respectively.