Inhibitory effect of soil humic compounds on the proteolytic enzyme pronase

Abstract

Neutralized solutions of soil humic acids inhibit the proteolytic activity of the enzyme pronase when tested against a variety of substrates. Protein hydrolysis was less sensitive than hydrolysis of dipeptide derivatives; 50% inhibition of benzyloxycarbonylglycylleucine hydrolysis was achieved with concentrations of humic acids as low as 1-2 µg/ml or less than 10-5M, on a carboxyl group basis. Humic acids, extracted from soils with different crop histories, showed only slight differences in their effectiveness as inhibitors of pronase activity. Their inhibitory power was comparable with that of other high molecular weight polyanions, e.g. polyacrylic acid and polycondensates derived from p-benzoquinone and catechol. Alginic acid was a relatively poor inhibitor. Preincubation of humic acids for various periods with either pronase or substrate (albumin or benzyloxycarbonylglycylleucine) had little or no effect on the subsequent inhibition of enzymic activity. However, inhibition is decreased by increasing substrate concentrations, following preincubation of humic acids and pronase. Both observations are consistent with a reversible inhibitory mechanism. Kinetic studies demonstrate that humic acids inhibit pronase activity towards albumin and N-benzyloxycarbonyl dipeptides by effectively reducing the affinity of pronase for the substrate, i.e. by increasing the Km value for the reaction. With benzoylarginine amide and benzoylarginine ethyl ester as substrates, the reaction velocity is lowered due to a reduction of the maximum velocity of the system. Both effects may possibly be explained by a conformational change in the enzyme structure due to combination with the humic acid molecules.