Legumain forms from plants and animals differ in their specificity

We purified forms of legumain from a plant source (seeds of kidney bean, Ph aseolus vulgaris) and a mammal (kidney of pig, Sus scropha) for comparison of their properties. Both forms were found to be stable only under moderate ly acidic pH conditions, and were maximally active at about pH 6; the plant enzyme was somewhat less stable and had a slightly higher pH optimum. With benzyloxycarbonyl-Xaa-Ala-Asn-aminomethylcoumarylamide substrates, the two forms of legumain showed distinctly different specificities for the P3 res idue, the plant legumain preferring amino acids with bulky hydrophobic side chains because of lower K-m values. Both forms of legumain were highly spe cific for hydrolysis of asparaginyl bonds in the arylamide substrates and i n neurotensin. Aspartyl bonds were hydrolysed about 100-fold more slowly wi th lower pH optima. Potential substrates containing other amino acids struc turally similar to asparagine were not hydrolysed. There were clear differe nces in specificity of hydrolysis of protein substrates. The plant legumain differed from pig legumain in its action on tetanus toxoid C-fragment, cle aving at Asn(97) but not at Asn(337), and produced more extensive digestion of phaseolin. The plant form of legumain was much more weakly inhibited by egg-white cystatin than was the mammalian form.