A basic residue at position 36p of the propeptide is not essential for thecorrect folding and subsequent autocatalytic activation of prochymosin

Position 36p in the propeptides of gastric aspartic proteinases is generall y occupied by lysine or arginine. This has led to the conclusion that a bas ic residue at this position, which interacts with the active-site aspartate s, is essential for folding and activation of the zymogen. Lamb prochymosin has been shown by cDNA cloning to possess glutamic acid at 36p. To investi gate the effect of this natural mutation which appears to contradict the pr oposed role of this residue, calf and lamb prochymosins and their two recip rocal mutants, K36pE and E36pK, respectively, were expressed in Escherichia coli, refolded in vitro, and autoactivated at pH 2 and 4.7. All four zymog ens could be activated to active chymosin and, at both pH values, the two p roteins with Glu36p showed higher activation rates than the two Lys36p form s. Glu36p was also demonstrated in natural prochymosin isolated from the fo urth stomach of lamb, as well as being encoded in the genomes of sheep, goa t and mouflon, which belong to the subfamily Caprinae. A conserved basic re sidue at position 36p of prochymosin is thus not obligatory for its folding or autocatalytic activation. The apparently contradictory results for porc ine pepsinogen A [Richter, C., Tanaka, T., Koseki, T & Yada, R.Y. (1999) Eu r J. Biochem. 261, 746-752] can be reconciled with those for prochymosin. L ys/Arg36p is involved in stabilizing the propeptide-enzyme interaction, alo ng with residues nearer the N-terminus of the propeptide, the sequence of w hich varies between species. The relative contribution of residue 36p to st ability differs between pepsinogen and prochymosin, being larger in the for mer.