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A basic residue at position 36p of the propeptide is not essential for the correct folding and subsequent autocatalytic activation of prochymosin
- Francky, Andrej, Francky, Bojana MozetiÄ, Å trukelj, Borut, Gruden, Kristina, Ritonja, Anka, KriÅ¾aj, Igor, Kregar, Igor, Pain, Roger H., PungerÄar, JoÅ¾e
- European journal of biochemistry 2001 v.268 no.8 pp. 2362-2368
- Escherichia coli, active sites, arginine, calves, chymosin, complementary DNA, genome, glutamic acid, goats, lysine, mouflon, mutants, mutation, pH, pepsinogen, proteins, sheep, stomach, swine
- Position 36p in the propeptides of gastric aspartic proteinases is generally occupied by lysine or arginine. This has led to the conclusion that a basic residue at this position, which interacts with the activeâsite aspartates, is essential for folding and activation of the zymogen. Lamb prochymosin has been shown by cDNA cloning to possess glutamic acid at 36p. To investigate the effect of this natural mutation which appears to contradict the proposed role of this residue, calf and lamb prochymosins and their two reciprocal mutants, K36pE and E36pK, respectively, were expressed in Escherichia coli, refolded in vitro, and autoactivated at pHâ2 and 4.7. All four zymogens could be activated to active chymosin and, at both pH values, the two proteins with Glu36p showed higher activation rates than the two Lys36p forms. Glu36p was also demonstrated in natural prochymosin isolated from the fourth stomach of lamb, as well as being encoded in the genomes of sheep, goat and mouflon, which belong to the subfamily Caprinae. A conserved basic residue at position 36p of prochymosin is thus not obligatory for its folding or autocatalytic activation. The apparently contradictory results for porcine pepsinogen A [Richter, C., Tanaka, T., Koseki, T. & Yada, R.Y. (1999) Eur. J. Biochem.261, 746â752] can be reconciled with those for prochymosin. Lys/Arg36p is involved in stabilizing the propeptideâenzyme interaction, along with residues nearer the Nâterminus of the propeptide, the sequence of which varies between species. The relative contribution of residue 36p to stability differs between pepsinogen and prochymosin, being larger in the former.