title: Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry.
abstract: Human M-proinsulin was cleaved by trypsin at the R(31)R(32)-E(33) and K(64)R(65)-G(66) bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K(59)R(60)-G(61) bond but at the B/C junction cleavage occurred at the R(31)R(32)-E(33) as well as the R(31)-R(32)E(33) bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k(cat.)/K(m) values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02±0.08×10(5)s(-1)M(-1)) and the cleavage of the K(29)-T(30) bond of M-insulin-RR (1.3±0.07×10(5)s(-1)M(-1)). However, the K(29)-T(30) bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k(cat.)/K(m) values around 1000s(-1)M(-1)). Hence, as the biosynthetic path follows the sequence; proinsulin→insulin-RR→insulin, the K(29)-T(30) bond becomes shielded, exposed then shielded again respectively.
authors.affiliation: School of Biological Sciences, University of the Punjab, New Campus, Lahore, 54590, Pakistan. annegardner80@yahoo.co.uk
authors.forename: Qurra-tul-Ann Afza
authors.initials: QA
authors.lastname: Gardner
authors.forename: Hooria
authors.initials: H
authors.lastname: Younas
authors.forename: Muhammad
authors.initials: M
authors.lastname: Akhtar
mesh_terms: D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011384:Proinsulin / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010455:Peptides; D011384:Proinsulin; D014357:Trypsin