Disulfide-linked propeptides stabilize the structure of zymogen and mature pancreatic serine proteases

Research output: Contribution to journalArticle

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Abstract

Chymotrypsinogen and proelastase 2 are the only pancreatic proteases with propeptides that remain attached to the active enzyme via a disulfide bridge. It is likely, although not proven, that these propeptides are functionally important in the active enzymes, as well as in the zymogens. A mutant chymotrypsin was constructed to test this hypothesis, but it was demonstrated that the lack of the propeptide had no effect on the catalytic efficiency, substrate specificity, or folding of the protein [Venekei, I., et al. (1996) FEBS Lett. 379, 139-142]. In this paper, we investigate the role of the disulfide-linked propeptide in the conformational stability of chymotrypsin(ogen). We compare the stabilities of the wild-type and mutant proteins (lacking propeptide-enzyme interactions) in their zymogen (chymotrypsinogen) and active (chymotrypsin) forms. The mutants exhibited a substantially increased sensitivity to heat denaturation and guanidine hydrochloride unfolding, and a faster loss of activity at extremes of pH relative to those of their wild-type counterparts. From guanidine hydrochloride denaturation experiments, we determined that covalently linked propeptide provides about 24 kJ/mol of free energy of extra stabilization (ΔΔG). In addition, the mutant chymotrypsinogen lacked the normal resistance to digestion by pepsin. This may also explain (besides keeping the zymogen inactive) the evolutionary conservation of the propeptide-enzyme interactions. Tryptophan fluorescence, circular dichroism, microcalorimetric, and activity measurements suggest that the propeptide of chymotrypsin restricts the relative mobility between the two domains of the molecule. In pancreatic serine proteases, such as trypsin, that lose the propeptide upon activation, this function appears to be accomplished via alternative interdomain contacts.

Original languageEnglish
Pages (from-to)12248-12257
Number of pages10
JournalBiochemistry
Volume38
Issue number38
DOIs
Publication statusPublished - Sep 21 1999

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Chymotrypsinogen
Enzyme Precursors
Serine Proteases
Disulfides
Enzymes
Denaturation
Guanidine
Pepsin A
Circular Dichroism
Substrate Specificity
Tryptophan
Trypsin
Free energy
Digestion
Conservation
Peptide Hydrolases
Stabilization
Hot Temperature
Fluorescence
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

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Disulfide-linked propeptides stabilize the structure of zymogen and mature pancreatic serine proteases. / Kardos, J.; Bódi, Árpád; Závodszky, P.; Venekei, I.; Gráf, L.

In: Biochemistry, Vol. 38, No. 38, 21.09.1999, p. 12248-12257.

Research output: Contribution to journalArticle

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