Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach

Abstract

Papain-like proteases contain an N-terminal pro-peptide in their zymogen form that is important for correct folding and spatio-temporal regulation of the proteolytic activity of these proteases. Catalytic removal of the pro-peptide is required for the protease to become active. In this study, we have generated three different mutants of papain (I86F, I86L and I86A) by replacing the residue I86 in its pro-peptide region, which blocks the specificity determining S2-subsite of the catalytic cleft of the protease in its zymogen form with a view to investigate the effect of mutation on the catalytic activity of the protease. Steady-state enzyme kinetic analyses of the corresponding mutant proteases with specific peptide substrates show significant alteration of substrate specificity—I86F and I86L have 2.7 and 29.1 times higher kcat/Km values compared to the wild-type against substrates having Phe and Leu at P2 position, respectively, while I86A shows lower catalytic activity against majority of the substrates tested. Far-UV CD scan and molecular mass analyses of the mature form of the mutant proteases reveal similar CD spectra and intact masses to that of the wild-type. Crystal structures of zymogens of I86F and I86L mutants suggest that subtle reorganization of active site residues, including water, upon binding of the pro-peptide may allow the enzyme to achieve discriminatory substrate selectivity and catalytic efficiency. However, accurate and reliable predictions on alteration of substrate specificity require atomic resolution structure of the catalytic domain after zymogen activation, which remains a challenging task. In this study we demonstrate that through single amino acid substitution in pro-peptide, it is possible to modify the substrate specificity of papain and hence the pro-peptide of a protease can also be a useful target for altering its catalytic activity/specificity.

Characterization of a novel cathepsin L-like protease from Taenia solium metacestodes for the immunodiagnosis of porcine cysticercosis

“…Here it consists of a signal peptide, a pro-peptide, and a mature catalytic domain. The pro-peptide has an important role in the correct folding and spatio-temporal regulation of the enzyme's proteolytic activity (Dutta et al, 2016). In addition, pro-peptides contain the highly conserved consensus sequences of ERFNIN (Karrer et al, 1993) and GNFD.…”

“…The signal peptide is cleaved off by signal peptidases following transportation. The pro-peptide acts as an intramolecular chaperone which in turn catalyzes the folding of the catalytic domain of the protease (Dutta et al, 2016). These pro-peptides contain highly conserved elements such as the consensus sequence ERFNIN motif that is present in the α2 helix of cathepsin L in numerous species.…”

Reframing prosegment‐dependent folding and limits on natural protein folding landscapes from an evolutionary perspective

“…Several enzymes in this family are potential drug targets, including plasmepsin II, beta‐secretase/BACE1, cathepsin D, and renin, which are implicated in malaria, Alzheimer's disease, breast cancer, and hypertension, respectively 29 . More generally, mutations to the PS domain can direct folding into slightly altered conformations, even following PS removal, implying a “protein memory” effect 27,57 . Based on this phenomenon, pro‐sequence engineering 58 has been used to modulate the stability, specificity, and/or catalytic efficiency of proteins involved in industrial applications such as papain, keratinase, and nerve growth factor proteins 27,28,59 .…”

“…29 More generally, mutations to the PS domain can direct folding into slightly altered conformations, even following PS removal, implying a "protein memory" effect. 27,57 Based on this phenomenon, pro-sequence engineering 58 has been used to modulate the stability, specificity, and/or catalytic efficiency of proteins involved in industrial applications such as papain, keratinase, and nerve growth factor proteins. 27,28,59 Note that papain and keratinase are proteases, while nerve growth factor is not, showing the potential for broader application of PS-folding knowledge.…”

“…2,7,[17][18][19][20][21][22][23] It is important to clarify, however, that not all proteases require a PS to fold properly. 8 Our understanding of protein frustration, 24 kinetic stability, 25 protein evolution, 26 and protein design principles 27,28 may be advanced by careful analysis of zymogen and mature protease folding.…”