Abstract
Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.
Highlights
Canonical serine protease inhibitors normally behave as “uncleavable” substrates; mesotrypsin targets these inhibitors as substrates
We use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates
To identify the subset of substitutions at these positions that are both necessary and sufficient to account for mesotrypsin resistance to inhibition and for inhibitor-cleaving activity, we introduced mesotrypsin residues into the corresponding positions of trypsin by site-directed mutagenesis
Summary
Canonical serine protease inhibitors normally behave as “uncleavable” substrates; mesotrypsin targets these inhibitors as substrates. In 1980, Laskowski and co-workers [7, 8] reported that an unusual trypsin-like enzyme from the starfish Dermasterias imbricata was able to cleave the reactive site bonds of several canonical serine protease inhibitors at highly accelerated rates This discovery came a few years too early to benefit from the genomic revolution, i.e. the gene and amino acid sequences of the enzyme were not determined and the molecular adaptations responsible for its catalytic capability remain a mystery. We have recently reported that mesotrypsin targets multiple endogenous human canonical inhibitors for cleavage with substratelike kinetics [6, 13], identifying a spectrum of likely physiological substrates that may enable mesotrypsin to function as a gatekeeper in the protease web At present, it is not clear how prevalent such enzymes are in nature nor how complex the evolutionary adaptation involved in achieving this unusual gain-of-function. Our results demonstrate that the canonical inhibitor-cleaving capability is a complex catalytic activity that is not automatically conferred upon enzymes with reduced inhibitor binding affinity, and our results suggest that in mesotrypsin the ability to cleave and inactivate trypsin inhibitors is a functional adaptation that may have evolved in response to positive selection pressure
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