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Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin.

ABSTRACT: An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P(1) (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'(2) favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P(1) and P'(2) substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin·APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.

SUBMITTER: Salameh MA

PROVIDER: S-EPMC2978618 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin.

Salameh Moh'd A MA Soares Alexei S AS Navaneetham Duraiswamy D Sinha Dipali D Walsh Peter N PN Radisky Evette S ES

The Journal of biological chemistry 20100922 47

An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor pr ...[more]

PMID: 20861008

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Project description:Enzymes are catalysts in biochemical reactions that, by definition, increase rates of reactions without being altered or destroyed. However, when that enzyme is a protease, a subclass of enzymes that hydrolyze other proteins, and that protease is in a multiprotease system, protease-as-substrate dynamics must be included, challenging assumptions of enzyme inertness, shifting kinetic predictions of that system. Protease-on-protease inactivating hydrolysis can alter predicted protease concentrations used to determine pharmaceutical dosing strategies. Cysteine cathepsins are proteases capable of cathepsin cannibalism, where one cathepsin hydrolyzes another with substrate present, and misunderstanding of these dynamics may cause miscalculations of multiple proteases working in one proteolytic network of interactions occurring in a defined compartment. Once rates for individual protease-on-protease binding and catalysis are determined, proteolytic network dynamics can be explored using computational models of cooperative/competitive degradation by multiple proteases in one system, while simultaneously incorporating substrate cleavage. During parameter optimization, it was revealed that additional distraction reactions, where inactivated proteases become competitive inhibitors to remaining, active proteases, occurred, introducing another network reaction node. Taken together, improved predictions of substrate degradation in a multiple protease network were achieved after including reaction terms of autodigestion, inactivation, cannibalism, and distraction, altering kinetic considerations from other enzymatic systems, since enzyme can be lost to proteolytic degradation. We compiled and encoded these dynamics into an online platform (https://plattlab.shinyapps.io/catKLS/) for individual users to test hypotheses of specific perturbations to multiple cathepsins, substrates, and inhibitors, and predict shifts in proteolytic network reactions and system dynamics.

Dataset Information

Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin.

Publications

Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin.

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