Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

Journal Article · Sat Dec 31 04:00:00 EST 2011 · Journal of Biological Chemistry

OSTI ID:1041744

Salameh, M; Soares, A; Navaneetham, D; Sinha, D; Walsh, P; Radisky, E

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{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 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{sub 1} and P'{sub 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.

• Research Organization: BROOKHAVEN NATIONAL LABORATORY (BNL)
• Sponsoring Organization: USDOE SC OFFICE OF SCIENCE (SC)
• DOE Contract Number: AC02-98CH10886
• OSTI ID: 1041744
• Report Number(s): BNL--97422-2012-JA
• Journal Information: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 47 Vol. 285; ISSN JBCHA3; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
AFFINITY
CATALYSIS
CATTLE
CLEAVAGE
CRYSTAL STRUCTURE
ENZYME INHIBITORS
FLEXIBILITY
HUMAN POPULATIONS
INACTIVATION
INTERACTIONS
KINETICS
MOBILITY
PRECURSOR
PROTEINS
PROTEOLYSIS
RESIDUES
STABILITY
THERMODYNAMICS
TRYPSIN
VULNERABILITY