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Binding, Proteolytic, and Crystallographic Analyses of Mutations at the Protease−Inhibitor Interface of the Subtilisin BPN‘/Chymotrypsin Inhibitor 2 Complex†,‡
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Abstract
A series of mutants of chymotrypsin inhibitor 2 (CI2), at residues that interact with the inhibited enzyme subtilisin BPN‘, were studied to determine the relative importance of intermolecular contacts on either side of the scissile bond. Mutants were tested for inhibition of subtilisin, rates of hydrolysis by subtilisin, and ability to acylate subtilisin. Additionally, crystal structures of the mutant CI2 complexes with subtilisin were obtained. Ordered water molecules were found to play an important role in inhibitor recognition, and features of the crystal structures, in combination with biochemical data, support a transition-state stabilization role for the P1 residue in subtilisin catalysis. Consistent with the proposed mechanism of inhibition, in which rapid acylation is followed by religation, leaving-group contacts with the enzyme were found to be more critical determinants of inhibition than acylating-group contacts in the mutants studied here.
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This article has been cited by 3 ACS Journal articles (3 most recent appear below).
Structure of a Switchable Subtilisin Complexed with a Substrate and with the Activator Azide
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Travis Gallagher, Biao Ruan, Mariya London, Molly A. Bryan and Philip N. BryanBiochemistry2009 48 (43), 10389-10394An engineered variant of the protease subtilisin from Bacillus amyloliquefaciens, in which the D32A mutation renders the enzyme’s activity dependent on the presence of certain small anions such as fluoride or azide, has been produced. This modified enzyme ...
Spacer Asn Determines the Fate of Kunitz (STI) Inhibitors, as Revealed by Structural and Biochemical Studies on WCI Mutants
Jhimli Dasgupta, Susmita Khamrui, Jiban K. Dattagupta, and Udayaditya SenBiochemistry2006 45 (22), 6783-6792Spacer Asn Determines the Fate of Kunitz (STI) Inhibitors, as Revealed by Structural and Biochemical Studies on WCI Mutants
Jhimli Dasgupta, Susmita Khamrui, Jiban K. Dattagupta, and Udayaditya SenBiochemistry2006 45 (22), 6783-6792The scaffold of serine protease inhibitors plays a significant role in the process of religation which resists proteolysis of the inhibitor in comparison to a substrate. Although the role of the conserved scaffolding Asn residue was previously implicated ...
Role of the Intramolecular Hydrogen Bond Network in the Inhibitory Power of Chymotrypsin Inhibitor 2,
Evette S. Radisky, Chia-Jung Karen Lu, Gene Kwan, and Daniel E. Koshland, Jr.Biochemistry2005 44 (18), 6823-6830Role of the Intramolecular Hydrogen Bond Network in the Inhibitory Power of Chymotrypsin Inhibitor 2,
Evette S. Radisky, Chia-Jung Karen Lu, Gene Kwan, and Daniel E. Koshland, Jr.Biochemistry2005 44 (18), 6823-6830A series of mutants of chymotrypsin inhibitor 2 (CI2), at residues involved in intramolecular interactions that shape and constrain the binding loop, were studied to determine their relative importance for inhibition of the serine protease subtilisin BPN‘,...
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History
- Published In Issue November 02, 2004
- Received June 9, 2004
Revised Manuscript Received August 23, 2004
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