Transient removal of proflavine inhibition of bovine β-trypsin by the bovine basic pancreatic trypsin inhibitor (Kunitz). A case for 'chronosteric effects'

E. Antonini, P. Ascenzi, M. Bolognesi, E. Menegatti, M. Guarneri

Research output: Contribution to journalArticlepeer-review

Abstract

The formation of the bovine β-trypsin-bovine basic pancreatic trypsin inhibitor (Kunitz) (BPTI) complex was monitored, making use of three different signals: proflavine displacement, optical density changes in the ultraviolet region, and the loss of the catalytic activity. The rates of the reactions indicated by the three different signals were similar at neutral pH, but diverged at low pH. At pH 3.50, proflavine displacement precedes the optical density changes in the ultraviolet and the loss of enzyme activity by several orders of magnitude in time. These data indicated that the bovine β-trypsin-BPTI complex formation is a multistage process and led to the prediction that, at pH 3.50, BPTI addition to the bovine β-trypsin-proflavine complex would remove proflavine inhibition and the enzyme would recover transiently its catalytic activity before being irreversibly inhibited by completion of BPTI binding. The kinetic evidences, here shown, verified this prediction, indicating that during the bovine β-trypsin-BPTI complex formation one transient intermediate occurs, which is not able to bind proflavine but may bind and hydrolyze the substrate. Thus, the observed peculiar catalytic behavior is in line with the proposed reaction mechanism for the bovine β-trypsin-BPTI complex formation, which postulates a sequence of distinct polar and apolar interactions at the contract area.

Original languageEnglish
Pages (from-to)4676-4678
Number of pages3
JournalJournal of Biological Chemistry
Volume258
Issue number8
Publication statusPublished - 1983

ASJC Scopus subject areas

  • Biochemistry

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