Keiko Kitagishi

Spectroscopic studies of pepsin and its complex with Streptomyces pepsin ingibitor

Binding of Streptomyces pepsin (EC 3.4.23.1) inhibitor to the active site of pepsin causes a characteristic ultraviolet difference spectrum having a trough around 298 nm which suggests that tryptophan residue(s) are involved in a decreased refractive index or different charge density. The fluoreschat of the pepsin-inhibitor complex. Relatively large circular dichroism (CD) spectrum change at 280--310 nm was observed upon binding of the inhibitor. Solvent perturbation difference spectra of pepsin alone and the pepsin-inhibitor. Solvent perturbation difference spectra of pepsin alone and the pepsin-inhibitor complex obtained with 20% ethylene glycol as perturbant showed that the exposed 2.5 tryptophan residues were not buried upon binding of the inhibitor, whereas 1.5 tyrosine residues were buried. It is speculated that the microenvironmental change around tryptophan residue(s) which are not located at the inhibitor binding site is induced upon binding of the inhibitor.

Kinetic study on the interaction of Rhizopus chinensis aspartic protease with Streptomyces pepsin inhibitor (acetylpepstatin)

The fluorescence of tryptophan residues of Rhizopus chinensis aspartic protease was quenched about 25% upon binding with an inhibitor, Streptomyces pepsin inhibitor (acetylpepstatin). The kinetics of binding between the enzyme and the inhibitor was studied by the fluorescence stopped-flow method. The concentration dependence of apparent rate constants was consistent with a two-step mechanism involving a fast bimolecular association followed by a slow unimolecular process. The unimolecular process was interpreted to be a conversion from a transient intermediate to the final complex in which the inhibitor is tightly bound to the active site of the enzyme. Fluorescence quenching occurred essentially in the unimolecular process, which suggests microenvironmental transition around at least one tryptophan residue in the enzyme-substrate complex.

Kinetic Studies on Binding of Mitochondrial and Cytosolic Aspartate Aminotransferases with Suicide Substrate, Gostatin

Binding of pig heart mitochondrial and cytosolic aspartate aminotransferases (m-AST and c-AST) with a specific inhibitor, gostatin (5-amino-2-carboxy-4-oxo-1,4,5,6-tetrahydropyridine-3-acetic acid) was studied kinetically, by monitoring the spectral change with a micro-stopped-flow apparatus and by following the inactivation of the enzyme activity. No appreciable difference in kinetics was observed between m-AST and c-AST. Any positive evidence for the existence of spectrophotometrically distinguishable intermediate was not obtained. Under the condition studied, the second-order rate constant of spectral change was about 1.5 to 2 times as large as that of inactivation.