Cell cycle-dependent caspase-like activity that cleaves p27(KIP1) is the beta(1) subunit of the 20S proteasome.

We previously described a caspase-like activity, which we termed KIPase that is implicated in the turnover of the mammalian cell cycle regulator p27(KIP1). KIPase cleaves a tetra-peptide substrate, Ac-DPSD-AMC, which mimics the target site in p27(KIP1), and inhibitors based on this tetra-peptide are ineffective against other known caspases. Here we describe the purification and characterization of KIPase, and trace its activity to the beta(1) subunit of the 20S proteasome.

Active site rearrangement of the 2-hydrazinopyridine adduct in Escherichia coli amine oxidase to an azo copper(II) chelate form: a key role for tyrosine 369 in controlling the mobility of the TPQ-2HP adduct.

Adduct I (lambda(max) at approximately 430 nm) formed in the reaction of 2-hydrazinopyridine (2HP) and the TPQ cofactor of wild-type Escherichia coli copper amine oxidase (WT-ECAO) is stable at neutral pH, 25 degrees C, but slowly converts to another spectroscopically distinct species with a lambda(max) at approximately 530 nm (adduct II) at pH 9.1. The conversion was accelerated either by incubation of the reaction mixture at 60 degrees C or by increasing the pH (>13).

The pig CYP2E1 promoter is activated by COUP-TF1 and HNF-1 and is inhibited by androstenone.

Functional analysis of the pig cytochrome P4502E1 (CYP2E1) promoter identified two major activating elements. One corresponded to the hepatic nuclear factor 1 (HNF-1) consensus binding sequence at nucleotides -128/-98 and the other was located in the region -292/-266. The binding of proteins in pig liver nuclear extracts to a synthetic double-stranded oligonucleotide corresponding to this more distal activating sequence was studied by electrophoretic mobility shift assay.

Solution structure of the two-iron rubredoxin of Pseudomonas oleovorans determined by NMR spectroscopy and solution X-ray scattering and interactions with rubredoxin reductase.

Here we provide insights into the molecular structure of the two-iron 19-kDa rubredoxin (AlkG) of Pseudomonas oleovorans using solution-state nuclear magnetic resonance (NMR) and small-angle X-ray scattering studies. Sequence alignment and biochemical studies have suggested that AlkG comprises two rubredoxin folds connected by a linker region of approximately 70 amino acid residues. The C-terminal domain (C-Rb) of this unusual rubredoxin, together with approximately 35 amino acid residues of the predicted linker region, was expressed in Escherichia coli, purified in the one-iron form and the structure of the cadmium-substituted form determined at high-resolution by NMR spectroscopy.

Probing the catalytic mechanism of Escherichia coli amine oxidase using mutational variants and a reversible inhibitor as a substrate analogue.

Copper amine oxidases are homodimeric enzymes containing one Cu(2+) ion and one 2,4,5-trihydroxyphenylalanine quinone (TPQ) per monomer. Previous studies with the copper amine oxidase from Escherichia coli (ECAO) have elucidated the structure of the active site and established the importance in catalysis of an active-site base, Asp-383. To explore the early interactions of substrate with enzyme, we have used tranylcypromine (TCP), a fully reversible competitive inhibitor, with wild-type ECAO and with the active-site base variants D383E and D383N.