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. Further analyses revealed that the activity of the beta(1) subunit is up-regulated as cells enter the cell cycle without concomitant change in the levels of the proteasome beta(1), beta(2) or beta(5) subunits. To our knowledge, this is the first description of cell cycle regulation of the caspase-like activity 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 active site base mutant forms of ECAO (D383N and D383E) showed spectral changes similar to WT when incubated at 60 degrees C. By contrast, in the Y369F mutant adduct I was not stable at pH 7, 25 degrees C, and gradually converted to adduct II, and this rate of conversion was faster at pH 9. To identify the nature of adduct II, we have studied the effects of pH and divalent cations on the UV-vis and resonance Raman spectroscopic properties of the model compound of adduct I (2). Strikingly, it was found that addition of Cu2+ to 2 at pH 7 gave a product (3) that exhibited almost identical spectroscopic signatures to adduct II. The X-ray crystal structure of 3 shows that it is the copper-coordinated form of 2, where the +2 charge of copper is neutralized by a double deprotonation of 2. These results led to the proposal that adduct II in the enzyme is TPQ-2HP that has migrated onto the active site Cu2+. The X-ray crystal structure of Y369F adduct II confirmed this assignment. Resonance Raman and EPR spectroscopy showed that adduct II in WT-ECAO is identical to that seen in Y369F. This study clearly demonstrates that the hydrogen-bonding interaction between O4 of TPQ and the conserved Tyr (Y369) is important in controlling the position and orientation of TPQ in the catalytic cycle, including optimal orientation for reactivity with substrate amines.

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. The minimum protein binding sequence was identified as TGTTCTGACCTCTGGG. Gel super-shift assays identified the protein binding to this site as chick ovalbumin upstream promoter transcription factor 1 (COUP-TF1). Androstenone inhibited promoter activity in transfection experiments only with constructs which included the COUP-TF1 binding site. Androstenone inhibited COUP-TF1 binding to synthetic oligonucleotides but did not affect HNF-1 binding. The results offer an explanation for the inhibition of CYP2E1 protein expression by androstenone in isolated pig hepatocytes and may be relevant to the low expression of hepatic CYP2E1 in those pigs which accumulate high levels of androstenone in vivo.

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. The structure shows that the C-Rb domain is similar in fold to the conventional one-iron rubredoxins from other organisms, whereas the linker region does not have any discernible structure. This tandem "flexible-folded" structure of the polypeptide chain derived for the C-Rb protein was confirmed using solution X-ray scattering methods. X-ray scattering studies of AlkG indicated that the 70-amino acid residue linker forms a structured, yet mobile, polypeptide segment connecting the globular N- and C-terminal domains. The X-ray scattering studies also showed that the N-terminal domain (N-Rb) has a molecular conformation similar to that of C-Rb. The restored molecular shape indicates that the folded N-Rb and C-Rb domains of AlkG are noticeably separated, suggesting some domain movement on complex formation with rubredoxin reductase to allow interdomain electron transfer between the metal centers in AlkG. This study demonstrates the advantage of combining X-ray scattering and NMR methods in structural studies of dynamic, multidomain proteins that are not suited to crystallographic analysis. The study forms a structural foundation for functional studies of the interaction and electron-transfer reactions of AlkG with rubredoxin reductase, also reported herein.

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. The formation of an adduct, analogous to the substrate Schiff base, between TCP and the TPQ cofactor in the active site of wild-type ECAO and in the D383E and D383N variants has been investigated over the pH range 5.5-9.4. For the wild-type enzyme, the plot of the binding constant for adduct formation (K(b)) against pH is bell-shaped, indicating two pK(a)s of 5.8 and approximately 8, consistent with the preferred reaction partners being the unprotonated active-site base and the protonated TCP. For the D383N variant, the reaction pathway involving unprotonated base and protonated TCP cannot occur, and binding must follow a less favoured pathway with unprotonated TCP as reactant. Surprisingly, for the D383E variant, the K(b) versus pH behaviour is qualitatively similar to that of D383N, supporting a reaction pathway involving unprotonated TCP. The TCP binding data are consistent with substrate binding data for the wild type and the D383E variant using steady-state kinetics. The results provide strong support for a protonated amine being the preferred substrate for the wild-type enzyme, and emphasize the importance of the active-site base, Asp-383, in the primary binding event.