Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology最新文献

Chondroitin AC lyase (chondroitinase EC 4.2.2.5), an eliminase from Flavobacterium heparinum, cleaves chondroitin sulfate glycosaminoglycans (GAGs) at 1,4 glycosidic linkages between N-acetylgalactosamine and glucuronic acid residues. Cleavage occurs through β-elimination in a random endolytic action pattern. Crystal structures of chondroitin AC lyase (wild type) complexed with oligosaccharides reveal a binding site within a narrow and shallow protein channel, suggesting several amino acids as candidates for the active site residues. Site-specific mutagenesis studies on residues within the active-site tunnel revealed that only the Arg to Ala 292 mutation (R292A) retained activity. Furthermore, structural data suggested that R292 was primarily involved in recognition of N-acetyl or O-sulfo moieties of galactosamine residues and did not directly participate in catalysis. The current study demonstrates that the R292A mutation affords ∼10-fold higher K_m values but no significant change in V_max, consistent with hypothesis that R292 is involved in binding the O-sulfo moiety of the saccharide residues. Change in chondroitin sulfate viscosity, as a function of its enzymatic cleavage, affords a shallower concave curve for the R292A mutant, suggesting its action pattern is neither purely random endolytic nor purely random exolytic. Product studies using gel electrophoresis confirm the altered action pattern of this mutant. Thus, these data suggest that the R292A mutation effectively reduces binding affinity, making it possible for the oligosaccharide chain, still bound after initial endolytic cleavage, to slide through the tunnel to the catalytic site for subsequent, processive, step-wise, exolytic cleavage.

Cytochrome c peroxidase (CcP) is a yeast mitochondrial enzyme that catalyzes the reduction of hydrogen peroxide to water by ferrocytochrome c. It was the first heme enzyme to have its crystallographic structure determined and, as a consequence, has played a pivotal role in developing ideas about structural control of heme protein reactivity. Genetic engineering of the active site of CcP, along with structural, spectroscopic, and kinetic characterization of the mutant proteins has provided considerable insight into the mechanism of hydrogen peroxide activation, oxygen–oxygen bond cleavage, and formation of the higher-oxidation state intermediates in heme enzymes. The catalytic mechanism involves complex formation between cytochrome c and CcP. The cytochrome c/CcP system has been very useful in elucidating the complexities of long-range electron transfer in biological systems, including protein–protein recognition, complex formation, and intracomplex electron transfer processes.

We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize hydrophobic, alanine-rich mutants of the basic region/leucine zipper (bZIP) protein GCN4. These bacterially expressed proteins were generated to probe how small, α-helical proteins bind specific DNA sites. Enzymatic digestion mapping combined with MALDI-TOF MS characterization of protein fragments allowed us to resolve mass discrepancies between the expected and observed molecular mass measurements. Changes in mass were attributed to posttranslational modifications (PTMs) by proteolytic cleavage of the initiating methionine residue, carbamylation at the amino terminus, oxidation of histidine side chains, and oxidative addition of β-mercaptoethanol (BME) at the cysteine side chain. Proteins can undergo a wide variety of co-translational modifications and PTMs during growth, isolation, and purification. Such changes in mass can only be detected by a high-resolution technique such as MALDI, which in conjunction with enzymatic digestion mapping, becomes a powerful methodology for characterization of protein structure.

The arylamidase activity of Zn-proteinase from Saccharomonospora canescens (NPS) was studied with series of peptide nitroanilides of varying amino acid sequence and N-acyl blocking groups. The partial mapping of the enzyme S₁, S₂, S₃, S₄ subsites shows that variations in all positions P₁ to P₄ in the substrate structure affect the catalytic efficiency. The importance of P₄–S₄ and P₁–S₁ interactions, which is a characteristic feature of the serine proteinases, is evidenced for the studied Zn-proteinases NPS and serralysin too. The presence of arylamidase activity in the case of Zn-proteinases—astacin EC 3.4.24.21 and serralysin EC 3.4.24.40 is correlated with some specific characteristics of their active site structure: penta-coordinated Zn²⁺ and a tyrosyl residue as a fifth ligand to the Zn²⁺. It is assumed that this tyrosyl residue plays a role in the productive binding and stabilization of the tetrahedral adduct formed during the reaction of enzyme-catalysed hydrolysis of peptide arylamides of corresponding length and sequence.

Fibronectin structural reorganization induced by temperature has been investigated by Fourier-transform infrared (FT-IR) spectroscopy and light-scattering experiments.

At 20 °C, from resolution enhanced by FT-IR spectra, 43% of β sheet, 31% of turn and 26% of unordered structures were estimated. Static and quasi-elastic light-scattering results do not change significantly between 20 and 34 °C. Just below 50 °C, a decrease of 1/3 of β sheet structures contents is observed, concomitantly with a corresponding increase of turn. The contribution of disordered structures is found to be temperature-independent.

Above 50 °C, our data reveals the formation of intermolecular hydrogen bonding leading to the formation of intermolecular β sheet structures. The IR band absorption at 1618 cm⁻¹ increases strongly as a function of temperature. The scattered intensity increases and becomes strongly q²-dependent. The dynamic structure factor is not a single exponential decay and becomes strongly dependent on the scattering angle. These results demonstrate that aggregation occurs in fibronectin solution. When temperature decreases, this aggregation is found irreversible.

Fibronectin aggregation is driven by the formation of intermolecular hydrogen bonds responsible for intermolecular β sheet structures.

The effects of guanidinium hydrochloride (GuHCl) on the functional and structural properties of a 75-kDa, functionally active hemocyanin (Hc) subunit isolated from the crab Carcinus aestuarii (holo-CaeSS2) were investigated. The holo form of the protein contains two copper ions in the active site and is capable of reversibly binding dioxygen. The present results are compared with those previously described for the corresponding functionally inactive subunit (apo-CaeSS2), devoid of the two active site copper ions (accompanying paper [R. Favilla, M. Goldoni, A. Mazzini, M. Beltramini, P. Di Muro, B. Salvato, paper published in this issue]). As with apo-CaeSS2, both equilibrium and kinetic unfolding measurements were carried out using light scattering (LS), circular dichroism, intrinsic and extrinsic fluorescence (IF and EF, respectively). In addition here, absorbance spectroscopy was exploited to evaluate oxygen binding by holo-CaeSS2. These data were combined with those relative to the protein copper content to obtain information on the stability of the active site as a function of denaturant concentration. The different techniques used revealed several unfolding transitions. At GuHCl<1 M, an appreciable increase of LS intensity was observed, about an order of magnitude lower than with apo-CaeSS2, suggesting some reversible protein aggregation. A first cooperative transition as a function of GuHCl was detected as an increase of intensity of the protein IF (C_1/2=1 M), followed by a second transition, characterised by a small intensity decrease and a red shift of the emission maximum (C_1/2=1.4 M). Cooperative transitions with C_1/2 values near 1.4 M GuHCl were also detected by following the decrement of: (a) EF intensity of anilino-1-naphtalenesulphonate (ANS) bound to the protein; (b) absorbance at 340 nm, typical of oxy holo-CaeSS2; (c) copper-to-protein stoichiometry. A transition at higher GuHCl (C_1/2=1.8 M) was also observed by far UV circular dichroism (far UV CD) and related to global unfolding. Unfolding kinetics was also studied using the fluorescence stopped-flow technique. All traces were best fitted by a sum of three or four exponential terms, depending on GuHCl concentration. A comprehensive unfolding model is proposed, which accounts for most of the complex behaviour of this protein subunit, including oxy and deoxy native and aggregation-prone intermediates, a highly fluorescent intermediate, molten globule-like apo and unfolded species.

Thymidylate synthase (TS) is a central target for the design of chemotherapeutic agents due to its vital role in DNA synthesis. Structural studies of binary complexes between Escherichia coli TS and various nucleotides suggest the chemotherapeutic agent FdUMP and the natural ligand dUMP bind similarly. We show, however, that FdUMP binding to human TS yields a substantially greater decrease in fluorescence than does dUMP. Because the difference in quenching due to ligand binding was approximately two-fold and this difference was not seen when using ecTS, the intriguing result indicated a significant difference in the mode of FdUMP binding to the human enzyme. We compared the binding affinities of dUMP, FdUMP, and TMP to TS from both species and found no significant differences for the individual ligands. Because binding affinities were not different among the ligands, the method of continuous variation was employed to determine binding stoichiometry. Similar to that found for dUMP binding to human and ecTS, FdUMP displayed single site occupancy with both enzymes. These results show that nucleotide binding differences exist for FdUMP and dUMP binding to the human enzyme. The observed differences are not due to differences in stoichiometry or ligand affinity. Therefore, although the crystal structure of human TS with various nucleotide ligands has not been solved, these results show that the differences observed using fluorescence methods result from as yet unidentified differential interactions between the human enzyme and nucleotide ligands.

The structural basis for the GTP-dependent co-translational targeting complex between the signal recognition particle (SRP) and its receptor is unknown. The complex has been shown to have unusual kinetics of formation, and association in vivo is likely to be dependent on catalysis by the SRP RNA. We have determined conditions for RNA-independent association of the ‘NG’ GTPase domains of the prokaryotic homologs of the SRP components, Ffh and FtsY, from Thermus aquaticus. Consistent with previous studies of the Escherichia coli proteins, the kinetics of association and dissociation are slow. The T. aquaticus FtsY is sensitive to an endogenous proteolytic activity that cleaves at two sites—the first in a lengthy linker peptide that spans the interface between the N and G domains, and the second near the N-terminus of the N domain of FtsY. Remarkably, this second cleavage occurs only on formation of the Ffh/FtsY complex. The change in protease sensitivity of this region, which is relatively unstructured in the FtsY but not in the Ffh NG domain, implies that it undergoes conformational change on formation of the complex between the two proteins. The N domain, therefore, participates in the interactions that mediate the GTP-dependent formation of the targeting complex.