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

The mechanism of action of a potent peptidic inhibitor fasciculin 2 (Fas2) on electric eel acetylcholinesterase (eleelAChE) has been examined in a three-level analysis. Classical steps included equilibration experiments for the evaluation of high affinity binding constant and the existence of residual hydrolytic activity in a solution of completely Fas2 saturated enzyme. The two rate constants for the association (k_on) and the dissociation (k_off) of Fas2 with free enzyme were determined by the time course of residual enzyme activity measurements. In the third step, with a nonclassical progress curve analysis, we found that the Fas2–enzyme complex exhibited hydrolytic activity in a butyrylcholinesterase-like kinetics. The switch appears to be a consequence of steric obstruction, but also the consequence of subtle rapid conformational changes around catalytic site, upon slow single-step binding of large Fas2 molecule at the peripheral site. An unusual unilateral effect of bound Fas2 is reflected by acylation-independent association and dissociation rates and might indeed be due to inability of small acylation agent to influence the binding of a large opponent.

o-Phthalaldehyde, a bifunctional cross-linking reagent, is commonly used as a probe for the active site of enzymes. In this study, the interaction of o-phthalaldehyde with camel lens ζ-crystallin was examined by activity and fluorescence measurements. Predictably, the oxidoreductase activity of ζ-crystallin was inhibited irreversibly by o-phthalaldehyde in a time- and concentration-dependent manner, and the presence of NADPH with the enzyme appeared to provide a high degree of protection against o-phthalaldehyde inactivation. Interaction of o-phthalaldehyde with ζ-crystallin resulted in formation of isoindole adduct, which exhibited characteristic fluorescence at 415 nm. However, neither inactivation nor modification of the enzyme showed the expected pseudo-first-order kinetics; both events were highly sequential reaching different levels of saturation at different concentrations of o-phthalaldehyde. The modified enzyme had a maximum stoichiometry of 1 mol isoindole/subunit, and bound NADPH to nearly the same extent as unmodified enzyme. Gel filtration experiments suggested that o-phthalaldehyde-modified ζ-crystallin had higher apparent molecular weight than unmodified enzyme, even though the enzyme remained largely monomeric as revealed by electrophoresis on denaturing gel. These results suggested that modification by o-phthalaldehyde might have been so intrusive as to sequentially modify the tetrameric structure of ζ-crystallin.

For insertion into lipidic media, most hydrosoluble proteins must cross the lipid–water interface and thus undergo conformational transitions. According to their chemical sequences these transitions may be restricted to changes involving only the tertiary structure, while for other proteins this environment modification will induce drastic changes such as the unfolding of large domains. The structural transitions are mainly governed by the presence of hydrophobic domains and/or by the existence of induced amphipathic properties.

Hexitol nucleic acids (HNA) as well as their 1,5-anhydrohexitol triphosphate building blocks were evaluated for their ability to be recognized by several DNA metabolizing enzymes. It was found that RNA polymerases can recognize the triphosphate of the adenine analogue. However, only the incorporation of a maximum of three consecutive building block analogues was possible under the applied experimental conditions. Terminal transferase was more successful succeeding in the elongation of a DNA primer with a maximum of 15 1,5-anhydrohexitol purine nucleotides. Furthermore, it was observed that the 1,5-anhydroaltritol triphosphate analogue of adenosine was a poor substrate for terminal transferase and that HNA could not act as a primer for this enzyme. Likewise, HNA did not function as a template for restriction enzymes, ligases or methylases.

The effects of guanidinium chloride (GuHCl) on the stability of the apo form of the 5S non-reassociating subunit of hemocyanin from the crab Carcinus aestuarii (apo-CaeSS2) were investigated, using a variety of optical spectroscopy techniques (light scattering (LS), fluorescence (IF and EF) and circular dichroism (CD)). The fluorescence of 8-anilino-1-naphtalene sulphonate (ANS) was strongly enhanced in the presence of apo-CaeSS2, in contast to holo-CaeSS2, suggesting the formation of a molten globule (MG)-like state, consequent to the removal of the two copper ions from the holo subunit. Other evidences, favouring the presence of this state in apo-CaeSS2, derive from an enhanced quenching of intrinsic fluorescence (IF) by acrylamide, a higher sensibility towards aggregation and a higher IF with respect to deoxy holo-CaeSS2. Aggregation of apo-CaeSS2 below 1.2 M GuHCl was detected by LS, suggesting the formation of an aggregation-prone intermediate, called I1. Due to this effect, fluorescence and CD data could only be collected above that denaturant concentration. Both IF (protein) and EF (ANS) fluorescence data were best fitted by a two-state cooperative transition, occurring between the intermediate I1 and the unfolded state U, with C_1/2 1.6–1.7 M. A similar two-state transition, with a slightly higher C_1/2 value (1.9 M), was also inferred from far-UV CD data, suggesting the possible formation of another intermediate. Partial refolding of apo-CaeSS2 by dilution was found to occur above 1.2 M GuHCl, i.e. up to the level of I1, since at lower denaturant concentration protein aggregation took place, as also observed in unfolding. All thermodynamic parameters, derived from data above 1.2 M GuHCl, are therefore referred to transitions between intermediate and unfolded states only. Unfolding kinetics, followed by fluorescence stopped-flow, was biphasic in the whole GuHCl range investigated (3–5 M), suggesting the formation of a transient intermediate, possibly related to that observed under equilibrium conditions.

It has been difficult to purify calpastatin without using a step involving heating to 90–100 °C. Preparations of calpastatin obtained after heating often contain several polypeptides that have been ascribed to proteolytic degradation. Because calpastatin is highly susceptible to proteolytic degradation and several different calpastatin isoforms can be produced by using different start sites of transcription/translation and/or alternative splicing from the single calpastatin gene, it is not clear whether the different polypeptides observed in purified calpastatin preparations are proteolytic fragments or calpastatin isoforms. It would be useful, therefore, to have a method for purifying calpastatin that does not involve heating. At low ionic strength, calpastatin from skeletal muscle extracts binds quantitatively to an immunoaffinity column made by coupling a monoclonal antibody (MAb) to the C-terminal end of calpastatin (epitope between amino acids 707 and 786) to agarose; the bound calpastatin can be eluted at pH 2.5. The C-terminal end of the calpastatin polypeptide was used because the known isoforms of calpastatin all contain domain IV. The eluted calpastatin, which retains all its calpain inhibitory activity, consists largely of a 125 kDa polypeptide (70%), and several smaller polypeptides that are labeled with a MAb to calpastatin. Expressed calpastatin constructs representing the full-length XL–IV calpastatin and domains L–IV, II–IV, III–IV, and IV also bind to the immunoaffinity column and can be purified. The immunoaffinity column is especially useful for purifying calpastatin from small tissue samples in a single step.

2-Oxoacid:ferredoxin oxidoreductase (OFOR) catalyzes the coenzyme A-dependent oxidative decarboxylation of 2-oxoacids, at an analogous metabolic position to 2-oxoacid dehydrogenase multienzyme complex. The enzyme from Sulfolobus sp. strain 7, a thermoacidophilic crenarchaeon, is a heterodimer comprising two subunits, a (632 amino acids) and b (305 amino acids). In contrast to other OFORs, the Sulfolobus enzyme shows a broad specificity for 2-oxoacids such as pyruvate and 2-oxoglutarate. Based on careful multiple alignment of this enzyme family and on the reported three-dimensional structure of the homodimeric pyruvate:ferredoxin oxidoreductase (POR) from Desulfovibrio africanus, we selected five amino acids, T256, R344 and T353 of subunit-a, and K49 and L123 of subunit-b, as candidate 2-oxoacid recognizing residues. To identify the residues determining the 2-oxoacid specificity of the enzyme family, we performed point mutations of these five amino acids, and characterized the resulting mutants. Analyses of the mutants revealed that R344 of subunit-a of the enzyme was essential for the activity, and that K49R and L123N of subunit-b drastically affected the enzyme specificity for pyruvate and 2-oxoglutarate, respectively. Replacement of the five residues resulted in significant changes in both K_m and V_max, indicating that these amino acids are clearly involved in substrate recognition and catalysis.

Triacylglycerol analogue p-nitrophenyl phosphonates specifically react with the active-site serine of lipolytic enzymes to give covalent lipase–inhibitor complexes, mimicking the first transition state which is involved in lipase-mediated ester hydrolysis. Here we report on a new type of phosphonate inhibitors containing a polarity-sensitive fluorophore to monitor micropolarity around the active site of the enzyme in different solvents. The respective compounds are hexyl and methyl dimethylamino-naphthalenecarbonylethylmercaptoethoxy-phosphonates. The hexyl phosphonate derivative was reacted with lipases from Rhizopus oryzae (ROL), Chromobacterium viscosum (CVL), and Pseudomonas cepacia (PCL). The resulting lipid–protein complexes were characterized in solution with respect to water penetration into the lipid binding site and the associated conformational changes of the proteins as a consequence of solvent polarity changes. We found that the accessibility of the lipid-binding site in all lipases studied was lowest in water. It was much higher when the protein was dissolved in aqueous ethanol. These biophysical effects may contribute to the previously observed dramatic changes of enzyme functions such as activity and stereoselectivity depending on the respective solvents.

Ferredoxin–NAD(P)⁺ reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacterium Chlorobium tepidum and purified to homogeneity. The molecular mass of the subunit is 42 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the native enzyme is approximately 90 kDa, estimated by gel-permeation chromatography, and is thus a homodimer. The enzyme contains one FAD per subunit and has absorption maxima at about 272, 385, and 466 nm. In the presence of ferredoxin (Fd) and reaction center (RC) complex from C. tepidum, it efficiently catalyzes photoreduction of both NADP⁺ and NAD⁺. When concentrations of NADP⁺ exceeded 10 μM, NADP⁺ photoreduction rates decreased with increased concentration. The inhibition by high concentrations of substrate was not observed with NAD⁺. It also reduces 2,6-dichlorophenol-indophenol (DPIP) and molecular oxygen with either NADPH or NADH as efficient electron donors. It showed NADPH diaphorase activity about two times higher than NADH diaphorase activity in DPIP reduction assays at NAD(P)H concentrations less than 0.1 mM. At 0.5 mM NAD(P)H, the two activities were about the same, and at 1 mM, the former activity was slightly lower than the latter.

The human UDP-glucuronosyltransferase 1A6 (UGT1A6) isoform is actively involved in the detoxication of phenolic compounds. In an effort to gain insight on active-site amino acids, we investigated the functional relevance of cysteinyl residues in the glucuronidation process. The enzyme was irreversibly inactivated upon exposure to thiol-specific reagents, especially N-phenylmaleimide. Site-directed mutagenesis of the conserved Cys126 into valine led to a fully inactive mutant, whereas conservative substitution with serine significantly restored the glucuronidation activity toward 4-methylumbelliferone used as a reference substrate. This mutant exhibited a reduced affinity toward the acceptor substrate, as evidenced by a 10-times increase in K_m value, compared to the wild-type enzyme. The two mutations did not alter the stability of UGT1A6 nor change the subcellular localization of the protein in the endoplasmic reticulum of recombinant cells. These results support the conclusion that Cys126 is an essential residue for the integrity of the substrate binding site of UGT1A6.