Vesna de Serrano

Different Modes of Binding of Mono-, Di-, and Trihalogenated Phenols to the Hemoglobin Dehaloperoxidase from Amphitrite ornata †

The hemoglobin dehaloperoxidase (DHP), found in the coelom of the terebellid polychaete Amphitrite ornata, is a dual-function protein that has the characteristics of both hemoglobins and peroxidases. In addition to oxygen transport function, DHP readily oxidizes halogenated phenols in the presence of hydrogen peroxide. The peroxidase activity of DHP is high relative to that of wild-type myoglobin or hemoglobin, but the most definitive difference in DHP is a well-defined substrate-binding site in the distal pocket, which was reported for 4-iodophenol in the X-ray crystal structure of DHP. The binding of 2,4,6-trihalogenated phenols is relevant since 2,4,6-tribromophenol is considered to be the native substrate and 2,4,6-trichlorophenol also gives high turnover rates in enzymatic studies. The most soluble trihalogenated phenol, 2,4,6-trifluorophenol, acts as a highly soluble structural analogue to the native substrate 2,4,6-tribromophenol. To improve our understanding of substrate binding, we compared the most soluble substrate analogues, 4-bromophenol, 2,4-dichlorophenol, and 2,4,6-trifluorophenol, using (1)H and (19)F NMR to probe substrate binding interactions in the active site of the low-spin metcyano adduct of DHP. Both mono- and dihalogenated phenols induced changes in resonances of the heme prosthetic group and an internal heme edge side chain, while (1)H NMR, (19)F NMR, and relaxation data for a 2,4,6-trihalogenated substrate indicate a mode of binding on the exterior of DHP. The differences in binding are correlated with differences in enzymatic activity for the substrates studied.

The activation of human [Glu1]plasminogen by human single-chain urokinase

Abstract The activation of human [Glu1]plasminogen ([Glu1]Pg) by single-chain human urokinase (SCUKase) displays a substantial lag phase at physiological levels of [GluP1Pg. Employing a monoclonal antibody that exhibits a high level of specificity for SCUKase, as compared to two-chain urokinase (TCUKase), we have demonstrated conclusively that during this lag phase a progressive loss of SCUKase occurs, most likely resulting from its conversion to TCUKase, in a reaction catalyzed by plasmin (HPm). The overall activation of [Glu1]Pg by SCUKase is inhibited by physiological levels of Cl− and stimulated by ϵ-amino caproic acid. Kinetic studies demonstrate that both these effects are based on first, the reaction of [Glu1]Pg with the TCUKase that is formed during the activation, and, second, the concomitant rate at which HPm is provided for the conversion of SCUKase to TCUKase. The results indicate that at physiological levels of [Glu1]Pg, its activation in the presence of SCUKase is regulated in one manner by the rate at which SCUKase is converted to TCUKase, in a process that is strongly influenced by physiological levels of Cl−. Finally, and importantly, we show that SCUKase possesses very little, if any, inherent ability to activate [Glu1]Pg at a rate that influences the kinetics of HPm generation under physiological conditions of [Glu1Pg and Cl− Concentrations.

Direct identification of lysine-33 as the principal cationic center of the ω-amino acid binding site of the recombinant kringle 2 domain of tissue-type plasminogen activator

We have generated site-specific mutants of the kringle 2 domain of tissue-type plasminogen activator [( K2tPA]) in order to identify directly the cationic center of the protein that is responsible for its interaction with the carboxyl group of important omega-amino acid effector molecules, such as epsilon-amino caproic acid (EACA). Molecular modeling of [K2tPA], docked with EACA, based on crystal structures of the kringle 2 region of prothrombin and the kringle 4 domain of human plasminogen, clearly shows that Lys33 is the only positively charged amino acid in [K2tPA] that is sufficiently proximal to the carboxyl group of the ligand to stabilize this interaction. In order to examine directly the importance of this particular amino acid residue in this interaction, we have constructed, expressed, and purified three recombinant (r) mutants of [K2tPA], viz., Lys33Thr, Lys33Leu, and Lys33Arg, and found that only the last variant retained significant ability to interact with EACA and several of its structural analogues at neutral pH. In addition, another mutated r-[K2tPA], i.e., Lys33His, interacts very weakly with omega-amino acids at neutral pH and much more strongly at lower pH values where His33 would be expected to undergo protonation. This demonstrates that any positively charged amino acid at position 33 satisfies the requirement for mediation of significant bindings to this class of molecules. Since, in other kringles, positively charged residues at amino acid sequence positions homologous to Lys68, Arg70, and Arg71 of [K2tPA] have been found to participate in kringle interactions with EACA-like compounds, we have also examined the binding of EACA, and some of its analogues, to three additional r-[K2tPA] variants, i.e., Lys68Ala, Arg70Ala, and Arg71Ala. In each case, binding of these omega-amino acids to the variant kringles was observed, with only the Lys68Ala variant showing a slightly diminished capacity for this interaction. These investigations provide clear and direct evidence that Lys33 is the principal cationic site in wild-type r-[K2tPA] that directly interacts with the carboxyl group of omega-amino acid effector molecules.

Appearance of plasminogen activator activity during a synchronous cycle of a rat adenocarcinoma cell line, PA-III

A study of the appearance of plasminogen activators during the cell cycle of a rat prostate adenocarcinoma cell line, PA-III, was undertaken. After release from a hydroxyurea (HU) block, the length of the cell cycle was determined and found to be 20-25 h, with the S-G2-M portions comprising approx. 10-15 h and the G1 phase occurring over a similar 10-15-h period. Assays for tissue-like plasminogen activator (t-PA) and urokinase-like plasminogen activator (u-PA) in cell-conditioned medium revealed an increased appearance of both enzymes shortly after the S phase of the cycle. The pattern of production of these activators remained the same under differing conditions of cell synchronization.

Structural and Kinetic Study of an Internal Substrate Binding Site in Dehaloperoxidase-Hemoglobin A from Amphitrite ornata

X-ray crystal structures of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata soaked with substrate, 2,4,6-tribromophenol (2,4,6-TBP), in buffer solvent with added methanol (MeOH), 2-propanol (2-PrOH), and dimethyl sulfoxide (DMSO) reveal an internal substrate binding site deep in the distal pocket above the α-edge of the heme that is distinct from the previously determined internal inhibitor binding site. The peroxidase function of DHP A has most often been studied using 2,4,6-trichlorophenol (2,4,6-TCP) as a substrate analogue because of the low solubility of 2,4,6-TBP in an aqueous buffer solution. Previous studies at low substrate concentrations pointed to the binding of substrate 2,4,6-TCP at an external site near the exterior heme β- or δ-edge as observed in the class of heme peroxidases. Here we report that the turnover frequencies of both substrates 2,4,6-TCP and 2,4,6-TBP deviate from Michaelis-Menten kinetics at high concentrations. The turnover frequency reaches a maximum in the range of 1400-1700 μM, with a decrease in rate at higher concentrations that is both substrate- and solvent-dependent. The X-ray crystal structure is consistent with the presence of an internal active site above the heme α-edge, in which the substrate would be oxidized in two consecutive steps inside the enzyme, followed by attack by H2O via a water channel in the protein. The physiological role of the internal site may involve interactions with any of a number of aromatic toxins found in benthic ecosystems where A. ornata resides.

Influence of various structural domains of fibrinogen and fibrin on the potentiation of plasminogen activation by recombinant tissue plasminogen activator

Fibrinogen, fibrin, and related fragments have varying stimulatory effects on the initial rate of the activation of human plasminogen ([Glu1]Pg) by recombinant tissue plasminogen activator (rt-PA). A detailed analysis of this enhancement was undertaken using various purified and complexed forms of the known domains of fibrin(ogen) with a view to gaining additional knowledge regarding the substructures of fibrinogen and fibrin that are important for their stimulatory capacities. Both arvin-mediated fibrin, as well as fibrinogen fragments generated as a result of its cleavage with CNBr, stimulate the activation in a biphasic manner, most likely as a result of changes in the promoter molecule accompanying the denaturation processes that are normally employed to either solubilize or generate these particular promoters. Using purified fibrinogen and fibrin fragments, it was found that fragment E, which binds to [Glu1]Pg, does not enhance the activation reaction, while fragment D1 has a potentiating effect. This suggests that the binding of [Glu1]Pg to fibrin(ogen) alone is not, in itself, sufficient for stimulation of activation to occur, but that the rt-PA-fibrin(ogen) interaction is fundamental to this same process. All purified and mixtures of fragments containing the fragment D domain (e.g., D2E, X-oligomer, fragment X) stimulate the reaction to a greater degree than fibrinogen and fragment D1. It is concluded that the fibrinogen D domain is asine qua non for the enhancement reaction, while structures containing the E domain had a symbiotic effect on enhancement.

The Role of the Distal Histidine in H2O2 Activation and Heme Protection in both Peroxidase and Globin Functions

The distal histidine mutations of dehaloperoxidase-hemoglobin A (DHP A) to aspartate (H55D) and asparagine (H55N) have been prepared to study the role played by the distal histidine in both activation and protection against oxidation by radicals in heme proteins. The H55D and H55N mutants of DHP A have ~6-fold and ~11-fold lower peroxidase activities than wild type enzyme toward the oxidation of 2,4,6-trichlorophenol (TCP) to yield 2,6-dichloroquinone (DCQ) in the presence of H(2)O(2). The origin of the lower rate constants may be the solvent-exposed conformations of distal D55 and N55, which would have the dual effect of destabilizing the binding of H(2)O(2) to the heme iron, and of removing the acid-base catalyst necessary for the heterolytic O-O bond cleavage of heme-bound H(2)O(2) (i.e., compound 0). The partial peroxidase activity of H55D can be explained if one considers that there are two conformations of the distal aspartate (open and closed) by analogy with the distal histidine. We hypothesize that the distal aspartate has an active conformation in the distal pocket (closed). Although the open form is observed in the low-temperature X-ray crystal structure of ferric H55D, the closed form is observed in the FTIR spectrum of the carbonmonoxy form of the H55D mutant. Consistent with this model, the H55D mutant also shows inhibition of TCP oxidation by 4-bromophenol (4-BP). Consistent with the protection hypothesis, compound ES, the tyrosyl radical-containing ferryl intermediate observed in WT DHP A, was not observed in H55D.

Examination of the secondary structure of the kringle 4 domain of human plasminogen.

The structure of a small region of human plasminogen (F4), consisting of amino acid residues Val354-Ala439 and containing its kringle 4 (K4) domain (residues Cys357-Cys434), has been predicted from Chou-Fasman calculations and hydropathy profiles, and compared to circular dichroism (CD) measurements on the isolated fragment. Calculations, by the Chou-Fasman method, of the probabilities of various types of secondary structures that exist in this region reveal that no helical structures are present. Of the total of 86 amino acid residues present in this K4-containing peptide region, 37% can adopt conformations of beta-pleated sheets, 48% of the amino acids can exist in beta-turns, and 15% of the residues can be present as coils. The structure of F4 in dilute aqueous solution has been experimentally evaluated by CD measurements. At pH = 7.4, in dilute salt solutions, a total of 64% beta-structures, 30% beta-turns, and 6% coiled structures is estimated to be present in this peptide region. Consideration of the marginal stability of many of the conformational regions of F4, as predicted by Chou-Fasman calculations, suggests that secondary structural flexibility is present in this fragment, which could result in ready adoption of new conformations. The hydropathy profile of F4 has been determined and suggests that this polypeptide is highly hydrophilic, especially in the regions of residues His387-Tyr396 and Cys406-Lys413. Thus, it appears as though a large portion of the surface of F4 can be exposed to solvent in its native conformation.

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine 86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D [Protein Data Bank (PDB) entry 3MYN ] and M86E (PDB entry 3MYM ) mutants reveal the structural origins of a stable catalytic triad in DHP A. A decrease in the rate of H(2)O(2) activation as well as a lowered reduction potential versus that of the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine in binding to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is five-coordinate, and this structure resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a six-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a six-coordinate low-spin adduct, which inhibits the ligation and activation of H(2)O(2) as required for peroxidase activity. The results suggest that the proximal charge relay in peroxidases regulate the redox potential of the heme Fe but that the trans effect is a carefully balanced property that can both activate H(2)O(2) and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, we studied three M86 mutants, M86A, M86D, and M86E, by spectroelectrochemistry and nuclear magnetic resonance spectroscopy of (13)C- and (15)N-labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the N(δ) hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.

A Model for the Flexibility of the Distal Histidine in Dehaloperoxidase-Hemoglobin A Based on X-ray Crystal Structures of the Carbon Monoxide Adduct

Dehaloperoxidase hemoglobin A (DHP A) is a multifunctional hemoglobin that appears to have evolved oxidative pathways for the degradation of xenobiotics as a protective function that complements the oxygen transport function. DHP A possesses at least two internal binding sites, one for substrates and one for inhibitors, which include various halogenated phenols and indoles. Herein, we report the X-ray crystallographic structure of the carbonmonoxy complex (DHPCO). Unlike other DHP structures with 6-coordinated heme, the conformation of the distal histidine (H55) in DHPCO is primarily external or solvent exposed, despite the fact that the heme Fe is 6-coordinated. As observed generally in globins, DHP exhibits two distal histidine conformations (one internal and one external). In previous structural studies, we have shown that the distribution of H55 conformations is weighted strongly toward the external position when the DHP heme Fe is 5-coordinated. The large population of the external conformation of the distal histidine observed in DHPCO crystals at pH 6.0 indicates that some structural factor in DHP must account for the difference from other globins, which exhibit a significant external conformation only when pH < 4.5. While the original hypothesis suggested that interaction with a heme-Fe-bound ligand was the determinant of H55 conformation, the current study forces a refinement of that hypothesis. The external or open conformation of H55 is observed to have interactions with two propionate groups in heme, at distances of 3.82 and 2.73 Å, respectively. A relatively weak hydrogen bonding interaction between H55 and CO, combined with strong interactions with heme propionate (position 6), is hypothesized to strengthen the external conformation of H55. Density function theory (DFT) calculations were conducted to test whether there is a weaker hydrogen bond interaction between H55 and heme bonded CO or O2. Molecular dynamics simulations were conducted to examine how the tautomeric forms of H55 affect the dynamic motions of the distal histidine that govern the switching between open and closed conformations. The calculations support the modified hypothesis suggesting a competition between the strength of interactions with heme ligand and the heme propionates as the factors that determine the conformation of the distal histidine.