Factors controlling the substrate specificity of peroxidases: kinetics and thermodynamics of the reaction of horseradish peroxidase compound I with phenols and indole-3-acetic acids

The rates of oxidation of reducing substrates by heme peroxidases have previously been thought to be controlled only by their ease of oxidation. In the present study, we have compared the kinetics and thermodynamics of the oxidation of indole-3-acetic acid and derivatives and of phenols by horseradish peroxidase. Different dependencies of the reaction rates on the thermodynamic driving force reveal substrate specificity controlled by the enzyme-substrate complexes dissociation constants (Michaelis-Menten constants) and by the reorganization energies of electron-transfer within those complexes.     

Nitrogenase reactivity: effects of pH on substrate reduction and CO inhibition

Molybdenum nitrogenase is composed of two separately purified proteins designated the iron protein (Fe protein) and the molybdenum-iron protein (MoFe protein), with the latter containing the substrate reduction site which is a metal cluster designated the iron-molybdenum cofactor (FeMo cofactor). In addition to its physiological substrates H+ and N2, nitrogenase reduces a number of nonphysiological substrates (e.g. C2H2 and N3-) and interacts with a number of similar molecules (e.g. CH3NC and CO) that serve as specific inhibitors. Despite their great diversity, all substrates are reduced by multiples of two electrons and require equivalent numbers of electrons and protons. Although the electron donor to a substrate is believed to be FeMo cofactor, the nature of the proton donor is unknown and might be different for different substrates. Here we report a three-component buffer assay system that eliminates variables of buffer type, ionic strength, and ATP and reductant availability and that is compatible with the nitrogenase system in the pH range 5.0-9.8. Preincubated studies and studies of the effects of pH on H2 evolution under Ar, H2 evolution under N2, H2 evolution under CO, and C2H2 reduction show that there is a group with a pK of ca. 6.3 that must be deprotonated for substrate reduction to occur and that there is a group with a pK of ca. 9.0 that must be protonated for substrate reduction to occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of two electron-transfer sites in ascorbate peroxidase using chemical modification, enzyme kinetics, and crystallography

Chemical and mutagenic modification combined with X-ray crystallography has been used to probe the ascorbate binding site in ascorbate peroxidase (APX). Chemical modification of the single Cys residue in APX with Ellman's reagent (DTNB) blocks the ability of APX to oxidize ascorbate but not other small aromatic phenolic substrates. DTNB-modified APX (APX-TNB) exhibits only 1.3% wild-type activity when ascorbate is used as the substrate but full activity when aromatic substrates, guaiacol or pyrogallol, are used. Stopped-flow studies show that APX-TNB reacts normally with peroxide to give compound I but that the rates of reduction of both compounds I and II by ascorbate are dramatically slowed. Conversion of Cys32 to Ser leads to approximately 70% drop in ascorbate peroxidase activity with no effect on guaiacol peroxidase activity. These results indicate that uncharged aromatic substrates and the anionic ascorbate molecule interact with different sites on APX. The 2.0 A X-ray crystal structure of APX-TNB shows clear electron density for the TNB group covalently attached to Cys32 in all four molecules of the asymmetric unit, indicating complete and specific modification. It appears that the ascorbate site is blocked by DTNB modification which is well removed from the exposed delta-heme edge where aromatic substrates are thought to bind. This is the first experimental evidence indicating that ascorbate oxidation does not occur at the exposed heme edge but at an alternate binding site in the vicinity of Cys32 near Arg172 and the heme propionates.     

Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1

The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 beta-CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-A resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation. We hypothesize that the new maltohexaose conformation is related to the enhanced alpha-cyclodextrin production of the CGTase. The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site-directed mutagenesis. Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (beta- and gamma-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of alpha-CD. Glu258 is involved in catalysis in CGTases as well as alpha-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu258 in the CGTase from T. thermosulfurigenes EM1 were changed. Phe284 was replaced by Lys and Asn327 by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme. This suggests that the pH optimum curve of CGTase is determined only by residue Glu258.     

trans-2-phenylcyclopropylamine is a substrate for and inactivator of horseradish peroxidase

Horseradish peroxidase (HRP) is well known for mediating the electron-transfer oxidation of electron-rich aromatic 'donors' such as phenols and anilines, but has not been described to oxidize aliphatic amines. We here confirm the inability of HRP to oxidize typical aliphatic amines, even those which would exist significantly as free bases at the operative pH. In contrast, trans-2-phenylcyclopropylamine (2-PCPA) is both a substrate (turnover product is cinnamaldehyde) and a time-dependent inactivator of HRP. These activities of 2-PCPA are consistent with either a concerted or rapid sequential one-electron-oxidation/ring-opening to give an intermediate capable of covalent binding to the enzyme. 2-PCPA is the first known example of a simple aliphatic amine which serves as a substrate for HRP under turnover conditions.     

Rat 7,8-dihydro-8-oxoguanine DNA glycosylase: substrate specificity, kinetics and cleavagemechanism at an apurinic site

Reactive oxygen species produce different lesions in DNA. Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative products implicated in mutagenesis. This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells. We have isolated and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1). Expression of the cDNA in the fgp mutY Escherichia coli double mutant allowed the purification of the untagged rOGG1 protein. It excises 8-oxoG from DNA with a strong preference for duplex DNA containing 8-oxoG:C base pairs. rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively. When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' to the lesion. However, rOGG1 acts on a substrate containing an apurinic site by a beta-delta elimination reaction and proceeds through a Schiff base intermediate. Expression of rOGG1 in E.coli fpg mutY suppresses its spontaneous mutator phenotype.     

Disability, dependency, and demoralization.

Explores the relationships between degree of functional disability, demoralization, and suicidal ideation. Analysis is guided by a theoretical framework that suggests that the relationship between disability and demoralization is a function of the level of dependency and the invasiveness of the dependency into the personal/private aspects of the individual's life. Data were derived from a state-wide sample of 4,745 individuals residing in Colorado. The sample was sorted into 1 of 5 dependency/invasiveness classes on the basis of their responses to a level-of-disability checklist. Using the Center for Epidemiological Studies Depression Scale as a measure of demoralization, results indicate a strong relationship between demoralization and the disability/invasiveness classes. Significant differences were also found in terms of suicidal ideation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Bicarbonate is required for the peroxidase function of Cu, Zn-superoxide dismutase at physiological pH

Cu,Zn-superoxide dismutase (SOD1) acts as a peroxidase in the presence of H2O2 at high pH (pH > 9). The high pH species of H2O2, HO2-, was previously implicated as the reactive species. However, recent EPR studies of the enzyme performed in the physiological pH range 7.4-7.6 with the spin trap 5,5'-dimethyl-1-pyrolline-N-oxide attributed the intense EPR signal of 5, 5'-dimethyl-1-pyrolline-N-oxide-OH obtained from SOD1 and H2O2 to the peroxidase activity of the enzyme. The present study establishes that this intense signal is obtained only in the presence of bicarbonate. To explore the critical role of HCO3-, a comprehensive EPR investigation of the radical production and redox state of the active site copper was performed. The results indicate that HCO3- competes with other anions for the anion-binding site of SOD1 (Arg141) but does not bind directly to the copper. Structurally different anions that bind to Arg141 did not stimulate, but rather blocked, peroxidase function, ruling out an effect due to mere anion binding. However, the structurally similar anions HSeO3- and HSO3- mimic HCO3- in stimulating peroxidase function. These data suggest that HCO3- bound to Arg141 anchors the neutral H2O2 molecule at the active site copper, enabling its redox cleavage. Thus, SOD1 acquires peroxidase activity at physiological pH only in the presence of HCO3- or structurally similar anions. Alterations in pH that shift the HCO3-/CO2 equilibrium as occur in disease processes such as ischemia, sepsis, or shock would modulate the peroxidase function of SOD1.     

Understanding the potential and pH dependency of high-strength β-titanium alloy environmental crack initiation

An explanation for the strong dependency of crack initiation of precracked high-strength β-titanium alloys in room-temperature 0.6 M NaCl on applied potential and bulk-solution pH is presented. It is proposed that environment-assisted cracking (EAC) susceptibility in neutral aqueous NaCl results from (1) film rupture due to plastic deformation at actively deformed crack tips, (2) accelerated dissolution of titanium, (3) crack tip acidification by hydrolysis of titanium ions, (4) crack tip potential excursions toward bare metal open-circuit potentials (OCPs) during film rupture due to large ohmic voltages in the crack solution, (5) accelerated crack tip proton or water reduction concurrent with titanium dissolution, (6) bare surface-dominated hydrogen ingress into a fracture process zone, and (7) crack initiation by hydrogen embrittlement. Evidence for each of the above stages of the crack initiation scenario is presented, with emphasis on crack tip electrode kinetics and ohmic voltage calculations which govern process zone-controlled hydrogen uptake. The seven stages are consistent with the strong dependencies of crack initiation and growth in precracked high-strength β-titanium alloys on (1) solution pH, (2) applied potential, and (3) strain rate, and they explain the “apparent” EAC resistance of smooth- and blunt-notch specimens. The latter lack both occluded crack tip geometries to promote acidification and ohmic voltage drops below reversible hydrogen, as well as localization of dynamic plastic strain. Hydrogen uptake is, subsequently, limited.     

Salicylic acid is a reducing substrate and not an effective inhibitor of ascorbate peroxidase

This communication describes the interactions of salicylic acid (SA) with plant ascorbate peroxidase (APX). Contrary to a recent report (Durner, J., and Klessig, D. F. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11312-11316) we show conclusively that ascorbate oxidation by APX is not inhibited by SA (10 mM), but that SA is a slow reducing substrate of this enzyme. The suggestion that SA-dependent inhibition of APX in planta may result in the elevation of H2O2 levels, which in turn acts as a second messenger in systemic acquired resistance signaling, is therefore not tenable. We conclude that APX remains a key antioxidant during systemic acquired resistance following pathogenic infection of plants. The transient products of SA oxidation by APX appear to be SA free radicals that undergo subsequent chemistry. APX-dependent oxidation of SA could be essential for diminishing the detrimental effects of this phenolic acid on plant cells.     

Effect of pH on tobacco anionic peroxidase stability and its interaction with hydrogen peroxide

The dendritic localization of mRNAs has been proposed to underlie the structural and functional polarity of neurons, as well as certain aspects of synaptic plasticity. Even though there is no conclusive evidence that such a localization is a physiological requirement, studies of mRNA localization in relation to function in other cell types and recent experiments on synaptic plasticity suggest that this proposal may be correct.     

Structural interactions between horseradish peroxidase C and the substrate benzhydroxamic acid determined by X-ray crystallography

The three-dimensional structure of recombinant horseradish peroxidase in complex with BHA (benzhydroxamic acid) is the first structure of a peroxidase-substrate complex demonstrating the existence of an aromatic binding pocket. The crystal structure of the peroxidase-substrate complex has been determined to 2.0 A resolution with a crystallographic R-factor of 0.176 (R-free = 0. 192). A well-defined electron density for BHA is observed in the peroxidase active site, with a hydrophobic pocket surrounding the aromatic ring of the substrate. The hydrophobic pocket is provided by residues H42, F68, G69, A140, P141, and F179 and heme C18, C18-methyl, and C20, with the shortest distance (3.7 A) found between heme C18-methyl and BHA C63. Very little structural rearrangement is seen in the heme crevice in response to substrate binding. F68 moves to form a lid on the hydrophobic pocket, and the distal water molecule moves 0.6 A toward the heme iron. The bound BHA molecule forms an extensive hydrogen bonding network with H42, R38, P139, and the distal water molecule 2.6 A above the heme iron. This remarkably good match in hydrogen bond requirements between the catalytic residues of HRPC and BHA makes the extended interaction between BHA and the distal heme crevice of HRPC possible. Indeed, the ability of BHA to bind to peroxidases, which lack a peripheral hydrophobic pocket, suggests that BHA is a general counterpart for the conserved hydrogen bond donors and acceptors of the distal catalytic site. The closest aromatic residue to BHA is F179, which we predict provides an important hydrophobic interaction with more typical peroxidase substrates.     

Assessment of dependency, agreeableness, and their relationship.

Agreeableness is central to the 5-factor model conceptualization of dependency. However, 4 meta-analyses of the relationship of agreeableness with dependency have failed to identify a consistent relationship. It was the hypothesis of the current study that these findings might be due in part to an emphasis on the assessment of adaptive, rather than maladaptive, variants of agreeableness. This hypothesis was tested by using experimentally altered NEO Personality Inventory—Revised (Costa & McCrae, 1992) items that were reversed with respect to their implications for maladaptiveness. The predicted correlations were confirmed with the experimentally altered version with measures of dependent personality disorder, measures of trait dependency (including 2 measures of adaptive dependency), and measures of dependency from alternative dimensional models of personality disorder. The theoretical implications of the findings and suggestions for future research are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Determinants of chemical dependency treatment placement: Clinical, economic, and logistic factors.

Studied 319 adult alcoholics with or without concurrent drug use disorders to determine the relative efficacy of inpatient, outpatient, and inpatient-to-outpatient treatment and to identify patient characteristics associated with differential outcome by treatment type. Successful 6-mo follow-up of 73% of the Ss revealed a 67% abstinence rate, with no significant differences by treatment setting. The routine use of standardized instruments and procedures for diagnosis and assessment is recommended, along with changes in service delivery systems and insurance coverage, as steps toward optimal treatment placement. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Violent versus nonviolent husbands: Differences in attachment patterns, dependency, and jealousy.

Two studies were conducted to compare the attachment patterns, dependency, and jealousy of violent and maritally distressed husbands with that of nonviolent distressed and nonviolent-nondistressed husbands. In Study 1, participants completed the Adult Attachment Scale, Spouse Specific Dependency Scale, and Interpersonal Jealousy Scale. In Study 2, participants completed the Relationship Styles Questionnaire, Rempel Trust Scale, and Adult Attachment Interview. Results were generally consistent with hypotheses that, relative to nonviolent husbands, violent men would evidence more insecure, preoccupied, and disorganized attachment (e.g., anxiety about abandonment, discomfort with closeness, and difficulty in classifying attachment); more dependency on and preoccupation with their wives; and more jealousy and less trust in their marriage. In addition, the findings suggest that researchers need to more carefully compare various measures of attachment. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Musk xylene: analysis, occurrence, kinetics, and toxicology

1,3-Dimethyl-2,4,6-trinitro-5-tert.-butylbenzene (musk xylene, MX), a synthetic musk, is often used in fragrances and soaps to substitute the natural musk. MX belongs to the common group of nitromusk compounds. The main environmental intake of MX occurs after sewage introduction. The consumption of fish and drinking water as well as the use of body care and perfumed household products could lead to an ingestion of this substance in humans. Although the acute oral and dermal toxicity of MX is low, some hint for the carcinogenic potential of MX was found in one animal experiment. These findings and the high potential of MX as environmental contaminant, it is stable against biological and chemical degradation and it is highly lipophil, raised considerable attention in the field of environmental medicine. Biological monitoring and the toxicology of MX, which previously has been described to occur in human milk, human fat tissue, as well as human blood samples, are of central interest. The aim of this article is to summarize the data on the analysis, occurrence, kinetics, and toxicology of MX. As there is a lack of knowledge on human toxicity and human carcinogenicity of MX, a final evaluation of the toxicological data with regard to public health is still impossible. Nevertheless, in view of the published data about MX, there is no evidence for any substantial human risk at the moment.     

Differences in pH optima and calcium requirements for maturation of the prohormone convertases PC2 and PC3 indicates different intracellular locations for these events

PC2 and PC3, which is also known as PC1, are subtilisin-like proteases that are involved in the intracellular processing of prohormones and proneuropeptides. Both enzymes are synthesized as propolypeptides that undergo proteolytic maturation within the secretory pathway. An in vitro translation/translocation system from Xenopus egg extracts was used to investigate mechanisms in the maturation of pro-PC3 and pro-PC2. Pro-PC3 underwent rapid (t1/2 < 10 min) processing of the 88-kDa propolypeptide at the sequence RSKR83 to generate the 80-kDa active form of the enzyme. This processing was blocked when the active site aspartate was changed to asparagine, suggesting that an autocatalytic mechanism was involved. In this system, processing of pro-PC3 was optimal between pH 7.0 and 8.0 and was not dependent on additional calcium. These results are consistent with pro-PC3 maturation occurring at an early stage in the secretory pathway, possibly within the endoplasmic reticulum, where the pH would be close to neutral and the calcium concentration less than that observed in later compartments. Processing of pro-PC2 in the Xenopus egg extract was much slower than that of pro-PC3 (t1/2 = 8 h). It exhibited a pH optimum of 5.5-6.0 and was dependent on calcium (K0.5 = 2-4 mM). The enzymatic properties of pro-PC2 processing were similar to that of the mature enzyme. Further studies using mutant pro-PC2 constructs suggested that cleavage of pro-PC2 was catalyzed by the mature 68-kDa PC2 molecule. The results were consistent with pro-PC2 maturation occurring within a late compartment of the secretory pathway that contains a high calcium concentration and low pH.     

Solution 1H NMR investigation of the heme cavity and substrate binding site in cyanide-inhibited horseradish peroxidase

Solution two-dimensional 1H NMR studies have been carried out on cyanide-inhibited horseradish peroxidase isozyme C (HRPC-CN) to explore the scope and limitations of identifying residues in the heme pocket and substrate binding site, including those of the "second sphere" of the heme, i.e. residues which do not necessarily have dipolar contact with the heme. The experimental methods use a range of experimental conditions to obtain data on residue protons with a wide range of paramagnetic relaxivity. The signal assignment strategy is guided by the recently reported crystal structure of recombinant HRPC and the use of calculated magnetic axes. The goal of the assignment strategy is to identify signals from all residues in the heme, as well as proximal and distal, environment and the benzhydroxamic acid (BHA) substrate binding pocket. The detection and sequence specific assignment of aromatic and aliphatic residues in the vicinity of the heme pocket confirm the validity of the NMR methodologies described herein. Nearly all residues in the heme periphery are now assigned, and the first assignments of several "second sphere" residues in the heme periphery are reported. The results show that nearly all catalytically relevant amino acids in the active site can be identified by the NMR strategy. The residue assignment strategy is then extended to the BHA:HRPC-CN complex. Two Phe rings (Phe 68 and Phe 179) and an Ala (Ala 140) are shown to be in primary dipolar contact to BHA. The shift changes induced by substrate binding are shown to reflect primarily changes in the FeCN tilt from the heme normal. The present results demonstrate the practicality of detailed solution 1H NMR investigation of the manner in which substrate binding is perturbed by either variable substrates or point mutations of HRP.     

Motivational influences on impression formation: Outcome dependency, accuracy-driven attention, and individuating processes.

How might being outcome dependent on another person influence the processes that one uses to form impressions of that person? We designed three experiments to investigate this question with respect to short-term, task-oriented outcome dependency. In all three experiments, subjects expected to interact with a young man formerly hospitalized as a schizophrenic, and they received information about the person's attributes in either written profiles or videotapes. In Experiment 1, short-term, task-oriented outcome dependency led subjects to use relatively individuating processes (i.e., to base their impressions of the patient on his particular attributes), even under conditions that typically lead subjects to use relatively category-based processes (i.e., to base their impressions on the patient's schizophrenic label). Moreover, in the conditions that elicited individuating processes, subjects spent more time attending to the patient's particular attribute information. Experiment 2 demonstrated that the attention effects in Experiment 1 were not merely a function of impression positivity and that outcome dependency did not influence the impression formation process when attribute information in addition to category-level information was unavailable. Finally, Experiment 3 manipulated not outcome dependency but the attentional goal of forming an accurate impression. We found that accuracy-driven attention to attribute information also led to individuating processes. (PsycINFO Database Record (c) 2010 APA, all rights reserved)