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PubMedDate not supplied

Modification of arginine residues in calf intestinal alkaline phosphatase.

Calf intestinal alkaline phosphatase is inactivated by 2,3-butanedione and phenylglyoxal. The reaction with either reagent results in a biphasic loss of enzymatic activity. Inactivation by 2,3-butanedione in borate buffer can be reversed after gel-filtration in Tris buffer but no enzyme reactivation is observed after phenylglyoxal treatment. Phosphate, ATP and NADH protect the enzyme from both compounds while no protection is displayed by L-phenylalanine. The selective chemical modification indicates that two differently reacting types of arginines are present in the active site domains of the dimeric enzyme.

Alkaline Phosphatase↗
PubMed2006

What factors influence the ratio of C-H hydroxylation versus C=C epoxidation by a nonheme cytochrome P450 biomimetic?

Density functional calculations on a nonheme biomimetic (Fe=O(TMCS+) have been performed and its catalytic properties versus propene investigated. Our studies show that this catalyst is able to chemoselectively hydroxylate C=H bonds even in the presence of C=C double bonds. This phenomenon has been analyzed and found to occur due to Pauli repusions between protons on the TMCS ligand with protons attached to the approaching substrate. The geometries of the rate determining transition states indicate that the steric hindrance is larger in the epoxidation transition states than in the hydroxylation ones with much shorter distances; hence the hydroxylation pathway is favored over the epoxidation. Although, the reactant experiences close lying triplet and quintet spin states, the dominant reaction mechanism takes place on the quintet spin state surface; i.e., Fe=O(TMCS)+ reacts via single-state reactivity. Our calculations show that this spin state selectivity is the result of geometric orientation of the transition state structures, whereby the triplet ones are destabilized by electrostatic repulsions between the substrate and the ligand while the quintet spin transition states are aligned along the ideal axis. The reactivity patterns and geometries are compared with oxoiron species of dioxygenase and monoxygenase enzymes. Thus, Fe=O(TMCS)+ shows some similarities with P450 enzyme reactivity: it chemoselectively hydroxylates C=H bonds even in the presence of a C=C double bond and therefore is an acceptable P450 biomimetic. However, the absolute barriers of substrate oxidation by Fe=O(TMCS)+ are higher than the ones obtained with heme enzymes, but the chemoselectivity is lesser affected by external perturbations such as hydrogen bonding of a methanol molecule toward the thiolate sulfur or a dielectric constant. This is the first oxoiron complex whereby we calculated a chemoselective hydroxylation over epoxidation in the gas phase.

Algorithms↗
PubMed1984

Effects of volume and surface property in hydrolysis by acetylcholinesterase. The trimethyl site.

beta-Substituted ethyl acetates, XCH2CH2OCOCH3, have been prepared, and their hydrolysis by acetylcholinesterase has been studied. Log of enzymic reactivity, normalized for intrinsic reactivity in hydrolysis by hydroxide, log (kcat/Km)n, rises linearly with increasing refraction volume, MR (or RD25), for substrates with beta-X = H, Cl, Br, CH3CH2, (CH3)2CH, (CH3)2S+, (CH3)3N+, and (CH3)3C. Larger substituents may be accommodated, (CH3)3Si and (CH3CH2)3N+, with no further increase in rate. Substrates with beta-substituents CH3S, CH3S(O), (CH3)3N+(OH), and CH3S(O2) are less reactive than consistent with the relation with MR by factors of 5-40, indicating that hydrophobic surface and desolvation of the substrate--enzyme interface may be necessary for maximum reactivity correlated with MR. Values of log (kcat/Km)n for substrates with beta-substituents X = CH3S, Cl, Br, CH3CH2, (CH3)2CH, (CH3)3C, and (CH3)3Si rise linearly with increasing hydrophobicity, pi, but reactivity of substrates with X = (CH3)3N+ and (CH3)2S+ are more reactive than consistent with a relation to pi by factors of 300 and 40 and with X = CH3S(O2), CH3S(O), and (CH3)2N+(OH), by factors of 7-100. Reactivity appears related to (i) volume of the beta-substituent and its fit in its subsite, which is trimethyl rather than anionic, and (ii) the hydrophobicity of its surface.

Acetates↗
PubMed1988

Age-related modifications in rat cardiac phosphoglycerate kinase. Rejuvenation of the old enzyme by unfolding-refolding.

The occurrence of age-related modifications in functional and structural properties of several enzymes has been documented; however, the molecular basis of this phenomenon is still mostly unexplained. In the present work a comparative study of phosphoglycerate kinase preparations isolated from hearts of young and old rats was undertaken. Marked age-related effects were revealed in the heat-inactivation kinetics of the enzyme, similar to the ones previously found in purified muscle phosphoglycerate kinase. In view of the previously reported failure of immunotitration to distinguish between phosphoglycerate kinase forms in crude heart extracts from young and old rats, it appears likely that the modifications in old rat heart phosphoglycerate kinase are in a domain which is not involved in antibody binding, and may be localized in the interior of the enzyme. These age-related modifications were completely relieved by extensive unfolding of the enzyme in 2 M guanidine hydrochloride, followed by enzyme reactivation upon dilution of the denaturant. The refolding products of young and old enzymes displayed identical heat-inactivation kinetics as native young phosphoglycerate kinase. It is concluded that the age-related alterations in rat cardiac phosphoglycerate kinase, like those found in the muscle enzyme, are purely conformational and hence develop postsynthetically.

Aging↗
PubMed1976

Sucrase-isomaltase deficiency. Absence of an inactive enzyme variant.

Deficiency of sucrase-isomaltase, an intestinal enzyme complex that is essential for digestion of nutritionally important carbohydrates, appears to be inherited as an autosomal recessive in 0.2 per cent of North Americans. The molecular basis of the deficiency has not been elucidated. To characterize the enzyme defect quantitatively, papain-solubilized intestinal biopsies were analyzed for total enzyme protein by radioimmunoassay and for enzyme activity by hydrolytic assay. Normal intestine had a close correlation between immunologically reactive enzyme and enzymic activity. In contrast, seven patients with sucrase-isomaltase deficiency were found to have complete absence of the enzyme protein by radioimmunoassay even though up to 10 times more intestinal protein was present than with normal tissue. This absence of an inactive enzyme variant can be explained by a major (no-sense) mutation of the structural gene or by a complete repression of the regulatory mechanism that controls structural gene function.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1989

Non-michaelian behavior of 5 alpha-reductase in human prostate.

An in-depth analysis of the kinetics of 5 alpha-reductase in human prostatic tissue gave findings inconsistent with the claim that the enzyme is michaelian. In both hyperplastic and malignant tissue, the time-course of the conversion of testosterone (T) into dihydrotestosterone (DHT) was non-linear under conditions ensuring less than 15% conversion of substrate and cofactor. An initial rapid phase of conversion was followed by a long steady-state phase. This time-dependent change in conversion rate was not due to enzyme denaturation, fast inhibition by substrate or product effects. It resulted from a true slow transient kinetic process induced in the reactive enzyme by the substrates. Under our experimental conditions at pH 5.5, 5 alpha-reductase appeared to undergo a conformational change from an initially highly reactive form to a less reactive form. Since this "hysteretic" behavior was correlated with apparently negative cooperativity in enzyme kinetics, we postulate that, as previously described for other key metabolic enzymes, regulation of 5 alpha-reductase activity in the prostate depends on the molecular flexibility of the enzyme and on changes in the cooperativity of different enzyme forms over time. This original non-michaelian behavior may explain the conflicting kinetics reported so far in the literature for this enzyme. The clinical implications of 5 alpha-reductase hysteresis and its involvement in the damping of DHT production within the prostate are discussed.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1996

Chemical modification of enzymes: kinetic aspects.

Important information on enzyme ligand interactions can be obtained when analyzing the kinetics of chemical modification reactions. In this article, several kinetic models of inactivation are discussed, along with the determination of enzyme-ligand dissociation constants and of the pK of enzyme reactive groups from chemical modification kinetic data.

Enzyme Activation↗
PubMed1993

The inactivation of dihydroxy-acid dehydratase in Escherichia coli treated with hyperbaric oxygen occurs because of the destruction of its Fe-S cluster, but the enzyme remains in the cell in a form that can be reactivated.

The enzyme dihydroxy-acid dehydratase previously has been shown to be inactivated in vivo in Escherichia coli within minutes of exposure to hyperbaric O2. In this paper, we show its inactivation is due to the destruction of its catalytically active [4Fe-4S] cluster. The inactivation is not followed by an appreciable decrease in the amount of dihydroxy-acid dehydratase protein as determined by Western blots. Thus, the protein from the inactivated enzyme remains unproteolyzed in the cells. Dihydroxy-acid dehydratase activity recovers after the cells treated with hyperbaric O2 are returned to ambient oxygen. Since this recovery in activity is not accompanied by a significant increase in dihydroxy-acid dehydratase protein and is not prevented by chloramphenicol, it appears primarily to be due to reactivation of the previously inactivated enzyme. The reactivation occurs by reconstitution of the enzyme's Fe-S cluster. These results demonstrate that this enzyme can cycle between forms in which the Fe-S cluster is either present or absent. The facile ability to cycle between these two forms would be compatible with a regulatory role in addition to a catalytic role for this enzyme.

Chloramphenicol↗
PubMed1983

Guanidoacetate methyltransferase from rat liver: purification, properties, and evidence for the involvement of sulfhydryl groups for activity.

Guanidoacetate methyltransferase (EC 2.1.1.2) has been purified about 800-fold from rat liver. The purified preparation shows a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme is estimated to be 25,000 and 26,000 by Sephadex gel molecular-exclusion chromatography and by electrophoresis in polyacrylamide gradient gel, respectively. The sodium dodecyl sulfate-denatured enzyme also has a molecular weight of 26,000; thus, the enzyme is a monomeric protein. Guanidoacetate methyltransferase as isolated is catalytically inactive, but is readily reactivated by incubation with a thiol. The reactivated enzyme, which contains 3 mol of sulfhydryl groups/mol of enzyme, is again inactivated by oxidized glutathione. This inactivation is accompanied by the disappearance of two sulfhydryl residues. The relationship between the loss of enzyme activity and the number of residues disappeared indicates that the integrity of these sulfhydryl residues is critical for activity. The oxidized enzyme fails to bind the substrate S-adenosylmethionine as evidenced by the equilibrium dialysis study. Alkylation of the nonoxidizable sulfhydryl by N-ethylmaleimide shows that this residue is also essential for activity. UV absorption, fluorescence, and CD spectra show no difference between the reduced and oxidized enzymes, but the former is more susceptible to proteolytic attack by trypsin. The enzyme has an isoelectric pH of 5.3, and is most active at pH 9.0. From the CD spectrum, an alpha helix content of 15% is calculated. The Km values for guanidoacetate and S-adenosylmethionine are 97.5 and 6.73 microM, respectively, at pH 8.0 and 37 degrees C.

Amino Acids↗
PubMed1977

Appearance of peroxidase reactivity within the rough endoplasmic reticulum of blood monocytes after surface adherence.

Rabbit blood monocytes, which contain no cytochemically demonstrable peroxidase, develop peroxidatic activity in the RER and perinuclear cisternae within 2 h after adherence to serum- or fibrin-coated surfaces. A similar reactivity appears in surface-adherent human and rat blood monocytes. In both localization and characteristics, this enzyme reactivity in monocytes resembles that normally seen in the resident peritoneal macrophages of the rabbit, as well as in several types of tissue macrophages in other species. Thus this observation supports the concept, presently based on the kinetic data of other investigators, that blood monocytes are the precursors of such cells. Moreover, the appearance of new enzyme activity after adherence may reflect alterations in cellular metabolism resulting from plasma membrane:surface interactions.

Animals↗
PubMed2003

Perturbation of folding and reassociation of lactate dehydrogenase by proline and trimethylamine oxide.

Investigations of protein-solute interactions typically show that osmolytes favor native conformations. In this study, the effects of representative compatible and counteracting osmolytes on the reactivation of lactate dehydrogenase from two different conformational states were explored. Contrary to expectations, proline and trimethylamine oxide inhibited both the initial time course and the extent of reactivation of lactate dehydrogenase from bovine heart following denaturation in guanidine hydrochloride, as well as following inactivation at pH 2.3. Reactivation of acid-dissociated porcine heart lactate dehydrogenase was inhibited by both proline and trimethylamine oxide (2 M). In all instances, trimethylamine oxide was the more effective inhibitor of reactivation. Analysis of the catalytic properties of the reactivating enzyme provided evidence that the molecular species that was enzymatically active during the initial stages of reactivation of acid-inactivated porcine heart lactate dehydrogenase reflects a non-native conformation. Proline and trimethylamine oxide stabilize polypeptides through exclusion from the polypeptide backbone; the inhibition of renaturation/reassociation described here is probably due to attenuation of this stabilizing influence through favorable interactions of the osmolytes with sidechains of residues that lie at the interfaces of the monomers and dimers that associate to form the active tetramer. In addition, these osmolytes may stabilize non-native intermediates in the folding pathway. The high viscosity of solutions containing more than 3 m proline was a major factor in the inhibition of reassociation of acid-dissociated porcine heart lactate dehydrogenase as well as other viscosity-dependent transformations that may occur during reactivation following unfolding in guanidine hydrochloride.

Animals↗
PubMed1981

[Mechanism of glycogen depletion in the liver in endotoxin-treated rats].

Studied were the rate and mechanism of glycogen breakdown in rats treated with Salmonella abortus ovis endotoxin. It was found that tissue homogenates from treated animals showed higher glycogenolytic activity in vitro as compared with homogenates from untreated animals, which demonstrated the direct mechanism of endotoxin action. The higher lytic activity with treated animals seemed to be associated with the higher activity of glycogen-phosphorylase, which showed peak values at the third hour following treatment. The higher activity of glycogen-phosphorylase was, in turn, the result of the enzyme reactivation under the effect of the endotoxin, and was not linked with the synthesis or new enzyme molecules.

Animals↗
PubMed1986

The affinity alkylators, 11 alpha-bromoacetoxyprogesterone and estrone 3-bromoacetate, modify a common histidyl residue in the active site of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase.

Purified human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase (native enzyme) was completely inactivated by the affinity alkylator, estrone 3-bromoacetate, in the presence of cofactor (NADPH). The inactivated enzyme was reactivated to 100% activity by base-catalyzed hydrolysis of the steroidal ester-enzyme conjugate and then repurified by dialysis. Control enzyme in mixtures which contained estrone in place of alkylator was treated the same as the reactivated enzyme. 11 alpha-Bromo[2'-14C]acetoxyprogesterone, an active site-directed affinity alkylator of the enzyme, produced 5.0-fold less radiolabeled 3-(carboxymethyl)histidine and S-(carboxymethyl)cysteine plus 1.4-fold more 1,3-bis(carboxymethyl)-histidine in the reactivated enzyme than in the control enzyme. The lesser amount of S-(carboxymethyl)cysteine and greater amount of 1,3-bis(carboxymethyl)histidine resulted from nonspecific interactions between the reactivated enzyme and the progestin radioalkylator. The nonradiolabeled 3-(carboxymethyl)histidine originally produced by estrone 3-bromoacetate in the enzyme active site hindered radioalkylation of this amino acid by 11 alpha-bromo[2'-14C]acetoxyprogesterone to yield 5-fold less radiolabeled 3-(carboxymethyl)histidine in the reactivated enzyme relative to control enzyme. Thus, the estrogen and progestin affinity alkylators modified a common histidyl residue in the active site. These studies are direct evidence that the estradiol 17 beta-dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities reside at a common locus on a single protein.

Alkylation↗
PubMed2006

Effect of pyridostigmine, pralidoxime and their combination on survival and cholinesterase activity in rats exposed to the organophosphate paraoxon.

Pyridostigmine (PSTG) is a carbamate inhibitor of cholinesterases. Carbamates are known to confer some protection from the lethal effects of (some) organophosphorus compounds. Recently, based on animal data, the FDA approved oral PSTG for pre-exposure treatment of soman. The purpose of the study was to quantify in vivo the effect of PSTG pre-treatment on survival in rats exposed to the organophosphate paraoxon (POX) with and without subsequent reactivator (pralidoxime) treatment. POX is a highly toxic non-neuropathic ethyl organophospate. Pralidoxime (PRX) is the enzyme reactivator used by some NATO armies. The prospective, controlled animal (rat) study included Group 1 that received 1 micromol POX ( approximately LD(75)); Group 2 that received 1 micromol PSTG followed 30 min later by 1 micromol POX; Group 3 that received 1 micromol PSTG followed 30 min later by 1 micromol POX and 50 micromol PRX; Group 4 that received 1 micromol POX and 50 micromol PRX; Group 5 that received 1 micromol PSTG; Group 6 that received 50 micromol PRX and Group 7 that received 1 micromol PSTG followed 30 min later by 50 micromol PRX. Each group contained six rats. The experiment was repeated twelve times (12 cycles). All substances were applied i.p. From surviving animals of eight cycles tail blood was taken for red blood cell acetylcholinesterase (RBC-AChE) measurements. The animals were monitored for 48 h and mortality (survival time) was recorded. RBC-AChE activities were determined. Mortality was analysed using Kaplan-Meier plots. Both PSTG and PRX statistically significantly decreased organophosphate induced mortality in the described model. While the same applies to their combination the decrease in mortality when using both PSTG and PRX is less than that achieved with their single use (but not significantly so). While certainly further work using different organophosphorus compounds and animal species are needed before a final conclusion is reached, the animal data presented does not support the combined use of PSTG and PRX.

Animals↗
PubMed1981

Glycosyl transferase activities are associated with the surface membrane in adult Schistosoma mansoni.

Incubation of live adult Schistosoma mansoni in a variety of media released tegumental material containing membrane bound alkaline phosphatase, mannosyl transferase and galactosyl transferase activities. Centrifugation of the tegumental material released by incubation of worms in phosphate-buffered saline in sucrose density gradients yielded a pellet and four fractions, two of which consisted mainly of surface membranes. The distribution of the enzymes in the gradient, espeically in the two surface membrane-containing subfractions was similar. Application of the "digitonin shift" technique showed that the membranes containing the enzyme reactivities were moved to an equal extent into a denser part of the sucrose gradient. Thus the enzymes are located on the same or similar cholesterol-containing membranes. It is concluded that the transferases, like the alkaline phosphatases, are located in the surface membranes of S. mansoni and the consequences of this location for the host-parasite interaction are discussed.

Alkaline Phosphatase↗
PubMed1978

Evidence for multiple reactive forms of papain.

The pH-dependence of the second-order rate constants of acylation and alkylation reactions of the -SH group of papain were determined by using neutral and charged reactants under identical conditions. From these pH rate profiles, in contrast to previous claims, different pKa values were obtained with different groups of reactants. In the case of charged reactants, like chloroacetate (pKa = 3.6) and arginine derivatives (pKa = 4.3), the pKa differences can be attributed to electrostatic effects. However, the fact that a pKa of 3.25 was found with methyl and ethyl bromoacetate, and a pKa of 4.0 was obtained with bromoacetamide and a number of neutral substrates, is inconsistent with the theories put forth hitherto for the meaning of such pKa values, because they all consider only one reactive enzyme form. The different pKa values obtained here with neutral reactants are explained in terms of various reactive papain forms. The perturbation of pKa by electrostatic effects was examined by reacting simple thiol compounds containing different charges, like 2-mercaptoacetate, 2-mercaptoethylamine, 2-mercaptoethanol and glutathione, with the neutral chloroacetamide and with the negatively charged chloroacetate. Differences in pKa can be interpreted in terms of intramolecular and intermolecular electrostatic interactions.

Acylation↗
PubMed1988

The effects of lauryl maltoside on the reactivation of several enzymes after treatment with guanidinium chloride.

The present study confirms the previous reports that detergents can facilitate the reactivation of guanidinium chloride (GdmCl) denatured rhodanese (Tandon, S. and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618; Tandon, S. and Horowitz, P. (1987) J. Biol. Chem. 262, 4486-4491). Here, we report the effect of the detergent, lauryl maltoside, on the reactivation of several enzymes other than rhodanese. For this study we used five different enzymes each having a single polypeptide chain, namely: adenosine deaminase; 3-phosphoglyceric phosphokinase; myokinase; 3 alpha-hydroxysteroid dehydrogenase; and phosphoglucomutase. The regain of enzyme activity was used to monitor refolding. Like rhodanese, these enzymes were denatured in 6 M GdmCl and diluted into a buffer containing various concentrations of lauryl maltoside. The effect of lauryl maltoside on reactivating these proteins depended on the specific enzyme used. For example, in the presence of lauryl maltoside, reactivation of adenosine deaminase increased to 98%, while phosphoglucomutase could not be reactivated significantly. The critical micelle concentration (CMC) of lauryl maltoside was measured under the present experimental conditions using 2-(p-toluidinyl)naphthalene 6-sulfonate (TNS) as an apolar fluorescent probe, and gave a value of 0.085 mg.ml-1 in 10 mM sodium phosphate (pH 7.4). The reactivating effect of lauryl maltoside was not generally related to its CMC. In some cases an induction period was observed before the enzyme attained its steady-state velocity. This might suggest the presence of intermediate(s) in the refolding pathway that could have been stabilized by the detergent. These findings indicate that 'non-denaturing' detergents may be useful for assisting reactivation of enzymes, although the optimum conditions will have to be determined for each individual case.

Enzyme Inhibitors↗
PubMed1996

Using the process of reactive immunization to induce catalytic antibodies with complex mechanisms: aldolases.

The process of reactive immunization has been used to induce efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes. Reactive immunogens are those that react chemically during induction of the immune response. This same reaction is used later in catalysis. In essence one immunizes with the equivalent of a mechanism-based inhibitor. The difference is that instead of inhibiting a mechanism, a mechanism is induced. This advance allows the experimenter to dictate the exact mechanism by which catalytic antibodies proceed. The hapten used in the present study is a 1,3-diketone that both traps the requisite lysine residue to initiate formation of the enamine and induces a binding pocket that overcomes the entropic barrier of this bimolecular reaction.

Acetone↗