The structural basis of anomalous kinetics of rabbit liver aryl sulfatase A

Rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) is inactivated during the hydrolysis of nitrocatechol sulfate and the rate of formation of turnover-modified aryl sulfatase A depends on the initial velocity of the enzymatic reaction. Organic solvents such as ethanol and dioxane favor the anomalous kinetic behavior. The turnover-modified enzyme can apparently be reactivated by arsenate, phosphate, pyrophosphate, and sulfate in the presence of nitrocatechol sulfate. The apparent dissociation constants of these ions in the reactivation of the enzyme are similar to their K_i values. Sulfite, which is a competitive inhibitor, does not reactivate the turnover-modified enzyme. Thus, all known activators are competitive inhibitors but not all competitive inhibitors are effective as activators. Inactivation of aryl sulfatase A during hydrolysis of ³⁵S-labeled substrate at pH values near the pH optimum (pH 5–6) is accompanied by the incorporation of radioactivity into the protein molecule and the turnover-modified enzyme is thereby covalently labeled. The stoichiometry of the incorporation of radioactivity corresponds to 2 g atom of sulfur per mole of enzyme monomer, or 1 g atom of sulfur per equivalent peptide chain. It is also shown that isolated turnover-modified rabbit liver aryl sulfatase A has lost approximately 76% of its secondary structure as compared to the native enzyme. The specific activity of the inactive enzyme is also decreased by 82%. Turnover-modified rabbit liver aryl sulfatase A is partially reactivated by sulfate ions in the presence of nitrocatechol sulfate. However, circular dichroism measurements and fluorescence spectra of the isolated “reactivated” turnover-modified enzyme indicate only a further loss of secondary structure. The specific activity of this “reactivated” enzyme is in fact decreased. The loss in secondary structure and the enzyme activity of the “reactivated” aryl sulfatase A is prevented in the presence of sulfate ions. Turnover-modified rabbit liver aryl sulfatase A behaves as a very fragile molecule.     

The monomer -- dimer association of rabbit lver aryl sulfatase A and its relationship to the anomalous kinetics.

The polymerization of aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) has been studied by frontal gel chromatography on Sephadex G-200 and Bio-Gel A-5m under various conditions of pH, ionic strength, and temperature. The aryl sulfatase A molecule exists as a monomer and as a dimer at pH 7.5 and pH 4.5, respectively. The extent of dissociation is markedly pH-, protein concentration-, and ionic strength-dependent. Only a small effect of temperature was observed. The enthalpy change (ΔH^o) for the dissociation was ?2.5 ± 1 kcal/mol at pH 5.5–5.6, and the entropy change for dissociation of the enzyme dimer to two monomeric units was ?47 cal mol^?1 deg^?1. Sulfate ion has little effect on the extent of dissociation of the enzyme at pH 5.6. The present studies suggest that the dissociation of rabbit liver aryl sulfatase A is regulated by the ionization of amino acid residues whose apparent pK is between pH 5 and 6. The driving force for the association of the subunits of the enzyme is primarily ionic and/or ionic/hydrogen bond formation. The small enthalpy change and the fact that dissociation is strongly favored by an increase in the ionic strength suggest that hydrophobic interactions play only a minor role in stabilizing the dimeric quaternary structure relative to the monomeric state. The monomeric form of the enzyme exhibits the anomalous kinetics often observed with sulfatase A but the dimer does not show anomalous kinetics. Since aryl sulfatase A is probably in the dimeric form in the lysosome, the anomalous kinetics of the enzyme are unlikely to be of physiological importance in the intact lysosome.     

A spectrophotometric determination of sulfate ion and its application in studies of substrate purity and of aryl sulfatase A kinetics.

A modification of the method of hydroxyproline determination in proteins was devised. The modification consists of the hydrolysis of proteins in 72% perchloric acid of 100°C for 2 hr instead of 20 hr, autoclaving in 6 n HCl or 2 n Ba(OH)₂. Determination of hydroxyproline by the modified method does not require any additional chromatographic purification, standardizes conditions of the assay, and increases the yield in a number of routine assays.     

O-methylhydroxylamine as a reagent for NAD+ modification in urocanase.

The inhibition of urocanase from Pseudomonas putida by O-methylhydroxylamine has been characterized as being due to the formation of an adduct between CH₃ONH₂ and NAD⁺, the latter of which has been recently shown to be a tightly bound coenzyme for this urocanase. Inhibition is maximal at pH 8.5 and is blocked by the presence of the substrate analog imidazole propionate. Loss of catalytic activity corresponds directly with the binding of 1 mol of ¹⁴CH₃ONH₂ per mole of enzyme, and partial reversibility of the modification, achieved by dialysis at pH 7.5, is accompanied by concomitant restoration of enzymatic activity. No incorporation of ¹⁴CH₃ONH₂ into urocanase is seen when enzyme-bound NAD⁺ is first converted to NADH or when NAD⁺ is removed by prior treatment of urocanase with 8 m urea. Stability and spectral properties of the CH₃ONH · NAD adduct are consistent with previous data reported for the product of the hydroxylamine reaction with NAD⁺. It is concluded that other urocanases which exhibit inhibition by hydroxylamine may likewise contain NAD⁺ as an essential coenzyme and that the use of ¹⁴CH₃ONH₂ as a reversible modification reagent for NAD⁺ should prove helpful for studies on the role of NAD⁺ in the urocanase catalytic process.     

Chemical modification of neamine.

The aminocyclitol antibiotic neamine has been modified chemically by removing one or two hydroxyl groups from the 2-deoxystreptamine moiety to give 5- and 6- deoxyneamines (5 and 10), as well as 5,6-dideoxyneamine (15). Their antimicrobial activities were determined against several microorganisms, including kanamycin-resistant strains.     

Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification.

Photochemical oxidation of Escherichia coli 50 S ribosomal subunits in the presence of methylene blue or Rose Bengal causes rapid loss of peptidyl transferase activity. Reconstitution experiments using mixtures of components from modified and unmodified ribosomes reveal that both RNA and proteins are affected, and that among the proteins responsible for inactivation there are both LiCl-split and core proteins. The proteins L2 and L16 from the split fraction and L4 from the core fraction of unmodified ribosomes were together nearly as effective as total unmodified proteins in restoring peptidyl transferase activity to reconstituted ribosomes when added with proteins from modified ribosomes. These three proteins are therefore the most important targets identified as responsible for loss of peptidyl transferase activity on photo-oxidation of 50 S ribosomal subunits.     

Inactivation of purine nucleoside phosphorylase by modification of arginine residues.

Treatment of purine nucleoside phosphorylase (EC 2.4.2.1), from either calf spleen or human erythrocytes, with 2,3-butanedione in borate buffer or with phenylglyoxal in Tris buffer markedly decreased the enzyme activity. At pH 8.0 in 60 min, 95% of the catalytic activity was destroyed upon treatment with 33 mM phenylglyoxal and 62% of the activity was lost with 33 mm 2,3-butanedione. Inorganic phosphate, ribose-1-phosphate, arsenate, and inosine when added prior to chemical modification all afforded protection from inactivation. No apparent decrease in enzyme catalytic activity was observed upon treatment with maleic anhydride, a lysine-specific reagent. Inactivation of electrophoretically homogeneous calf-spleen purine nucleoside phosphorylase by butanedione was accompanied by loss of arginine residues and of no other amino acid residues. A statistical analysis of the inactivation data vis-à-vis the fraction of arginines modified suggested that one essential arginine residue was being modified.     

A sensitive cyclic nucleotide phosphodiesterase assay for transient enzyme kinetics

A rapid and sensitive method was developed for the quantitative determination of alpha-tocopherol in tissues and plasma of rats and mice. Tissue and plasma were extracted in acetone and chromatographed on a reverse-phase C18 column with 2% water in methanol. Fluorescence and ultraviolet detection were used for tissue and plasma alpha-tocopherol levels, respectively. Extraction of tissues and plasma was found to be more complete in acetone than in other solvent systems analyzed. The average recovery of alpha-tocopherol added to tissue samples was 97%. As little as 0.1 g of tissue or 0.1 ml plasma can be accurately used for analysis. The method is sensitive to 0.05 micrograms alpha-tocopherol/g tissue.     

The kinetics of photooxidation of c-type cytochromes by Rhodospirillum rubrum reaction centers.

The kinetics of photooxidation of c-type cytochromes from horse heart, Rhodospirillum rubrum, and Rhodopseudomonas capsulata by purified reaction centers from R. rubrum have been investigated. The kinetic mechanism was found to be complex with a second-order step (complex formation) followed by a rate limiting first-order step. Based on studies of the reaction as a function of pH, ionic strength, and detergent concentration, it appears that the complex formation step is largely electrostatically controlled with only portions of the surfaces of the interacting molecules participating. Further, the first-order process observed at high cytochrome concentration appears to result from solvent reorganization and/or a conformational change following complex formation. Based on data analysis in terms of outersphere electron transfer, it is proposed that another first-order process exists which is not rate limiting and is the electron transfer step. Finally, it was found that the detergent concentration can have a profound effect on both the oxidation-reduction potential of the cytochromes and the kinetics of photooxidation. These results limit the detergent concentration range over which experiments can be conducted and interpreted.     

An assay for iduronate sulfatase (Hunter corrective factor)

Acetylation of benzyl α-D-mannopyranoside with acetic anhydride-sodium acetate at room temperature gave crystalline benzyl 2,3,6-tri-O-acetyl-α-D-manno-pyranoside (25%) and benzyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (≈65%). Similar esterification of benzyl β-D-glucopyranoside yielded the crystalline benzyl 2,4,6-triacetate (66%), whereas the corresponding galactopyranoside gave the crystalline 3,4,6-, 2,3,6-, and 2,4,6-triacetates (3, 25, and 9%. respectively). The structures of these compounds were established by methylation with diazomethane-boron trifluoride etherate and were confirmed by n.m.r. studies.     

The use of the acetylenic function for direct chemical modification of proteins

The possibility of using the acetylenic function for chemical modification of proteins was tested on chlorotetrolic acid and methyl chlorotetrolate. These compounds react under mild conditions with different functional groups analogous to those present in proteins. The ease with which the same nucleophile adds to the triple bond and substitutes the chlorine was compared, and the stability of the addition products was checked. The modification of amino groups by addition across the triple bond can be reversed in acidic medium. In reactions with small model molecules and with proteins, the tested compounds behave like bifunctional protein reagents.     

Computer modeling of muscle phosphofructokinase kinetics

The kinetics of the phosphofructokinase reaction were studied by computer modeling. A general random order, two-state allosteric model, of which the Monod--Wyman--Changeux model is a limiting case, was found to most accurately reproduce the experimental observations of Pettigrew & Frieden (1979 a,b). A simplified model with Hill coefficients was found to fit almost as well. In these models substrates bind preferentially to and stabilize the enzyme in the R state, and ATPH3-, the inhibitory species, binds preferentially to and stabilizes the enzyme in the T state. Enzymatic activity is regulated by conversion from the R to the T state, which is effected by protonation, especially of the uncomplexed enzyme, but the experimental data are inadequate for accurate estimation of the pKa of the enzyme. Random order binding of substrates is an important cause of sigmoidal kinetics. Additional experiments that would aid in the discrimination among rival models are described.     

Alternative diaspirins for modification of hemoglobin and sickle hemoglobin

Studies of modification of hemoglobin and of sickle hemoglobin by alternative aspirins have been extended to a series of new bis esters with a variety of substituted bridging diacids and to a group of mono esters with polar acyl groups. Rates of hydrolysis of these alternative aspirins have also been examined, and they reveal that a careful balance between stability and reactivity is essential for optimal activity. Four-carbon bridging groups have been found to be particularly effective, two of these raising the minimum gelling concentration of sickle hemoglobin by as much as 100%.     

Pre-steady-state kinetics of beef heart mitochondrial ATPase

The pre-steady-state kinetics of beef heart mitochondrial ATPase (F1) were examined. F1 was found to exhibit hysteretic behavior when hydrolyzing ATP. The hysteretic property was expressed as an activation process which occurred when the enzyme was mixed with its substrate, MgATP. Many catalytic turnovers were required before the activation was complete. The lag in hydrolysis increased hyperbolically as the concentration of enzyme increased. Passage of F1 through Sephadex G25 eliminated the activation process. Several kinetically distinct possibilities for explaining these data, including multiple nucleotide dissociations, enzyme conformational changes, and regulatory site interactions, are discussed. The enzyme was apparently able to recognize nucleotide in a noncatalytic manner, as evidenced by the fact that F1 preincubated with ADP in the absence of substrate achieved partial activation (smaller lag times) before being introduced to substrate. ADP is also a time-dependent inhibitor, exhibiting a slow hysteretic inhibition in addition to immediate competitive inhibition.     

Properties of human neutral bronchial mucins after modification of the peptide or the carbohydrate moieties.

Trypsin and pronase treatment of purified human neutral bronchial mucins released small fragments from the C-terminal end of these molecules and resulted in slight increases in their sedimentation coefficient presumably reflecting conformational changes. The antigenic determinant of neutral bronchial mucins which appears to be located on this C-terminal fragment is destroyed by pronase or by treatments such as periodate oxidation or galactose oxidase-bromine oxidation which modify the carbohydrate moieties. Thus, both amino acid and carbohydrate residues are involved in the structure of the antigenic determinant.     

Effects of electron mediator charge properties on the reaction kinetics of hydrogenase from Chlamydomonas

Anions modulate hydrogenase activity in cell-free preparations of Chlamydomonas reinhardtii, and this modulation is greatly influenced by the charge properties of the redox agent included to mediate electron transfer to hydrogenase. With cationic methyl viologen as the electron mediator, anions stimulate the maximum velocity of H2 production (e.g., a 320% increase in the presence of 1 M NaCl) but have little effect on the Km for methyl viologen. Conversely, when hydrogenase activity is mediated by polyanionic metatungstate or ferredoxin, H2 production is strongly inhibited by anions (e.g., 70-77% inhibition by 0.2 M NaCl). This inhibition is primarily due to a reduced affinity of hydrogenase for these mediators (as evidenced by a large increase in Km values), rather than a change in the maximum velocity of the reaction. Anions have little effect on the kinetics of hydrogenase activity mediated by zwitterionic sulfonatopropyl viologen, a redox agent with a nearly neutral net charge. These results suggest the presence of a cationic region near the active site of hydrogenase. This cationic region, probably due to lysine and/or arginine residues, may serve in vivo to facilitate the interaction between hydrogenase and ferredoxin, the polyanionic, physiological electron mediator.     

A microassay for the peptidase activity of enzymes utilizing ribonuclease S peptide as a substrate

A microassay for the peptidase activity of proteins obtained in minute amounts was devised. The method uses ribonuclease S peptide as a substrate. The substrate when cleaved is unable to reconstitute an active ribonuclease S complex. Therefore the loss in activity of the reconstituted complex is a measure of the peptidase activity. The method was previously tested with known peptidases such as clastase (9), chymotrypsin (8), and trypsin. In this work the peptidase activity of a protein related to a sperm-decapitating factor (1) is evidenced.     

No evidence of covalent modification of glutamine synthetase in the thermophilic phototropic bacterium Chloroflexus aurantiacus

Abstract Regulation of glutamine synthetase (GS) in the thermophilic green phototrophic bacterium, Chloroflexus aurantiacus , was studied. The enzyme was partially purified from cells grown photosynthetically in media with limiting (1 mM) or non-limiting (10 mM) NH⁺₄-concentrations. GS preparations from both cell types were indistinguishable in respect to pH-optimum of GS-transferase activity, sensitivity to feedback modifiers (AMP, L-alanine, glycine) and lack of Mg-inhibition of transferase activity. In contrast to results obtained with a GS preparation from the facultatively phototrophic bacterium, Rhodopseudomonas sphaeroides , the catalytic properties of Chloroflexus GS did not change during incubation with snake venom phosphodiesterase suggesting the absence of in vivo regulation of Chloroflexus GS by adenylylation/deadenylylation.     

Light-dependent chemical modification of thylakoid membrane proteins with carboxyl-directed reagents

Spinach chloroplast thylakoid membranes were chemically modified with membrane penetrating reagents reactive toward protein carboxyl groups, a carbodiimide and the nucleophiles [¹⁴C]glycine ethyl ester or [³H]serotonin. The reagents, being weak bases, were accumulated within the inner aqueous space in the light, due to the low pH inside. Both the accumulation and the low pH stimulating effect on the carbodiimide activation step contributed to a greater labeling in the light compared to dark, and uncouplers inhibited most of the light-dependent increase. Hence, it is likely that the proteins showing the light-dependent, uncoupler-sensitive labeling have those parts located within the inner aqueous space or within the membrane itself. While many membrane proteins which separated on sodium dodecyl sulfate-polyacrylamide gels (12.5–25% gradient) showed some increased labeling in the light, the most conspicuous were the four polypeptides of the chlorophyll $\text{a}\text{b}$ light-harvesting complex. The light-harvesting complex was purified from dark- and light-treated, labeled membranes. The resultant preparation showed about a sixfold, light-dependent, uncoupler-sensitive labeling increase compared to dark conditions. Polypeptides near 6 and 8 kdalton showed light-dependent, uncoupler-resistent increases in carboxyl group modification, which could be due to localized acidic conditions near sites of proton release.