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PubMed1972

Mechanism of allopurinol-mediated increase in enzyme activity in man.

Allopurinol therapy in man interferes with pyrimidine biosynthesis de novo by inhibition of one or both of the two enzymes, orotate phosphoribosyltransferase (OPRT) and orotidylic decarboxylase (ODC), responsible for the conversion of orotic acid to uridine-5'-monophosphate. Inhibition of this pathway in vivo is followed in 1-3 wk by an increase in the activity of both of these enzymes in erythrocytes and of ODC in circulating leukocytes. This drug-mediated increase in enzyme activity in erythrocytes could not be attributed to enzyme stabilization or induction in vivo but appeared to be due to enzyme "activation." "Activation" of the OPRT enzyme was directly demonstrated in erythrocytes studied in vitro after incubation with oxipurinol, and to a lesser extent, with allopurinol. No evidence for "activation" of the ODC enzyme was demonstrated in vitro. This response to allopurinol therapy provides an excellent model for examining the mechanism of increased enzyme activity in response to drug administration.

Allopurinol↗

PubMed1985

Stimulation of bull seminal RNase by various basic proteins.

The activity of purified bull seminal RNase was markedly stimulated by various basic proteins. At the half concentration of substrate RNA, basic proteins such as histones, high-mobility group chromosomal proteins and cytochrome c stimulated the enzyme activity 4-6 fold. Other non-basic proteins such as bovine serum albumin and human gamma-globulin were far less effective. In addition to enzyme-stimulating activity, basic proteins showed a marked enzyme-stabilizing activity, indicating the presence of a strong interaction between the enzyme and basic proteins.

Animals↗

PubMed1995

High-alkaline protease from Bacillus PB92 entrapped in calcium alginate gel. Physicochemical and microscopic studies.

High-alkaline protease (HAP) has been entrapped in Manugel DMB (an alginate gel) and assayed with two sizes and types of substrates: neutral protein casein and synthetic chromogenic tripeptide substrate, Z-Gly-Pro-Cit-PNA. Increasing the concentration of calcium chloride used for capsule formation decreased the measured enzyme activity with both substrates. Capsules were found to be stable in water for long periods of time, but they dissolved in both phosphate and carbonate-bicarbonate buffers. The pH vs activity profiles of encapsulated enzyme showed pH optima between 10 and 11 with both substrates. The calcium alginate matrix surrounding the enzyme was quite effective in stabilizing the enzyme at 20-25 degrees C and even more so at 4 degrees C. Enzyme stability at 50 degrees C was quite impressive, some enzyme activity being evident even after remaining for 1 wk at this temperature in water. Increasing concentrations of sodium dodecyl sulfate (SDS) were also found to inhibit the protease progressively, whereas a polyhexamethylene biguanidium chloride (PHMBH+Cl-) and SDS:PHMBH+Cl- combination showed the opposite effect. Optical microscopy, especially polarized light microscopy, provided a sensitive physical means of ascertaining some of the structural properties (sphericity, disorganization or organization, distinct layer enveloping the capsules, intensity of the maltese cross) of the capsules with and without enzyme before and after different chemical treatments and the presence or absence of the substrate.

Alginates↗

PubMed2001

Monovalent cation-induced conformational change in glucose oxidase leading to stabilization of the enzyme.

Glucose oxidase (GOD) from Aspergillus niger is an acidic dimeric enzyme having a high degree of localization of negative charges on the enzyme surface and dimer interface. We have studied the effect of monovalent cations on the structure and stability of GOD using various optical spectroscopic techniques, limited proteolysis, size exclusion chromatography, differential scanning calorimetry, and enzymic activity measurements. The monovalent cations were found to influence the enzymic activity and tertiary structure of GOD, but no effect on the secondary structure of the enzyme was observed. The monovalent cation-stabilized GOD was found to have a more compact dimeric structure but lower enzymic activity than the native enzyme. The enzyme's K(m) for D-glucose was found to be slightly enhanced for the monovalent cation-stabilized enzyme (maximum enhancement of about 35% for LiCl) as compared to native GOD. Comparative denaturation studies on the native and monovalent cation-stabilized enzyme demonstrated a significant resistance of cation-stabilized GOD to urea (about 50% residual activity at 6.5 M urea) and thermal denaturation (Delta T(m) maximum of 10 degrees C compared to native enzyme). However, pH-induced denaturation showed a destabilization of monovalent cation-stabilized GOD as compared to the native enzyme. The effectiveness of monovalent cations in stabilizing GOD structure against urea and thermal denaturation was found to follow the Hofmeister series: K(+) > Na(+) > Li(+).

Aspergillus niger↗

PubMed1997

Kinetic isotope effect characterization of the transition state for oxidized nicotinamide adenine dinucleotide hydrolysis by pertussis toxin.

Pertussis toxin from Bordatella pertussis catalyzes the ADP ribosylation of several G-proteins, using NAD+ as a substrate. In the absence of an acceptor protein, the toxin acts as a NAD+ glycohydrolase. Pertussis toxin is one of the virulent factors for whooping cough and therefore a target for site-specific inhibitors based on the transition state structure. A family of kinetic isotope effects was determined for the hydrolysis reaction, using NAD+ labeled with 3H, 14C, and 15N as substrates. Primary isotope effects were 1.021 +/- 0.001 for [1'N-14C]NAD+ and 1.021 +/- 0.004 for [1N-15N]NAD+, and the double-primary effect of [1'N-14C,1N-15N]NAD+ was 1.049 +/- 0.004. Secondary kinetic isotope effects were 1.207 +/- 0.010 for the [1'N-3H]-, 1.144 +/- 0.005 for the [2'N-3H]-, 0.989 +/- 0.001 for the [4'N-3H]-, and 1.019 +/- 0.004 for the [5'N-3H]NAD+, respectively. Commitment to catalysis was excluded by isotope trapping experiments, and the experimental kinetic isotope effects were independent of pH. The measured isotope effects are therefore intrinsic. The isotope effects are remarkable because they indicate an oxocarbenium-like ribose ring at the transition state but a stiffer than expected vibrational environment for C1' at the reaction center. On the basis of these isotope effects, a bond order vibrational analysis was performed to locate a transition state structure consistent with the isotope effects. The kinetic isotope effects predict a residual bond order to the nicotinamide leaving group of 0.11, corresponding to a distance of 2.14 A. Participation of the water nucleophile is weak, consistent either with an S(N)1-like transition state with no water interaction or with the water oxygen no closer than 3.5 A from the reaction center. The positive charge of the ribose oxocarbenium is stabilized by delocalization between the C1'-O4' and C1'-C2' bonds. The enzyme contacts restrict the vibrational environment of the reaction coordinate requiring increased bonding force constants for the enzyme-stabilized transition state. NAD+ analogues with the nicotinamide ribose replaced by an iminoribitol ring, mimicking the flattened ribose ring of the transition state, are expected to be transition state inhibitors.

Carbon Radioisotopes↗

PubMed1997

CGP 48664, a potent and specific S-adenosylmethionine decarboxylase inhibitor: effects on regulation and stability of the enzyme.

Mammalian S-adenosylmethionine decarboxylase (AdoMetDC) catalyses a regulatory important step in the biosynthesis of polyamines and is a potential target for therapeutic agents against various parasitic diseases and proliferative disorders. In the present study we examined the effects of a newly synthesized AdoMetDC inhibitor. 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664), on polyamine metabolism in the mouse leukaemia cell line L1210. Treatment of the cells with 2 microM CGP 48664 led to a depletion of cellular spermidine and spermine. The putrescine content, in contrast, was markedly increased. Cells seeded in the presence of the inhibitor showed a significant decrease in growth rate, which was fully reversed by the addition of 2 microM spermidine or 1 microM spermine. The syntheses of ornithine decarboxylase and AdoMetDC were greatly increased in cells treated with CGP 48664. These increases were not correlated with similar changes in the mRNA levels, indicating the involvement of a translational mechanism. CGP 48664 was demonstrated to be a very poor competitor of spermidine uptake in the L1210 cells. L1210 cells deficient in polyamine transport were as sensitive to the antiproliferative effect of the inhibitor as were the parental cells, indicating that CGP 48664 did not enter the cells by the polyamine transport system. In addition to inhibiting AdoMetDC, CGP 48664 stabilized the enzyme against degradation. In the present study we also demonstrated that aminoguanidine (AMG), which is frequently used in cellular systems to inhibit any action of serum polyamine oxidase, apparently inhibits AdoMetDC by an irreversible mechanism that markedly stabilizes the enzyme against proteolytic degradation. CGP 48664 and the parental compound methylglyoxal bis(guanylhydrazone), which is also a potent inhibitor of AdoMetDC, contain one or two AMG-like moieties; the importance of these residues in the inhibition of AdoMetDC is discussed.

Adenosylmethionine Decarboxylase↗

PubMed1991

Protein engineering to change thermal stability for food enzymes.

In this review we have briefly indicated how the present state of knowledge allows proteins to be mutated to increase or decrease stability. We have discussed experiments on both model proteins and those of relevance to the food industry, and show how hydrophobic forces are a major driving force for folding as well as having a major role in thermostability. We have also indicated the large contribution that hydrogen bonding, electrostatic interactions and, in a less well predicted way, disulphide bridges make to thermostability.

Enzyme Stability↗

PubMed1987

Adenosine deaminase converts purine riboside into an analogue of a reactive intermediate: a 13C NMR and kinetic study.

The 13C NMR spectra of [2-13C]- and [6-13C]purine ribosides have been obtained free in solution and bound to the active site of adenosine deaminase. The positions of the resonances of the bound ligand are shifted relative to those of the free ligand as follows: C-2, -3.7 ppm; C-6, -73.1 ppm. The binary complexes are in slow exchange with free purine riboside on the NMR time scale, and the dissociation rate constant is estimated to be 13.5 s-1 from the slow exchange broadening of the free signal. In aqueous solution, protonation of purine riboside at N-1 results in changes in 13C chemical shift relative to those of the free base as follows: C-2, -4.9 ppm; C-6, -7.9 ppm. The changes in chemical shift that occur when purine riboside binds to the enzyme indicate that the hybridization of C-6 changes from sp2 to sp3 in the binary complex with formation of a new bond to oxygen or sulfur. A change in C-2 hybridization can be eliminated as can protonation at N-1 as the sole cause of the chemical shift changes. The kinetic constants for the adenosine deaminase catalyzed hydrolysis of 6-chloro- and 6-fluoropurine riboside have been compared, and the reactivity order implies that carbon-halogen bond breaking does not occur in the rate-determining step. These observations support a mechanism for the enzyme in which formation of a tetrahedral intermediate is the most difficult chemical step. Enzymic stabilization of this intermediate may be an important catalytic strategy used by the enzyme to lower the standard free energy of the preceding transition state.

Adenosine Deaminase↗

PubMed1976

Purification and some properties of a monoacylglycerol-hydrolyzing enzyme of rat adipose tissue.

A monoacylglycerol-hydrolyzing enzyme has been purified 2500-fold from rat adipose tissue. The key step was the solubilization of the enzyme, presumably as an enzyme-detergent complex, by sonication with a nonionic polyoxyethylene alcohol detergent. The purification was achieved by ion exchange and gel chromatography, and isoelectric focusing, in the presence of detergent. By sodium dodecyl sulfate gel electrophoresis the enzyme protein was more than 85% pure. This method indicated a minimum molecular weight of 32,900. The preliminary amino acid composition, excluding tryptophan, could best be fitted with a value of 31,800. The purified enzyme had a pI of 7.2, an estimated Stokes radius of 39 A by gel chromatography and a pH optimum of 8.0. Enzyme stability was highly dependent on presence of detergent and free sulfhydryl groups. The enzyme was responsible for the main monoacylglycerol- but only a small part of the p-nitrophenylacetate-hydrolyzing activity of crude adipose tissue extracts and hydrolyzed 1(3)- and 2-monooleoylglycerol at equal rates. Under the assay conditions used it did not catalyze the hydrolysis of emulsified trioleoylglycerol, micellar or emulsified dioleoylglycerol, emulsified cholesterol oleate or micellar lysophosphatidylcholine. It is possible that the enzyme may be a specific monoacylglycerol hydrolase.

Adipose Tissue↗

PubMed1986

Enzymatic semisynthesis of [LeuB30] insulin.

Experimental conditions for the preparation of [LeuB30] insulin by coupling of des-AlaB30 insulin with Leu-OBu(t) were determined using Achromobacter protease I and trypsin as catalysts. Successful coupling required a large excess of the amine component (0.8 M), a high concentration of organic cosolvent (35-50%) and neutral pH of the reaction mixture. The coupling yield of Achromobacter protease I after 24 h at 37 degrees C was almost the same or a little higher than that at 25 degrees C. With trypsin, the coupling yield at 37 degrees C after 24 h was considerably lower than at 25 degrees C. This was partly ascribed to the difference in concentration of organic cosolvent at 37 degrees C and 25 degrees C; 35% and 50%, respectively, or possibly of enzyme stability at these temperatures. The maximum product yield was about 90% with both enzymes under optimal conditions. A preparative scale experiment was performed with Achromobacter protease I; the yield of [LeuB30] insulin was 51% using porcine insulin as the starting material. This semisynthetic insulin was identified by HPLC and amino acid analysis. No difference was observed in CD spectra between [LeuB30] insulin and human insulin.

Alcaligenes↗

PubMed1986

[Interaction of hexokinase II isoenzyme from rat skeletal muscles with lecithin liposomes].

It was found that in the presence of Mg2+ (pH 7.5) rat skeletal muscle hexokinase isozyme II is firmly adsorbed on mitochondrial and artificial phospholipid membranes (lecithin liposomes). In both cases the adsorption isotherm has similar quantitative and qualitative characteristics, which points to the absence of specific binding sites on the membranes. Under these conditions, immobilization of hexokinase on various membranes is concomitant with similar changes in the enzyme stability upon storage as well as with the pH-dependence of the enzyme activity. It was demonstrated that the bound hexokinase form has a greater value of V, an increased affinity for glucose and a decreased sensitivity to the inhibitory action of glucose-6-phosphate as compared to the free form. Besides, this form is in a greater degree subjected to the inhibitory influence of ADP with respect to glucose. In this case, the enzyme affinity for ATP and the Ki value for ADP with respect to ATP is practically the same both for the free and membrane-bound forms. The data obtained suggest that the phospholipid component of mitochondrial membranes participates in the enzyme binding in the presence of Mg2+. It was assumed that the model system used in the present study, i.e., hexokinase-Mg2+-liposomes, may be successfully used for the analysis of an adsorption mechanism of regulation of hexokinase activity in the cell.

Adsorption↗

PubMedDate not supplied

Characterization of fish Cu/Zn-superoxide dismutase and its protection from oxidative stress.

Copper/zinc superoxide dismutase was cloned from the zebrafish ( Danio rerio). The full coding region of the zebrafish superoxide dismutase (ZSOD) complementary DNA was ligated with pET-20b(+) and successfully expressed in Escherichia coli strain AD494(DE3)pLysS. The active enzyme was purified by His tagging. The ZSOD yield was 6 mg from 0.2 L of E. coli culture, and the specific activity was 2000 U/mg as assayed using a RANSOD kit. The enzyme stability was characterized by reaction to temperature, pH, and detergent treatment. The results showed enzyme activity was still active after heat treatment at 70 degrees C for 10 minutes, resistant to pH treatment from 2.3 to 12, and resistant to treatment with sodium dodecyl sulfate (SDS) under 4%. In addition, the recombinant ZSOD was used to protect fish from 100 ppm of paraquat-induced oxidative injury by soaking fish larva in 55 micro g/ml SOD enzyme. The results were significant.

Animals↗

PubMed2001

Binding thermodynamics of the transition state analogue coformycin and of the ground state analogue 1-deazaadenosine to bovine adenosine deaminase.

Binding of the transition state analogue coformycin and the ground state analogue 1-deaazadenosine to bovine adenosine deaminase have been thermodynamically characterized. The heat capacity changes for coformycin and 1-deazaadenosine binding are -4.7 +/- 0.8 kJ/mole-K and -1.2 +/- 0.1 kJ/mole-K, respectively. Since the predominant source of heat capacity change in enzyme interactions are changes in the extent of exposure of nonpolar amino acid side chains to the aqueous environment and the hydrophobic effect is the predominant factor in native structure stabilization, we propose that the binding of either class of ligand is associated with a stabilizing enzyme conformational change with coformycin producing the far greater effect. Analysis of the T dependence of the second order rate constant for formation of the enzyme/coformycin complex further reveals that the conformational change is not rate limiting. We propose that the enzyme may facilitate catalysis via the formation of a stabilizing conformation at the reaction transition state.

Adenosine Deaminase↗

PubMed2005

Photophysics of tryptophan fluorescence: link with the catalytic strategy of the citrate synthase from Thermoplasma acidophilum.

The formation of all major intermediates in the reaction catalyzed by the citrate synthase from Thermoplasma acidophilum is accompanied by changes in tryptophan fluorescence. The largest change is the strong quenching observed on formation of the binary complex with substrate, oxaloacetate (OAA). The four tryptophan residues present in the enzyme have been changed to nonfluorescent ones in various combinations without major perturbations in protein stability, enzyme mechanism, or other physical properties. W348, residing in the hydrophobic core of the protein behind the active site wall ca. 9 A from OAA, is responsible for the majority of the protein's intrinsic fluorescence and all of the quenching that accompanies OAA binding. Lifetime studies show that all of the quenching results from excited-state processes. The lack of solvent isotope effects on the quantum yields excludes a quenching mechanism involving proton transfer to an acceptor. There are no significant changes in fluorescence properties in single site mutants of residues near W348 that change conformation and/or interactions when OAA binds. This result excludes these changes from a direct role. Electron transfer from the indole excited state to some acceptor is the major quenching mechanism; the reduced quenching observed in the 5F-W-substituted protein strengthens this conclusion. Using the X-ray structures of the unliganded enzyme and its OAA binary complex, hybrid quantum mechanics-molecular dynamics (QM-MM) calculations show that OAA itself is the most likely quencher with the OAA carbonyl as the electron acceptor. This conclusion is strengthened by the ability of an alpha-keto acid model compound, trimethylpyruvate, to act as a diffusional quencher of indole fluorescence in solution. The theoretical calculations further indicate that the positive electrostatic potential surrounding the OAA carbonyl within the enzymes' active site is essential to its ability to accept an electron from the excited state of W348. These same environmental factors play a major role in activating OAA to react with the carbanion of acetyl-CoA. Since carbonyl polarization plays a role in the catalytic strategies of numerous enzymes whose reactions involve this functional group, tryptophan fluorescence changes might be useful as a mechanistic probe for other systems.

Amino Acid Substitution↗

PubMed1977

[On the changes in alpha-chymotrypsin stability after its modification by polyelectrolytes].

Reversible thermal denaturation of alpha-chymotrypsin, its electrostatic complexes with carboxyl-containing polymers and the enzyme covalently bound with those polymers were studied. It was shown that the enzyme stability is affected by matrix, which manifests itself in a simultaneous decrease in enthalpy and enthropy of the reversible denaturation process and a simultaneous decrease in activation enthalpy and enthropy of denaturation. Modification and complexing of chymotrypsin with polymers has practically no effect on the activation parameters of renaturation. Differences in the original states of alpha-chymotrypsin and its derivatives and similarity of their activated states are proposed. The formation of ionic complexes of enzyme or covalent binding to polymers results in alteration of the protein native state similar to the denaturated state.

Chymotrypsin↗

PubMedDate not supplied

Yeast alcohol dehydrogenase bound to membranes: surface and microenvironment effects on activity and stability.

The enzyme, yeast alcohol dehydrogenase, was adsorbed to porous nitrocellulose and nylon membranes. The two membranes provide different surface chemistries as indicated by the results of the streaming potential, enzyme adsorption, and fluorescein isothiocyanate adsorption experiments. The stability of the enzyme, as determined by continually measuring the extent of coenzyme reduction as a function of time, appeared to be much less for the enzyme adsorbed to the positively charged membrane surface. Moreover, the enzyme adsorbed to the positively charged membrane was the least responsive to pulses of the reducing agent, dithiothreitol, and appeared to exhibit the highest transition temperature when subjected to differential scanning calorimetry analysis. These results indicate that the entropically spreading process observed for other adsorbed proteins may be occurring and the process is more rapid and extensive when enzyme is adsorbed to the nylon than the nitrocellulose membrane. In addition to the relative stability of the enzyme on two different surfaces being examined, the effect of the microenvironment on modulating the activity of the enzyme was investigated by using the reversibility of the enzyme-catalyzed reaction as a probe of the average local environment of the enzyme. It was found that a threshold buffer concentration existed that, once exceeded, the effect of proton production by the reaction could be suppressed.

Alcohol Dehydrogenase↗

PubMed1986

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: chromatographic, electrophoretic, and sedimentation behavior of active and inactive enzyme forms.

This paper describes the study of a highly purified pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Short-term storage (24 h at 4 degrees C) of the purified enzyme in the absence of dithiothreitol, a sulfhydryl reducing agent, led to considerable losses of enzyme activity. Most of the lost activity could be regained, however, by incubating the enzyme with 50 mM dithiothreitol. Enzyme stabilization by dithiothreitol and reactivation by dithiothreitol were enhanced in the presence of phosphate buffer. Severe enzyme inhibition was produced by micromolar concentrations of sulfhydryl group reagents. Chromatographic, electrofocusing, and sucrose gradient centrifugation experiments revealed that the enzyme has a molecular weight of about 26,000, an isoelectric point of 4.7, and a sedimentation coefficient of 2.5. These experiments were also carried out with enzyme preparations which had been almost completely inactivated by means of dialysis to remove dithiothreitol. Enzyme preparations of this type displayed at least one additional enzyme form. This form(s) was inactive but capable of being partially reactivated by dithiothreitol. The inactive form(s) exhibited the same apparent molecular weight as the native enzyme but possessed a higher isoelectric point (5.7). A working hypothesis was presented which states (1) that inactive enzyme forms arise because of disulfide bond formation, (2) that enzyme sulfhydryl groups are less susceptible to oxidation in the presence of phosphate buffer, and (3) that enzyme reactivation by dithiothreitol results from the regeneration of critical enzyme sulfhydryls.

Animals↗

PubMed1988

Purification and characterization of a bacterial nitrophenol oxygenase which converts ortho-nitrophenol to catechol and nitrite.

A nitrophenol oxygenase which stoichiometrically converted ortho-nitrophenol (ONP) to catechol and nitrite was isolated from Pseudomonas putida B2 and purified. The substrate specificity of the enzyme was broad and included several halogen- and alkyl-substituted ONPs. The oxygenase consisted of a single polypeptide chain with a molecular weight of 58,000 (determined by gel filtration) or 65,000 (determined on a sodium dodecyl sulfate-polyacrylamide gel). The enzymatic reaction was NADPH dependent, and one molecule of oxygen was consumed per molecule of ONP converted. Enzymatic activity was stimulated by magnesium or manganese ions, whereas the addition of flavin adenine dinucleotide, flavin mononucleotide, or reducing agents had no effect. The apparent Kms for ONP and NADPH were 8 and 140 microM, respectively. 2,4-Dinitrophenol competitively (Ki = 0.5 microM) inhibited ONP turnover. The optimal pH for enzyme stability and activity was in the range of 7.5 to 8.0. At 40 degrees C, the enzyme was totally inactivated within 2 min; however, in the presence of 1 mM ONP, 40% of the activity was recovered, even after 10 min. Enzymatic activity was best preserved at -20 degrees C in the presence of 50% glycerol.

Catalysis↗