Active Site Sulfhydryl Group Research Articles

Inactivation of Jack Bean Urease by Nitidine Chloride from Zanthoxylum nitidum: Elucidation of Inhibitory Efficacy, Kinetics and Mechanism.

Urease is a metalloenzyme that catalyzes the hydrolysis of urea into ammonia and carbon dioxide, which has a negative impact on human health and agriculture. In this study, the inactivation of jack bean urease by nitidine chloride (NC) was investigated to elucidate the inhibitory effect, kinetics, and underlying mechanism of action. The results showed that NC acted as a concentration- and time-dependent inhibitor with an IC50 value of 33.2 ± 4.8 μM and exhibited a similar inhibitory effect to acetohydroxamic acid (IC50 = 31.7 ± 5.8 μM). Further kinetic analysis demonstrated that NC was a slow-binding and non-competitive inhibitor for urease. Thiol-blocking reagents (dithiothreitol, glutathione, and l-cysteine) significantly retarded urease inactivation, while Ni2+ competitive inhibitors (boric acid and sodium fluoride) synergetically suppressed urease with NC, suggesting that the active site sulfhydryl groups were possibly obligatory for NC blocking urease. Molecular docking simulation further argued its inhibition mechanism. Additionally, NC-induced deactivation of urease was verified to be reversible since the inactivated enzyme could be reactivated by glutathione. Taking together, NC was a non-competitive inhibitor targeting the thiol group at the active site of urease with characteristics of concentration dependence, reversibility, and slow binding, serving as a promising novel urease suppressant.

Insight into the inhibitory effects of Zanthoxylum nitidum against Helicobacter pylori urease and jack bean urease: Kinetics and mechanism.

Zanthoxylum nitidum (Roxb.) DC. is a traditional Chinese medicine characterised by anti-inflammatory and anti-Helicobacter pylori, which is widely used to treat H. pylori-induced gastric disease in China. However, the underlying mechanism related to its anti-H. pylori activity remains unclear. Urease plays a crucial role in the colonisation and survival of H. pylori. The root aqueous extract of Z. nitidum against H. pylori urease (HPU) and jack bean urease (JBU) was investigated to illuminate the inhibitory potency, kinetics and potential mechanism. Z. nitidum components were determined by UPLC. The enzyme inhibitory effects of Z. nitidum were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. Urease inhibition kinetics were determined by Lineweaver-Burk plots. Sulfhydryl group reagents and Ni2+-binding inhibitors were used in the mechanism study. Moreover, the molecular docking technique was used to investigate the binding conformations of the main compounds of Z. nitidum on Urease. According to UPLC results, the major components of Z. nitidum were magnoflorine, sanguinarine, nitidine chloride, chelerythrine, skimmianine and L-Sesamin. Z. nitidum has higher enzyme inhibitory activity on HPU (IC50=1.29±0.10mg/mL) than on JBU (IC50=2.04±0.27mg/mL). Enzyme inhibitory kinetic analysis revealed that the type of Z. nitidum inhibition against HPU was a slow-binding and mixed-type, whereas a slow-binding and non-competitive type inhibited JBU. Further mechanism study indicated that the active site of sulfhydryl group might be the target of inhibition by Z. nitidum. The molecular docking study indicated that the above six main components of Z. nitidum exhibited stronger affinity to HPU than to JBU through interacting with the key amino acid residues located on the mobile flap or interacting with the active site Ni2+. Results indicated that these components are potential active ingredients directed against urease. Z. nitidum inactivated urease in a concentration-dependent manner through slow-binding inhibition and binding to the urease active site sulfhydryl group. Our investigation might provide experimental evidence for the traditional application of Z. nitidum in the treatment of H. pylori-associated gastric disorders.

WITHDRAWN: Coptisine, an alkaloid from Rhizoma Coptidis, inhibits Helicobacter pylori urease activity by targeting its active site and maturation process

// Cailan Li 1, * , Ping Huang 2, * , Kambo Wong 3 , Yifei Xu 2 , Lihua Tan 1 , Hanbin Chen 4 , Qiang Lu 5 , Chaodan Luo 1 , Chunlai Tam 3 , Lixiang Zhu 2 , Ziren Su 1, 6 and Jianhui Xie 7 1 Guangdong Provincial Key Laboratory of New Drug Development and Research of Chinese Medicine, Mathematical Engineering Academy of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, P. R. China 2 School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, P. R. China 3 School of Life Sciences, Center for Protein Science and Crystallography, The Chinese University of Hong Kong, Hong Kong, P. R. China 4 The First Affiliated Hospital of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, P. R. China 5 Key Laboratory of Ministry of Education, Research Center of Chinese Herbal Resources and Engineering, Guangzhou University of Chinese Medicine, Guangzhou, P. R. China 6 Dongguan Mathematical Engineering Academy of Chinese Medicine, Guangzhou University of Chinese Medicine, Dongguan, P. R. China 7 Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, The Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou, P. R. China * These authors have contributed equally to this work Correspondence to: Ziren Su, email: suziren@gzucm.edu.cn Jianhui Xie, email: xiejianhui888@hotmail.com Keywords: coptisine; Helicobacter pylori urease; sulfhydryl group; nickel ion; UreG Received: May 18, 2017 Accepted: January 01, 2018 Published: January 02, 2018 ABSTRACT In this study, we examined the anti- Helicobactor pylori effects of the main protoberberine-type alkaloids in Rhizoma Coptidis. Coptisine exerted varying antibacterial and bactericidal effects against three standard H. pylori strains and eleven clinical isolates, including four drug-resistant strains, with MICs ranging from 25 to 50 μg/mL and MBCs ranging from 37.5 to 125 μg/mL. Coptisine’s anti- H. pylori effects derived from specific inhibition of urease in vivo . In vitro , coptisine inactivated urease in a concentration-dependent manner through slow-binding inhibition and involved binding to the urease active site sulfhydryl group. Coptisine inhibition of H. pylori urease (HPU) was mixed type, while inhibition of jack bean urease (JBU) was non-competitive. Importantly, coptisine also inhibited HPU by binding to its nickel metallocenter. Besides, Coptisine interfered with urease maturation by inhibiting activity of prototypical urease accessory protein UreG and formation of UreG dimers and by promoting dissociation of nickel from UreG dimers. These findings demonstrate that coptisine inhibits urease activity by targeting its active site and inhibiting its maturation, thereby effectively inhibiting H. pylori . Coptisine may thus be an effective anti- H. pylori agent.

Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process

In this study, we examined the anti-Helicobactor pylori effects of the main protoberberine-type alkaloids in Rhizoma Coptidis. Coptisine exerted varying antibacterial and bactericidal effects against three standard H. pylori strains and eleven clinical isolates, including four drug-resistant strains, with minimum inhibitory concentrations ranging from 25 to 50 μg/mL and minimal bactericidal concentrations ranging from 37.5 to 125 μg/mL. Coptisine’s anti-H. pylori effects derived from specific inhibition of urease in vivo. In vitro, coptisine inactivated urease in a concentration-dependent manner through slow-binding inhibition and involved binding to the urease active site sulfhydryl group. Coptisine inhibition of H. pylori urease (HPU) was mixed type, while inhibition of jack bean urease was non-competitive. Importantly, coptisine also inhibited HPU by binding to its nickel metallocentre. Besides, coptisine interfered with urease maturation by inhibiting activity of prototypical urease accessory protein UreG and formation of UreG dimers and by promoting dissociation of nickel from UreG dimers. These findings demonstrate that coptisine inhibits urease activity by targeting its active site and inhibiting its maturation, thereby effectively inhibiting H. pylori. Coptisine may thus be an effective anti-H. pylori agent.

Epiberberine, a natural protoberberine alkaloid, inhibits urease of Helicobacter pylori and jack bean: Susceptibility and mechanism

In our previous study, Rhizoma Coptidis extract was found to exert more potent inhibitory effect than its major component berberine towards urease from Helicobacter pylori (HPU) and jack bean (JBU). In continuation of our work, the present study was designed to further comparatively investigate the urease inhibitory activities of five major protoberberine alkaloids in Rhizoma Coptidis, namely berberine, palmatine, coptisine, epiberberine, jateorhizine to identify the bioactive constituent, and illuminate the potential mechanism of action. Results indicated that the five protoberberine alkaloids acted as concentration-dependent inactivators of urease with IC50 values ranging between 3.0 and 5087μM for HPU and 2.3–>10,000μM for JBU, respectively. Notably, epiberberine (EB) was found to be the most potent inhibitor against both ureases with IC50 values of 3.0±0.01μM for HPU and 2.3±0.01μM for JBU, which was more effective than the standard urease inhibitor, acetohydroxamic acid (83±0.01μM for HPU and 22±0.01μM for JBU, respectively). Further kinetic analysis revealed that the type of EB inhibition against HPU was slow-binding and uncompetitive, with Ki of 10.6±0.01μM, while slow-binding and competitive against JBU with Ki of 4.6±0.01μM. Addition of thiol reagents, such as l-cysteine, glutathione and dithiothreitol, significantly abolished the inhibition, while Ni2+ competitive inhibitors, boric acid and sodium fluoride, synergetically inhibited urease with EB, indicating the obligatory role of the active site sulfhydryl group for the inhibition. In addition, binding of EB with the urease proved to be reversible, as about 65% and 90% enzymatic activity of HPU and JBU, respectively, could be restored by dithiothreitol application. These findings highlighted the potential role of Rhizoma Coptidis protoberberine alkaloids, especially EB, as a lead urease inhibitor in the treatment of diseases associated with ureolytic bacteria. Thus, EB had good potential for further development into a promising therapeutic approach for the treatment of urease-related diseases.

Inhibition of Helicobacter pylori and Its Associated Urease by Palmatine: Investigation on the Potential Mechanism.

In this paper, we evaluated the anti-Helicobacter pylori activity and the possible inhibitory effect on its associated urease by Palmatine (Pal) from Coptis chinensis, and explored the potential underlying mechanism. Results indicated that Pal exerted inhibitory effect on four tested H. pylori strains (ATCC 43504, NCTC 26695, SS1 and ICDC 111001) by the agar dilution test with minimum inhibitory concentration (MIC) values ranging from 100 to 200 μg/mL under neutral environment (pH 7.4), and from 75 to 100 μg/mL under acidic conditions (pH 5.3), respectively. Pal was observed to significantly inhibit both H. pylori urease (HPU) and jack bean urease (JBU) in a dose-dependent manner, with IC50 values of 0.53 ± 0.01 mM and 0.03 ± 0.00 mM, respectively, as compared with acetohydroxamic acid, a well-known urease inhibitor (0.07 ± 0.01 mM for HPU and 0.02 ± 0.00 mM for JBU, respectively). Kinetic analyses showed that the type of urease inhibition by Pal was noncompetitive for both HPU and JBU. Higher effectiveness of thiol protectors against urease inhibition than the competitive Ni2+ binding inhibitors was observed, indicating the essential role of the active-site sulfhydryl group in the urease inhibition by Pal. DTT reactivation assay indicated that the inhibition on the two ureases was reversible, further supporting that sulfhydryl group should be obligatory for urease inhibition by Pal. Furthermore, molecular docking study indicated that Pal interacted with the important sulfhydryl groups and inhibited the active enzymatic conformation through N-H ∙ π interaction, but did not interact with the active site Ni2+. Taken together, Pal was an effective inhibitor of H. pylori and its urease targeting the sulfhydryl groups, representing a promising candidate as novel urease inhibitor. This investigation also gave additional scientific support to the use of C. chinensis to treat H. pylori-related gastrointestinal diseases in traditional Chinese medicine. Pal might be a potentially beneficial therapy for gastritis and peptic ulcers induced by H. pylori infection and other urease-related diseases.

Glyceraldehyde-3-phosphate dehydrogenase: Aggregation mechanisms and impact on amyloid neurodegenerative diseases

The review analyses data on specific features of aggregation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and possible role of this enzyme in the development of neurodegenerative diseases. Different post-translational modifications of the enzyme are considered: oxidation, nitrosylation, and S-glutathionylation of the active site sulfhydryl groups, as well as phosphorylation, glycation and homocysteinylation of other amino acid residues. Modification of the sulfhydryl groups of the enzyme inhibits the enzymatic activity of GAPDH, resulting in slowdown of glycolysis, and may lead to the dissociation of the cofactor NAD from the active site of the enzyme. The resulting apo-GAPDH (without NAD) is less stable and prone to dissociation, denaturation, and subsequent aggregation. These processes could play a crucial role in the translocation of GAPDH subunits from the cytoplasm into the nucleus, which is linked to the induction of apoptosis. Phosphorylation and glycation of GAPDH are presumably involved in the regulation of protein-protein interactions and intracellular localization of the enzyme. Besides, glycation by dicarbonyl compounds and aldehydes may directly inhibit glycolysis. Homocysteinylation of GAPDH may stabilize aggregates of the enzyme by additional disulfide bonding. All types of post-translational modifications affect aggregation of GAPDH. A special attention is given to the role of chaperones in the amyloidogenic transformation of proteins and to confirmation of the hypothesis on blocking of the chaperones by misfolded protein forms. The denatured GAPDH forms were shown to interact directly with amyloidogenic proteins (alpha-synuclein and amyloid-beta peptide) and to play a crucial role in blocking of chaperone system.

Comparison of Helicobacter pylori Urease Inhibition by Rhizoma Coptidis, Cortex Phellodendri and Berberine: Mechanisms of Interaction with the Sulfhydryl Group.

Rhizoma Coptidis, Cortex Phellodendri, and berberine were reported to inhibit Helicobacter pylori. However, the underlying mechanism remained elusive. Urease plays a vital role in H. pylori colonization and virulence. In this work, aqueous extracts of Rhizoma Coptidis, Cortex Phellodendri of different origins, and purified berberine were investigated against H. pylori urease and jack bean urease to elucidate the inhibitory capacity, kinetics, and mechanism. Results showed that berberine was the major chemical component in Rhizoma Coptidis and Cortex Phellodendri, and the content of berberine in Rhizoma Coptidis was higher than in Cortex Phellodendri. The IC50 values of Rhizoma Coptidis were significantly lower than those Cortex Phellodendri and purified berberine, of which Coptis chinensis was shown to be the most active concentration- and time-dependent urease inhibitor. The Lineweaver-Burk plot analysis indicated that the inhibition pattern of C. chinensis against urease was noncompetitive for both H. pylori urease and jack bean urease. Thiol protectors (L-cysteine, glutathione, and dithiothreithol) significantly protected urease from the loss of enzymatic activity, while fluoride and boric acid showed weaker protection, indicating the active-site sulfhydryl group was possibly responsible for its inhibition. Furthermore, the urease inhibition proved to be reversible since C. chinensis-blocked urease could be reactivated by glutathione. The results suggested that the anti-urease activity of Rhizoma Coptidis was superior to that of Cortex Phellodendri and berberine, which was believed to be more likely to correlate to the content of total alkaloids rather than berberine monomer. The concentration- and time-dependent, reversible, and noncompetitive inhibition against urease by C. chinensis might be attributed to its interaction with the sulfhydryl group of the active site of urease.

Andrographolide sodium bisulphite-induced inactivation of urease: inhibitory potency, kinetics and mechanism.

BackgroundThe inhibitory effect of andrographolide sodium bisulphite (ASB) on jack bean urease (JBU) and Helicobacter pylori urease (HPU) was performed to elucidate the inhibitory potency, kinetics and mechanism of inhibition in 20 mM phosphate buffer, pH 7.0, 2 mM EDTA, 25 °C.MethodsThe ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of ASB was characterized with IC50 values. Lineweaver-Burk and Dixon plots for JBU inhibition of ASB was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni2+ binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode.ResultsThe IC50 of ASB against JBU and HPU was 3.28 ± 0.13 mM and 3.17 ± 0.34 mM, respectively. The inhibition proved to be competitive and concentration- dependent in a slow-binding progress. The rapid formation of initial ASB-JBU complex with an inhibition constant of Ki = 2.86 × 10−3 mM was followed by a slow isomerization into the final complex with an overall inhibition constant of Ki* = 1.33 × 10−4 mM. The protective experiment proved that the urease active site is involved in the binding of ASB. Thiol reagents (L-cysteine and dithiothreithol) strongly protect the enzyme from the loss of enzymatic activity, while boric acid and fluoride show weaker protection, indicating that the active-site sulfhydryl group of JBU was potentially involved in the blocking process. Moreover, inhibition of ASB proved to be reversible since ASB-inactivated JBU could be reactivated by dithiothreitol application. Molecular docking assay suggested that ASB made contacts with the important sulfhydryl group Cys-592 residue and restricted the mobility of the active-site flap.ConclusionsASB was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for the treatment of urease-related diseases.

Inactivation of jack bean urease by scutellarin: Elucidation of inhibitory efficacy, kinetics and mechanism

In the present study, the inactivation effect of scutellarin (SL) on jack bean urease was investigated to elucidate the inhibitory potency, kinetics and mechanism of inhibition. It was revealed that SL acted as a concentration- and time-dependent inactivator of urease characteristic of slow-binding inhibition with an IC50 of 1.35±0.15mM. The rapid formation of the initial SL–urease complex with an inhibition constant of Ki=5.37×10−2mM was followed by a slow isomerization into the final complex with the overall inhibition constant of Ki*=3.49×10−3mM. High effectiveness of thiol protectors, such as L-cysteine (L-cys), 2-mercaptoethanol (2-ME) and dithiothreitol (DTT) significantly slowed down the rate of inactivation, indicating the strategic role of the active site sulfhydryl group in the blocking process. While the insignificant protection by boric acid and fluoride from the inactivation further confirmed that the active site cysteine should be obligatory for urease inhibition, which was also rationalized by the molecular docking study. The inhibition of SL on urease proved to be reversible since SL-blocked urease could be reactivated by DTT application and multidilution. The results obtained indicated that urease inactivation resulted from the reaction between SL and the sulfhydryl group.

Kinetics and Mechanism Study of Competitive Inhibition of Jack-Bean Urease by Baicalin

Baicalin (BA) is the principal component of Radix Scutellariae responsible for its pharmacological activity. In this study, kinetics and mechanism of inhibition by BA against jack-bean urease were investigated for its therapeutic potential. It was revealed that the IC50 of BA against jack-bean urease was 2.74 ± 0.51 mM, which was proved to be a competitive and concentration-dependent inhibition with slow-binding progress curves. The rapid formation of initial BA-urease complex with an inhibition constant of K i = 3.89 × 10−3 mM was followed by a slow isomerization into the final complex with an overall inhibition constant of K i* = 1.47 × 10−4 mM. High effectiveness of thiol protectors against BA inhibition indicated that the strategic role of the active-site sulfhydryl group of the urease was involved in the blocking process. Moreover, the inhibition of BA was proved to be reversible due to the fact that urease could be reactivated by dithiothreitol but not reactant dilution. Molecular docking assay suggested that BA made contacts with the important activating sulfhydryl group Cys-592 residues and restricted the mobility of the active-site flap. Taken together, it could be deduced that BA was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for treatments on urease-related diseases.

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25°C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of Ki* = 4.5 × 10− 7 mM. The respective inhibition constant of TCoBQ was equal to: Ki* = 2.4 × 10− 6 mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.

Inhibition of jack bean urease by p-benzoquinone: elucidation of the role of thiols and reversibility of the process

p-Benzoquinone (pBQ) was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 °C. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. The influence of the inhibitor concentration and the preincubation time on the enzyme activity was elucidated. It was found that increase in pBQ concentration resulted in a linear decrease of urease activity. The dependence of the enzyme activity on the preincubation time showed that the rate of inhibition rapidly decreased at the beginning of the process in order to achieve the constant value. The inhibition became time independent in the studied time range. This observation is characteristic of a slow binding mechanism of inhibition. The protective experiment proved that the urease active site is involved in the binding of pBQ. High effectiveness of thiol protectors against pBQ inhibition indicates the strategic role of the active site sulfhydryl group in the blocking process. There were two methods used for reactivation of pBQ-inhibited urease. The dilution of the urease-pBQ complex in urea solution did not result in a regain of enzyme activity. Alternatively, the addition of dithiothreitol into the urease-pBQ mixture caused the instant and efficient reactivation of the enzyme. The experiments showed that the nature of the urease-pBQ complex is irreversible but the application of a specific thiol reagent can release the active enzyme from the complex.

Active-site sulfhydryl chemistry plays a major role in the misfolding of urea-denatured rhodanese.

Unfolded bovine rhodanese, a sulfurtransferase, does not regain full activity upon refolding due to the formation of aggregates and disulfide-linked misfolded states unless a large excess of reductant such as 200 mM beta-ME and 5 mg/ml detergent are present [Tandon and Horowitz (1990), J. Biol. Chem. 265, 5967]. Even then, refolding is incomplete. We have studied the unfolding and refolding of three rhodanese forms whose crystal structures are known: ES, containing the transferred sulfur as a persulfide; E, without the transferred sulfur, and carboxymethylated rhodanese (CMR), in which the active site was blocked by chemical modification. The X-ray structures of ES, E, and CMR are virtually the same, but their tertiary structures in solution differ somewhat as revealed by near-UV CD. Among these three, CMR is the only form of rhodanese that folds reversibly, requiring 1 mM DTT. A minimum three-state folding model of CMR (N<-->I<-->U) followed by fluorescence at 363 nm, (N<-->I) by fluorescence at 318 nm, and CD (I<-->U) is consistent with the presence of a thermodynamically stable molten globule intermediate in 5-6 M urea. We conclude that the active-site sulfhydryl group in the persulfide form is very reactive; therefore, its modification leads to the successful refolding of urea-denatured rhodanese even in the absence of a large excess of reductant and detergent. The requirement for DTT for complete reversibility of CMR suggests that oxidation among the three non-active-site SH groups can represent a minor trap for refolding through species that can be easily reduced.

Inhibition of membrane-bound succinate dehydrogenase by fluorescamine

Fluorescamine rapidly inactivated membrane-bound succinate dehydrogenase. The inhibition of the enzyme by this reagent was prevented by succinate and malonate, suggesting that the group modified by fluorescamine was located at the active site. The modification of the active site sulfhydryl group by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) did not alter the inhibitory action of fluorescamine. However, the protective effect of malonate against fluorescamine inhibition was abolished in the enzyme modified at the thiol.

Approaches to chemically modified enzymes as synthetic catalysts

Attempts to introduce new catalytic activities of potential use in synthetic transformations into enzyme active sites are described. Substitution of the naturally occurring zinc in carboxypeptidase A by several metals known to catalyse hydrogenation was investigated; a new protein characterised as a rhodium carboxypeptidase was isolated but it failed to show activity as a hydrogenation catalyst for the reduction of a series of dehydroamino acid amides. Horse liver alcohol dehydrogenase was investigated for its potential to act as an oxygen transferase via Lewis acid catalysis. A series of cyclohexenyl and phenylribosides together with new alkenyl(arenyl)oxymethylenoxyethanols was prepared for evaluation as substrates; in the course of this study, novel neighbouring group participation by the oxygen atom of a chloromethyl ether was observed. Although the binding of potential oxygen acceptors (alkenes and aromatic compounds) and oxygen donors (hydrogen peroxide and alkyl hydroperoxides) was demonstrated, oxygen transfer did not occur with the combinations investigated. In contrast to the failure of the above metalloenzymes to catalyse new reactions, papain modified at the active site sulfhydryl group by thiazolium salts and pyridinium salts was shown to exhibit reactions characteristic of the covalently attached cofactor. Thus the thiazolopapains acted as decarboxylation catalysts for pyruvate and, the pyridinopapains could be reduced to dihydropyridines which reduced electrophilic carbonyl substrates with small enantiomeric excess.

Reversible covalent binding of peptide nitriles to papain

The dissociation constants for reversible covalent binding of twelve peptide nitrile inhibitors to the active site of papain have been measured by means of flourescence titration. The binding constants generally the kinetic specificity constants ( k cat/ K m) for related papain substrates, supporting earlier suggestions that peptide nitriles behave as transition state analog inhibitors of papain. In ten cases the temperature dependence of binding was analyzed to determine the enthalpic and entropic contributions to the binding energy. A compensation plot of ΔH vs. TΔS resulted in two parallel lines, one for ‘specific’ nitriles (i.e., N- Ac- L -aa-NHCH 2CN ; aa = Phe, Leu, Met)_and the other for ‘non-specific’ nitriles (e.g., N- Ac- D-Phe-NHCH 2CN, PhCH 2CH 2CONHCH 2CN hippurylnitrile, etc.). For both specific and nonspecific nitriles representing an 1800-fold range of K d values (0.27 μM–490 μM), the solvent deuterium isotope effect on binding ( K d ( H 2O /K d ( D 2O ) = D K d ) was very close to 2.0. This isotope effect could be accounted for entirely by the simple protonic change which occurs upon the reversible addition of the active site sulfhydryl of papain to the nitrile group of the peptide derivative to form a covalent thioimidate linkage. In contrast, six closely related non-nitrile ligands containing identical peptide side chains but having C-terminal groups incapable of binding covalently to papain had unmeasureably high dissociation constants. Collectively, these results indicate that strong binding of peptide nitrile substrate analogs to papain requires a combination of (1) hydrophobic interaction (especially at the P2 position), (2) specific intermolecular hydrogen bonding and (3) covalent interaction of the nitrile with the active site sulfhydryl group.

The removal of exogenous thiols from proteins by centrigugal column chromatography

Centrifugal column chromatography was shown to provide a rapid, efficient, and useful means of separation of various low molecular weight thiols from proteins. The single chromatographic step procedure employed standard 5 ml plastic syringes containing Sephadex G-25 as the bed matrix and required less than 5 min to procedure average dilutions of 5000-, 980-, and 25-fold, respectively, from 5 to 200 mM initial concentrations of 2-mercaptoethanol, dithiothreitol, and reduced glutathione in the sample as measured by titration with 5,5′-dithiobis-(2-nitrobenzoic acid). Dihydrofolate reductase solutions of 0.07–0.08 mM were separated from 50 mM 2-mercaptoethanol, dithiothreitol, or reduced glutathione with a minimum 16 500-fold dilution of the thiol after centrifugal chromatography on two consecutive columns. Thymidylate synthase solutions of 0.06 mM were effectively separated from mM 2-mercaptoethanol or dithiothreitol with a minimum average 5900-fold dilution of the thiol after consecutive column chromatography. There was no change in either the physical or chemical properties of the enzyme throughout the course of the experiments as determined by activity, active site sulfhydryl group titration, and binding assays. Recoveries of protein obtained in the load fraction were usually in excess of 70% of the protein with virtually no dilution from the initial concentration. This method was developed in order to facilitate the study of the active site sulfhydryl groups in enzymes.

Sulfhydryl-directed triggering of conformational changes in the enzyme rhodanese.

The enzyme rhodanese in the form without transferred sulfur, (E), was inactivated by carboxymethylation with iodoacetic acid (E.IAA), and its conformation was compared with that of E inactivated by oxidative processes (Eox). Formation of E.IAA led to the exposure of binding sites for the fluorescent apolar probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (BisANS). The dissociation constant for BisANS decreased as the concentration of E.IAA decreased and ranged from approximately 200 microM at 1 mg/ml protein to approximately 2 microM at protein concentrations below 0.1 mg/ml. Centrifugation confirmed that E.IAA, but not the underivatized enzyme, could associate. E.IAA was proteolyzable by chymotrypsin or endoproteinase Glu C (V8), while rhodanese containing bound sulfur, ES, was totally refractory, and E was only clipped to a small extent. This constellation of consequences was only previously observed with oxidatively inactivated rhodanese. Fluorescence depolarization measurements of bound BisANS were consistent with exposure of apolar surfaces and association of the protein. The fluorescence spectra of BisANS bound to E.IAA or Eox were identical and distinct from the spectrum of BisANS bound to phenylglyoxal-inactivated ES. Digestion with chymotrypsin was followed using protein and BisANS fluorescence and showed a similar response for E.IAA and Eox. These results indicate that the consequences of forming Eox and E.IAA are very similar. Thus, reaction of the active site sulfhydryl group apparently triggers a conformational change leading to increased protein flexibility and increased exposure of hydrophobic surfaces. In the case of oxidation, the trigger might involve initial formation of an active site sulfenic acid which ultimately gives higher oxidation states that could include disulfides.

Identification and reactivity of the catalytic site of pig liver thioltransferase.

The active site cysteine of pig liver thioltransferase was identified as Cys22. The kinetics of the reaction between Cys22 of the reduced enzyme and iodoacetic acid as a function of pH revealed that the active site sulfhydryl group had a pKa of 2.5. Incubation of reduced enzyme with [1-14C]cysteine prevented the inactivation of the enzyme by iodoacetic acid at pH 6.5, and no stable protein-cysteine disulfide was found when the enzyme was separated from excess [1-14C]cysteine, suggesting an intramolecular disulfide formation. The results suggested a reaction mechanism for thioltransferase. The thiolated Cys22 first initiates a nucleophilic attack on a disulfide substrate, resulting in the formation of an unstable mixed disulfide between Cys22 and the substrate. Subsequently, the sulfhydryl group at Cys25 is deprotonated as a result of micro-environmental changes within the active site domain, releasing the mixed disulfide and forming an intramolecular disulfide bond. Reduced glutathione, the second substrate, reduces the intramolecular disulfide forming a transient mixed disulfide which is then further reduced by glutathione to regenerate the reduced enzyme and form oxidized glutathione. The rate-limiting step for a typical reaction between a disulfide and reduced glutathione is proposed to be the reduction of the intramolecular disulfide form of the enzyme by reduced glutathione.