Inhibition of Jack Bean Urease by N-(n-butyl)thiophosphorictriamide and N-(n-butyl)phosphorictriamide: Determination of the Inhibition Mechanism

N-(n-butyl)thiophosphorictriamide (NBPT) and its oxygen analogue N-(n-butyl)phosphorictriamide (NBPTO) were studied as inhibitors of jack bean urease. NBPTO was obtained by spontaneous conversion of NBPT into NBPTO. The conversion under laboratory conditions was slow and did not affect NBPT studies. The mechanisms of NBPT and NBPTO inhibition were determined by analysis of the reaction progress curves in the presence of different inhibitor concentrations. The obtained plots were time-dependent and characteristic of slow-binding inhibition. The effects of different concentration of NBPT and NBPTO on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships for a one-step enzyme-inhibitor interaction, qualified as mechanism A. The inhibition constants of urease by NBPT and NBPTO were found to be 0.15 μM and 2.1 nM, respectively. The inhibition constant for NBPT was also calculated by steady-state analysis and was found to be 0.13 μM. NBPTO was found to be a very strong inhibitor of urease in contrast to NBPT.     

新型磷酰胺类脲酶抑制剂对不同质地土壤尿素转化的影响

施用脲酶抑制剂是降低尿素水解、减少氨气挥发损失、提高作物氮(N)肥利用率的重要途径之一.采用室内恒温、恒湿模拟试验方法,在25 ℃黑暗条件下培养,研究新型磷酰胺类脲酶抑制剂N-丙基磷酰三胺(NPPT)的脲酶抑制效果,比较其与N-丁基磷酰三胺(NBPT)在不同尿素用量条件下不同质地土壤中对脲酶的抑制差异.结果表明: 在壤土和黏土中,尿素作用时间≤9 d,添加抑制剂可以将尿素水解时间延长3 d以上.砂土中,尿素分解过程相对缓慢,添加抑制剂显著降低土壤脲酶活性,抑制NH₄⁺-N生成.在培养期间,不同尿素用量条件下,脲酶抑制剂在不同质地土壤中的抑制效果表现为高施N量优于低施N量.培养第6天,在尿素用量250 mg N·kg^-1条件下,NBPT和NPPT在砂土中脲酶抑制率分别为56.3%和53.0%,在壤土中分别为0.04%和0.3%,在黏土中分别为4.1%和6.2%;尿素用量500 mg N·kg^-1,NBPT和NPPT在砂土中脲酶抑制率分别为59.4%和65.8%,在壤土中分别为14.5%和15.1%,在黏土中分别为49.1%和48.1%.不同质地土壤中脲酶抑制效果表现为砂土>黏土>壤土.不同抑制剂处理在培养期间土壤NH₄⁺-N含量呈现先上升后下降的趋势,而NO₃^--N含量和表观硝化率均呈现逐渐上升的趋势.与单施尿素处理相比,添加脲酶抑制剂NBPT和NPPT显著增加土壤中的残留尿素态N,降低NH₄⁺-N生成.新型脲酶抑制剂NPPT在不同质地土壤中的抑制效果与NBPT相似,是一款有效的脲酶抑制剂.     

Short term physiological implications of NBPT application on the N metabolism of Pisum sativum and Spinacea oleracea

The application of urease inhibitors in conjunction with urea fertilizers as a means of reducing N loss due to ammonia volatilization requires an in-depth study of the physiological effects of these inhibitors on plants. The aim of this study was to determine how the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) affects N metabolism in pea and spinach. Plants were cultivated in pure hydroponic culture with urea as the sole N source. After 2 weeks of growth for pea, and 3 weeks for spinach, half of the plants received NBPT in their nutrient solution. Urease activity, urea and ammonium content, free amino acid composition and soluble protein were determined in leaves and roots at days 0, 1, 2, 4, 7 and 9, and the NBPT content in these tissues was determined 48 h after inhibitor application. The results suggest that the effects of NBPT on spinach and pea urease activity differ, with pea being most affected by this treatment, and that the NBPT absorbed by the plant caused a clear inhibition of the urease activity in pea leaf and roots. The high urea concentration observed in leaves was associated with the development of necrotic leaf margins, and was further evidence of NBPT inhibition in these plants. A decrease in the ammonium content in roots, where N assimilation mainly takes place, was also observed. Consequently, total amino acid contents were drastically reduced upon NBPT treatment, indicating a strong alteration of the N metabolism. Furthermore, the amino acid profile showed that amidic amino acids were major components of the reduced pool of amino acids. In contrast, NBPT was absorbed to a much lesser degree by spinach plants than pea plants (35% less) and did not produce a clear inhibition of urease activity in this species.     

The physiological implications of urease inhibitors on N metabolism during germination of Pisum sativum and Spinacea oleracea seeds

The development of new nitrogen fertilizers is necessary to optimize crop production whilst improving the environmental aspects arising from the use of nitrogenous fertilization as a cultural practice. The use of urease inhibitors aims to improve the efficiency of urea as a nitrogen fertilizer by preventing its loss from the soil as ammonia. However, although the action of urease inhibitors is aimed at the urease activity in soil, their availability for the plant may affect its urease activity. The aim of this work was therefore to evaluate the effect of two urease inhibitors, namely acetohydroxamic acid (AHA) and N-(n-butyl) thiophosphoric triamide (NBPT), on the germination of pea and spinach seeds. The results obtained show that urease inhibitors do not affect the germination process to any significant degree, with the only process affected being imbibition in spinach, thus also suggesting different urease activities for both plants. Our findings therefore suggest an activity other than the previously reported urolytic activity for urease in spinach. Furthermore, of the two inhibitors tested, NBPT was found to be the most effective at inhibiting urease activity, especially in pea seedlings.     

Inhibition of jack bean urease by 1,4-benzoquinone and 2,5-dimethyl-1,4-benzoquinone. Evaluation of the inhibition mechanism

1,4-benzoquinone (BQ) and 2,5-dimethyl-1,4-benzoquinone (DMBQ) were studied as inhibitors of jack bean urease in 50 mM phosphate buffer, pH 7.0. The mechanisms of inhibition were evaluated by progress curves studies and steady-state approach to data achieved by preincubation of the enzyme with the inhibitor. The obtained reaction progress curves were time-dependent and characteristic of slow-binding inhibition. The effects of different concentrations of BQ and DMBQ on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. The rapid formation of an initial BQ-urease complex with an inhibition constant of Ki = 0.031 mM was followed by a slow isomerization into the final BQ-urease complex with the overall inhibition constant of Ki* = 4.5 x 10(-5) mM. The respective inhibition constants for DMBQ were Ki = 0.42 mM, Ki* = 1.2 x 10(-3) mM. The rate constants of the inhibitor-urease isomerization indicated that forward processes were rapid in contrast to slow reverse reactions. The overall inhibition constants obtained by the steady-state analysis were found to be 5.1 x 10(-5) mM for BQ and 0.98 x 10(-3) mM for DMBQ. BQ was found to be a much stronger inhibitor of urease than DMBQ. A test, based on reaction with L-cysteine, confirmed the essential role of the sulfhydryl group in the inhibition of urease by BQ and DMBQ.     

Molecular Modeling and docking of Wheat Hydroquinone Glucosyl transferase by using Hydroquinone,Phenyl phosphorodiamate and n-(n butyl) Phosphorothiocic Triamide as Inhibitors

In agriculture high urease activity during urea fertilization causes substantial environmental and economical problems by releasing abnormally large amount of ammonia into the atmosphere which leads to plant damage as well as ammonia toxicity. All over the world, urea is the most widely applied nitrogen fertilizer. Due to the action of enzyme urease; urea nitrogen is lost as volatile ammonia. For efficient use of nitrogen fertilizer, urease inhibitor along with the urea fertilizer is one of the best promising strategies. Urease inhibitors also provide an insight in understanding the mechanism of enzyme catalyzed reaction, the role of various amino acids in catalytic activity present at the active site of enzyme and the importance of nickel to this metallo enzyme. By keeping it in view, the present study was designed to dock three urease inhibitors namely Hydroquinone (HQ), Phenyl Phosphorodiamate (PPD) and N-(n-butyl) Phosphorothiocic triamide (NBPT) against Hydroquinone glucosyltransferase using molecular docking approach. The 3D structure of Hydroquinone glucosyltransferase was predicted using homology modeling approach and quality of the structure was assured using Ramachandran plot. This study revealed important interactions among the urease inhibitors and Hydroquinone glucosyltransferase. Thus, it can be inferred that these inhibitors may serve as future anti toxic constituent against plant toxins.     

Inhibition of Jack Bean Urease by 1,4-benzoquinone and 2,5-dimethyl-1,4-benzoquinone. Evaluation of the Inhibition Mechanism

1,4-benzoquinone (BQ) and 2,5-dimethyl-1,4-benzoquinone (DMBQ) were studied as inhibitors of jack bean urease in 50 mM phosphate buffer, pH 7.0. The mechanisms of inhibition were evaluated by progress curves studies and steady-state approach to data achieved by preincubation of the enzyme with the inhibitor. The obtained reaction progress curves were time-dependent and characteristic of slow-binding inhibition. The effects of different concentrations of BQ and DMBQ on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. The rapid formation of an initial BQ-urease complex with an inhibition constant of K i =0.031 mM was followed by a slow isomerization into the final BQ-urease complex with the overall inhibition constant of K*_i=4.5 × 10 ^?5 mM. The respective inhibition constants for DMBQ were K i =0.42 mM, K*_i =1.2 × 10 ^?3 mM. The rate constants of the inhibitor-urease isomerization indicated that forward processes were rapid in contrast to slow reverse reactions. The overall inhibition constants obtained by the steady-state analysis were found to be 5.1 × 10 ^?5 mM for BQ and 0.98 × 10 ^?3 mM for DMBQ. BQ was found to be a much stronger inhibitor of urease than DMBQ. A test, based on reaction with L-cysteine, confirmed the essential role of the sulfhydryl group in the inhibition of urease by BQ and DMBQ.     

Short-term effects of urea amended with the urease inhibitor N-(n-butyl) thiophosphoric triamide on perennial ryegrass

The current study investigated the short-term physiological implications of plant nitrogen uptake of urea amended with the urease inhibitor N-(n-butyl) thiophosphoric triamide (nBTPT) under both greenhouse and field conditions. ¹⁵N labelled urea amended with 0.0, 0.01, 0.1 and 0.5% nBTPT (w/w) was surface applied at a rate equivalent to 100 kg N ha^–1 to perennial ryegrass in a greenhouse pot experiment. Root, shoot and soil fractions were destructively harvested 0.75, 1.75, 4, 7 and 10 days after fertilizer application. Urease activity was determined in each fraction together with ¹⁵N recovery and a range of chemical analyses. The effect of nBTPT amended urea on leaf tip scorch was evaluated together with the effect of the inhibitor applied on its own on plant urease activity.nBTPT-amended urea dramatically reduced shoot urease activity for the first few days after application compared to unamended urea. The higher the nBTPT concentration the longer the time required for shoot activity to return to that in the unamended treatment. At the highest inhibitor concentration of 0.5% shoot urease activity had returned to that of unamended urea by 10 days. Root urease activity was unaffected by nBTPT in the presence of urea but was affected by nBTPT in the absence of urea.Transient leaf tip scorch was observed approximately 7–15 days after nBTPT + urea application and was greatest with high concentrations of nBTPT and high urea-N application rates. New developing leaves showed no visual sign of tip necrosis.Urea hydrolysis of unamended urea was rapid with only 1.3% urea-N remaining in the soil after 1.75 days. N uptake and metabolism by ryegrass was rapid with ¹⁵N recovery from unamended urea, in the plant (shoot + root) being 33% after 1.75 days. Most of the ¹⁵N in the soil following the urea+0.5% nBTPT application was still as urea after 1.75 days, yet ¹⁵N plant recovery at this time was 25% (root+shoot). This together with other evidence, suggests that if urea hydrolysis in soil is delayed by nBTPT then urea can be taken up by ryegrass as the intact molecule, albeit at a significantly slower initial rate of uptake than NH₄ ⁺-N. Protein and water soluble carbohydrate content of the plant were not significantly affected by amending urea with nBTPT however, there was a significant effect on the composition of amino acids in the roots and shoots, suggesting a difference in metabolism.Although nBTPT-amended urea affected plant urease activity and caused some leaf-tip scorch the effects were transient and short-lived. The previously reported benefit of nBTPT in reducing NH₃ volatilization of urea would appear to far outweigh any of the observed short-term effects, as dry-matter production of ryegrass is increased.     

L-Tyrosine beta-naphthylamide is a potent competitive inhibitor of tyramine N-(hydroxycinnamoyl)transferase in vitro

L-Tyrosine beta-naphthylamide, a synthetic substrate designed to measure tyrosine aminopeptidase activity, is a potent inhibitor of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) purified from elicited tobacco cell-suspension cultures. The inhibition is competitive, with the inhibitor binding reversibly to the tyramine binding site of the enzyme. Similar results were obtained with THT extracted from elicited potato cell-suspension cultures. Ki values were found to be 0.66 microM for the enzyme from tobacco and 0.3 microM for the enzyme from potato. L-Tyrosine 7-amido-4-methylcoumarin, a fluorogenic substrate for tyrosine aminopeptidases, the structure of which is close to that of L-tyrosine beta-naphthylamide. was also a powerful inhibitor, but slightly less effective with Ki values of 0.72 and 0.42 microM for tobacco and potato THT, respectively. L-Tyrosine beta-naphthylamide was rapidly hydrolysed when fed in vivo to tobacco or potato cell cultures or when incubated in crude enzymic extracts prepared from these cultures. This hydrolysis, which is presumably catalysed by aminopeptidases, precludes the use of L-tyrosine amides as inhibitors of THT in vivo.     

Synthesis of N-(beta-D-glucopyranosyl)- and N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl) amides as inhibitors of glycogen phosphorylase

2,3,4,6-Tetra-O-acetyl-beta-D-glucopyranosyl- and 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl azides were transformed into the corresponding per-O-acetylated N-(beta-D-glycopyranosyl) amides via a PMe(3) mediated Staudinger protocol (generation of N-(beta-D-glycopyranosyl)imino-trimethylphosphoranes followed by acylation with carboxylic acids, acid chlorides or anhydrides). The deprotected compounds obtained by Zemplén deacetylation were evaluated as inhibitors of rabbit muscle glycogen phosphorylase b. The best inhibitor of this series has been N-(beta-D-glucopyranosyl) 3-(2-naphthyl)-propenoic amide (K(i)=3.5microM).     

N-(Hydroxyaminocarbonyl)phenylalanine: a novel class of inhibitor for carboxypeptidase A

N-(Hydroxyaminocarbonyl)phenylalanine (1) was designed rationally as a new type of inhibitor for carboxypeptidase A (CPA). The designed inhibitor was readily prepared from phenylalnine benzyl ester in two steps and evaluated to find that rac-1 inhibits CPA in a competitive fashion with the Ki value of 2.09 microM. Surprisingly, inhibitor 1 having the D-configuration is more potent (Ki = 1.54 microM) than its antipode by about 3-fold. A possible explanation for the stereochemistry observed in the inhibition of CPA with 1 is presented.     

Use of urease inhibitors to reduce ammonia loss following application of urea to flooded rice fields

Ammonia (NH₃) volatilization is an important mechanism for nitrogen (N) loss from flooded rice fields following the application of urea into the floodwater. One method of reducing losses is to use a urease inhibitor that retards the hydrolysis of urea by soil urease and allows the urea to diffuse deeper into the soil. The two chemicals that have shown most promise are phenylphosphorodiamidate [PPD] and N(n-butyl)thiophosphorictriamide [NBPT], but they seldom work effectively. PPD decomposes rapidly when the pH departs from neutrality, and NBPT must be converted to the oxygen analogue for it to be effective. Our field studies in Thailand show that the activity of PPD can be prolonged, and NH₃ loss markedly reduced, by controlling the floodwater pH with the algicide terbutryn. A mixture of NBPT and PPD in the presence of terbutryn was even more effective than PPD alone. It appears that during the time when the PPD was effective, NBPT was being converted to the oxygen analogue. The combined urease inhibitor-algicide treatment reduced NH₃ loss from 10 to 0.4 kg N ha^-1.     

Interaction of N-(2-Nitro-4-Azidophenyl)-Serotonin with Two Types of Monoamine Oxidase in Rat Brain

The effects of N-(2-nitro-4-azidophenyl) serotonin (NAP-5-HT) on types A and B monoamine oxidase (MAO) in rat brain cortex were studied. In the dark this compound acted as a competitive inhibitor for both types A and B MAO (Ki values of 0.19 microM and 0.21 microM for types A and B MAO, respectively). Upon photolysis, NAP-5-HT became an irreversible inhibitor for only type B MAO. A 50% inhibition was obtained by irradiation of the enzyme in the presence of 35 nM NAP-5-HT. Furthermore the inhibition of type B MAO could be protected by including its substrate phenylethylamine during the irradiation. Under the same photolytic conditions photodependent inhibition of type A MAO by NAP-5-HT was not clearly observed. These results provide further evidence that there is a fundamental difference in the active site of the two types of MAO in brain. NAP-5-HT may be a useful photoaffinity probe for characterizing the active site of type B MAO.     

Combination of a urease inhibitor and a plant essential oil to control coliform bacteria, odour production and ammonia loss from cattle waste

AIM: To evaluate urea hydrolysis, volatile fatty acid (VFA) production (odour) and coliforms in cattle waste slurries after a urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) and a plant oil component (thymol) were added. METHODS AND RESULTS: Faeces from cattle fed a diet of 70% corn silage and 30% alfalfa haylage, urine and distilled water in the ratio 50 : 35 : 15 were blended at high speed for 1 min. Triplicate aliquots of 750 ml were amended with NBPT plus or minus thymol and reblended for 1 min, and were poured into 1.6 l wide-mouth jars covered 90% with a lid. After 56 days, thymol (2000 mg kg(-1) waste) in combination with NBPT (80 mg kg(-1) waste) retained 5.2 g of an initial 9.2 g of urea in cattle waste slurries, compared with less than 1 g of urea retained when NBPT was the only additive (P < 0.05). Another experiment using excreta from cattle fed 76.25% high moisture corn, 19.25% corn silage and a 4.5% supplement, blended at a low speed, gave a similar response with urea hydrolysis; and the two treatments, thymol alone and thymol in combination with NBPT, reduced VFA production (P < 0.01) and eliminated all coliform bacteria by day 1. A third experiment indicated coliforms disappeared in the no addition treatment after 8 days; however, they were viable at 6.6 x 10(4) CFU g(-1) waste beyond 35 days in the NBPT treatment. CONCLUSIONS: Thymol supplements the effect of NBPT by increasing the inhibitory period for hydrolysis of urea in cattle waste slurries and nitrogen retention in the waste. SIGNIFICANCE AND IMPACT OF THE STUDY: Thymol and NBPT offer the potential to reduce odour and pathogens in cattle manure, and increase the fertilizer value.     

Polyamines with N-(3-phenylpropyl) substituents are effective competitive inhibitors of trypanothione reductase and trypanocidal agents

Several N-(3-phenylpropyl)-substituted spermidine and spermine derivatives were prepared and found to be potent competitive inhibitors of Trypanosoma cruzi trypanothione reductase (seven compounds with Ki values < 5 microM are described). The most effective inhibitor studied was compound 12 with a Ki value of 0.151 microM. Six of the compounds described are also effective trypanocides with IC50 values < 1 microM.     

Synthesis of N-(beta-D-glucopyranosyl) monoamides of dicarboxylic acids as potential inhibitors of glycogen phosphorylase

O-peracetylated N-(beta-D-glucopyranosyl)imino trimethylphosphorane obtained in situ from 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl azide and PMe3 was reacted with saturated and unsaturated aliphatic and aromatic dicarboxylic acids, or their anhydrides, or monoesters to give the corresponding N-(beta-D-glucopyranosyl) monoamides of dicarboxylic acids or derivatives. The acetyl protecting groups were removed according to the Zemplén protocol to give a series of compounds which showed moderate inhibitory effects against rabbit muscle glycogen phosphorylase b. The best inhibitor was 3-(N-beta-D-glucopyranosyl-carbamoyl)propanoic acid (7) with Ki = 20 microM.     

Effect of <Emphasis Type="Italic">N</Emphasis>-(<Emphasis Type="Italic">n</Emphasis>-butyl) thiophosphoric triamide on urea metabolism and the assimilation of ammonium by <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

The use of urea as an N fertilizer has increased to such an extent that it is now the most widely used fertilizer in the world. However, N losses as a result of ammonia volatilization lead to a decrease in its efficiency, therefore different methods have been developed over the years to reduce these losses. One of the most recent involves the use of urea combined with urease inhibitors, such as N-(n-butyl) thiophosphoric triamide (NBPT), in an attempt to delay the hydrolysis of urea in the soil. The aim of this study was to perform an in-depth analysis of the effect that NBPT use has on plant growth and N metabolism. Wheat plants were cultivated in a greenhouse experiment lasting 4 weeks and fertilized with urea and NBPT at different concentrations (0, 0.012, 0.062, 0.125%). Each treatment was replicated six times. A non-fertilized control was also cultivated. Several parameters related with N metabolism were analysed at the end of growth period. NBPT use was found to have visible effects, such as a transitory yellowing of the leaf tips, at the end of the first week of treatment. At a metabolic level, plants treated with the inhibitor were found to have more urea in their tissues and a lower amino acid content, lower glutamine synthetase activity, and lower urease and glutamine synthetase content at the end of the study period, whereas their urease activity seemed to have recovered by this stage.     

脲酶/硝化抑制剂在黑土和褐土中对尿素氮转化的调控效果

本试验研究脲酶/硝化抑制剂不同组合在黑土和褐土中对尿素水解和硝化作用的调控效果,旨在筛选出适合东北黑土、褐土的高效抑制剂组合。采用室内恒温、恒湿培养试验,以不施氮肥(CK)和施用普通尿素肥料(U)为对照,研究分别添加脲酶抑制剂N-丁基硫代磷酰三胺(NBPT)及其与硝化抑制剂双氰胺(DCD)、3,4-二甲基吡唑磷酸盐(DMPP)、2-氯-6(三氯甲基)-吡啶(CP)、2-氨基-4-氯-6-甲基嘧啶(AM)、3-甲基吡唑(MP)组合制成的6种高效稳定性尿素在黑土和褐土中的尿素水解和氨氧化特征。在培养125 d内分别取土壤样品15次,通过测定2种土壤中尿素态氮、铵态氮和硝态氮含量,及氨氧化作用强度,计算硝化抑制率,确定最适合2种土壤的抑制剂或组合。结果表明: 尿素在黑土和褐土中水解时间约7 d,添加NBPT以及其与不同硝化抑制剂组合均能将尿素水解时间延长21 d以上。与U处理相比,添加抑制剂可显著增加土壤NH₄⁺-N含量,降低NO₃^--N生成量,维持土壤中高NH₄⁺-N含量的时间更久。黑土中,添加硝化抑制剂的处理均能显著抑制土壤硝化作用,有效硝化抑制时间超过125 d;DMPP、CP与NBPT配施使黑土NH₄⁺-N含量提高1.6～1.8倍,培养125 d时其硝化抑制率分别为47.9%和24.1%。褐土中,U处理培养80 d左右基本完成硝化过程,而添加硝化抑制剂使硝化过程延长至少30 d;DCD、DMPP与NBPT配施使土壤NH₄⁺-N含量提高2.1～3.4倍,培养125 d时其硝化抑制率分别为25.3%和23.2%。因此,尿素与NBPT+DMPP和NBPT+DCD制成的高效稳定性尿素分别在黑土和褐土中施用效果最好,其次分别是NBPT+CP和NBPT+DMPP。     

Enzymes of de novo pyrimidine biosynthesis in Babesia rodhaini

The pathway of de novo pyrimidine biosynthesis in the rodent parasitic protozoa Babesia rodhaini has been investigated. Specific activities of five of the six enzymes of the pathway were determined: aspartate transcarbamylase (ATCase: E.C. 2.1.3.2); dihydroorotase (DHOase: E.C. 3.5.2.3); dihydroorotate dehydrogenase (DHO-DHase: E.C. 1.3.3.1); orotate phosphoribosyltransferase (OPRTase: E.C. 2.4.2.10); and orotidine-5'-phosphate decarboxylase (ODCase: E.C. 4.1.1.23). Michaelis constants for ATCase, DHO-DHase, OPRTase, and ODCase were determined in whole homogenates. Several substrate analogs were also investigated as inhibitors and inhibitor constants determined. N-(phosphonacetyl)-L-aspartate was shown to be an inhibitor of the ATCase with an apparent Ki of 7 microM. Dihydro-5-azaorotate inhibited the DHO-DHase (Ki, 16 microM) and 5-azaorotate (Ki, 21 microM) was an inhibitor of the OPRTase. The UMP analog, 6-aza-UMP (Ki, 0.3 microM) was a potent inhibitor of ODCase, while lower levels of inhibition were found with the product, UMP (Ki, 120 microM) and the purine nucleotide, XMP (Ki, 95 microM). Additionally, menoctone, a ubiquinone analog, was shown to inhibit DHO-DHase.     

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 degrees 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 K(i)* = 4.5 x 10(-7) mM. The respective inhibition constant of TCoBQ was equal to: K(i)* = 2.4 x 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.