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Significance of sulfhydryl groups in the activity of urease from

pigeonpea (

Cajanus cajan

L.) seeds

Punit K. Srivastava, Arvind M. Kayastha *

School of Biotechnology,Faculty of Science,Banaras Hindu Uni6ersity,Varanasi221 005,India

Received 18 February 2000; received in revised form 12 July 2000; accepted 13 July 2000

Abstract

Titration of urease from pigeonpea (Cajanus cajan L.), a hexameric protein (mol. wt. 480 000; subunit mol. wt. 80 000), with 5,5%_{-dithiobis-(2-nitrobenzoate) (DTNB) reveals the presence of 5.82}90.13 ‘accessible’ sulfhydryl groups per molecule of the enzyme protein (i.e. about one ‘accessible’ SH group per subunit). Denatured enzyme was found to titrate for 12.190.1 SH groups per molecule (i.e. about two SH groups per subunit). Half of the ‘accessible’ groups react faster than the remaining at pH 8.5 as well as pH 7.5. However, the reaction was slower at pH 7.5 than 8.5. Time-dependent loss of enzyme activity with DTNB was also found to be biphasic. The enzyme was inactivated at low concentration ofp-chloromercuribenzoate (p-CMB),N-ethyl maleimide (NEM) and iodoacetamide. The inactivation reactions were biphasic, with half of the activity lost more rapidly than the remaining half. The loss of activity withp-CMB was linearly related to the blocking of accessible SH groups. Inactivation by

p-CMB is largely reversible by addition of excess of cysteine. Fluoride ion strongly protects the enzyme against NEM inactivation, however, substrate urea provides much weaker protection against SH group reagents. The significance of these results is discussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Urease; Pigeonpea; Active site groups; Thiol inactivation; Fluoride protection; Half-site reactivity

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1. Introduction

Urease (urea amidohydrolase; 3.5.1.5), catalyzes the hydrolysis of urea to form ammonia and car-bon dioxide. High concentrations of ammonia arising from these reactions, as well as the accom-panying pH elevation, have important implica-tions in medicine and agriculture. Urease serves as a virulence factor in human and animal infections of the urinary and gastrointestinal tracts (reviewed in [1]), while high urease activity during soil nitro-gen fertilization with urea causes loss of ammonia by volatilization, inducing plant damage by am-monia toxicity and soil pH increase [2]. The en-zyme also plays a critical role in the nitrogen

metabolism of many microorganism and plants [1,3]. Even before the medical and agricultural importance of urease was appreciated, it has been shown to be a historical enzyme. In 1926, Sumner crystallized the urease from jack bean seeds [4]. Nearly 50 years later, urease from jack bean was shown to possess nickel [5]. Although most recent bacterial urease related studies have focused on the structure, analysis of genes and enzyme, how-ever, interest in plant enzyme has continued [1].

The most extensively studied plant urease is the homohexameric protein from jack bean, which contains two-nickel ions per subunit [6]. However, bacterial urease from Klebsiella aerogenes is the best-characterized [1]. Recently 3-D structure of

K. aerogenes urease was determined at 2.2 A, re-solution [7]. Both enzymes possess a cysteine residue (Cys592 _{and Cys}319 _{in jack bean and} _K_.

aerogenes, respectively); however, the bacterial and * Corresponding author. Tel.: +91-542-368331; fax: +

91-542-368693/368174.

E-mail address:[email protected] (A.M. Kayastha).

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plant enzyme thiols differ in their chemical proper-ties [8,9].

The urease active site is conserved among all sources as shown by extensive identity in the protein sequences, a common requirement for nickel, and similarity in behavior towards in-hibitors and inactivating reagents. For example, the presence of a thiol in jack bean urease is supported by studies showing that alkylating and disulfide reagents inactivate the enzyme [8,10]. Similarly, disulfides inactivateK.aerogenes urease, apparently by disulfide exchange with the active site cysteine [9,11]. It has been suggested that the slow loss of jack bean urease activity in the pres-ence of b-mercaptoethanol and oxygen was due to the formation of mixed disulfide involving a thiol located at the active site [12]. Titration of native jack bean urease has been carried out with NEM [10,13] and 5,5%-dithiobis-(2-nitrobenzoate) (DTNB) [10] and a pKa value of 9.15 at 25°C has

been assigned to the unique cysteine [8]. The amino acid sequence has been reported for a 34-residue cyanogen bromide peptide, which contains this residue [14]. In contrast, urease from Staphy

-lococcus xylosus has a threonine, instead of cys-teine, at the active site; it is not inhibited by the SH group inhibitor and its DNA sequence encodes no cysteine residue at this position [15]. Very recent structural studies on urease from Bacillus pasteuriihave shown that Cys322_{plays a significant}

role in the catalytic process, even though it is not essential [16,17]. Such detailed structural informa-tion on both the binuclear Ni centre in the enzyme active site and the mode of thiol inhibition is vital for structubased rational design of urease re-lated drugs [16]. Alkylating reagents specific for thiol groups, like iodoacetate (IA), iodoacetamide (IAM), NEM and p-CMB, have been shown to inhibit several microbial ureases thus, the micro-bial ureases possess a thiol group [1].

Urease from dehusked pigeonpea (Cajanus cajan

L.) seeds has been purified to apparent homogene-ity, partially characterized [18,19], and shown to be an important enzyme for analytical purposes [20 – 23]. In continuation of our work on urease characterization from pigeonpea, the present pa-per describes the significance of sulfhydryl groups in the activity of urease. Relative reactivities of the functional groups (-SH) and site – site heterogene-ity within the urease hexamer molecule have also been described.

2. Materials and methods

2.1. Enzyme

Urease was purified from dehusked seeds of pigeonpea (Cajanus cajan L.) seeds as described earlier [18]. The isolated enzyme was more than 95% pure as judged by native and SDS-PAGE. The specific activity of the purified enzyme varied from batch to batch in the range 4500 – 5500 units/

mg protein. 2.2. Chemicals

DTNB, p-CMB, NEM, IAM, Bovine Serum Albumin (BSA) and triethanolamine buffer were obtained from Sigma Chemical Co. (St. Louis, MO, USA.). Tris Buffer, urea (enzyme grade), trichloroacetic acid (TCA) and sodium fluoride were from Sisco Research Labs. (Mumbai, India). Nessler’s reagent was procured from HiMedia Labs. (Mumbai, India). All other chemicals were of analytical grade. All solutions were prepared in triple distilled water from an all-quartz distillation assembly.

2.3. Enzyme and protein determination

For routine assay of urease activity, ammonia liberated in a fixed time interval at an enzyme-sat-urating concentration of urea was determined us-ing Nessler’s reagent as described earlier [19]. The yellow color produced was measured spectropho-tometrically at 405 nm. The amount of ammonia liberated in the test solution was calculated by calibrating the reagent with standard ammonium chloride solution. An enzyme unit has been defined as the amount of enzyme required to liberate 1 mmole of product ammonia per minute under our test conditions (0.1 M Urea, 0.05 M Tris-acetate buffer, pH 7.3, 37°C).

Protein was assayed by the method of Lowry et al. [24] with BSA as standard.

2.4. SH group assay

Enzyme SH groups were assayed using DTNB as the reagent [25]. The assay solution (1.05 ml) contained 50 mM TEA buffer, pH 8.5, DTNB (0.2 mM) and enzyme (0.75 mg/ml). The enzyme aliquot (0.2 ml) was added last and the reaction

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monitored at 412 nm (_412nm 1.39×104 _and

1.31×104 _M−1 _cm−1 _{at pH 8.5 and 7.5,}

respectively).

Simultaneously, in a similar set of experiment pigeonpea urease was incubated with DTNB (0.2 mM) at 37°C. Aliquots withdrawn at different time intervals were assayed for the residual activity of the enzyme.

2.5. Inacti6_{ation of urease with SH group}

modifying reagents

The enzyme was incubated with the desired concentration of specified reagent (p-CMB, NEM or IAM) in 50 mM Tris-acetate buffer, pH 7.3 at 37°C in the absence or presence of substrate urea. Aliquots withdrawn at different time intervals were transferred immediately to activity assay so-lution (2.0 ml, containing 0.9 ml of 50 mM Tris-acetate buffer, pH 7.3 and 1.0 ml of 0.2 M urea). In a separate set of experiments, the residual SH groups were assayed in the aliquots as described earlier.

For reactivation studies, the enzyme (0.75 mg/

ml) was incubated with p-CMB 100 mM for 11 min in Tris-acetate buffer, pH 7.3, at 37°C fol-lowed by addition of excess cysteine (1 mM) and the recovery of activity was monitored at different time intervals.

For experiments measuring fluoride ion protec-tion against NEM inactivaprotec-tion, the enzyme was incubated with NEM (250 mM) in the presence of sodium fluoride (500 and 750mM) in assay buffer at 37°C. Aliquots withdrawn at different time intervals were checked for residual activity as de-scribed above.

2.6. Analysis of kinetic data

The time course of absorbance change at 412 nm and inactivation of the enzyme activity were analyzed according to Eq. (1) and Eq. (2), respec-tively (given below). Initial estimates of the rate constants and amplitudes were obtained from semi-log plots as described earlier [26]. Their val-ues were refined by iterative curve fitting proce-dure [27].

(DA−DAt)=Afast·e

−k

fast·t+A_slow·e−kslow·t, (1)

where DAt is the corrected absorbance increase at

time ‘t’ and DA the corrected absorbance

in-crease when all the accessible SH groups have reacted with excess DTNB, kfast and kslow are the

pseudo-first order rate constants and Afast and

Aslow are amplitudes (expressed as absorbance

in-crease so that Afast+Aslow=DA) of the fast and

slow phases, respectively.

A_t₌A_fast·e−k

fast·t+A_slow·e−kslow·t, (2)

whereAtis the percent residual activity at time ‘t’,

Afast and Aslow are the amplitudes (expressed as

percent of initial activity) andkfastandksloware the

pseudo-first order rate constants of the fast and the slow phases of reaction, respectively.

3. Results

3.1. Assay of SH groups of urease

Free SH groups of freshly isolated urease were assayed by monitoring its reaction with excess DTNB, spectrophotometrically at 412 nm. The results are shown in Fig. 1. Note the presence of SH groups of different reactivities. There is almost a ‘burst’ of absorbance increase (initial 60 s), followed by a slower reaction (completed in 6 min) and a very slow absorbance increase which is linear with time and is not completed even after 15 min. If a correction is applied for the last category of (very slow reacting phase) groups by extrapola-tion to zero time (Fig. 1), the absorbance increase in the first two phases corresponds to the reaction of 5.8290.13, i.e. $6 ‘accessible’ SH groups per hexamer enzyme protein molecule (mol. wt. 480 kDa), i.e. one SH group per monomeric subunit. When the enzyme was denatured by SDS-heat treatment before adding DTNB, there was an instantaneous increase in absorbance with no fur-ther time-dependent change (data not shown). The absorbance increase corresponds to 12.190.1, i.e.

$12 SH groups per molecule, i.e. about two SH groups per monomeric subunit.

3.2. Kinetics of reaction of DTNB with the

‘accessible’ SH groups

A semi-log plot of the reaction of accessible SH groups of urease with DTNB shows biphasic ki-netics (Fig. 2A). For this plot, the difference be-tween the extrapolated value of the slow reaction and the experimentally observed absorbance

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in-crease at the corresponding time was taken as indicative of the residual ‘accessible’ SH groups. The semi-log plot clearly shows that some of the reactive SH groups react faster than the rest (fast and slow phases). The time course of the reaction of the ‘accessible’ SH groups of urease with excess DTNB can be represented by a rate equation consisting of two first-order terms, corresponding to the fast and slow phases of the reaction (Eq. (1), see Materials and methods). Similar biphasic kinetics of reaction of urease and DTNB was observed at pH 7.5 (data not shown), however the reaction at this pH was slower. The values of the amplitudes and rate constants at different pH are shown in Table 1.

When loss of enzyme activity was monitored with DTNB in a time-dependent manner, again biphasic kinetics was observed (Fig. 2B). The time-course of inactivation of pigeonpea urease with DTNB is consistent with (Eq. (2)) (see Materials and methods). The values of the amplitudes and rate constants are shown in Table 1.

In these experiments DTNB was always present in large excess, so that the observed kinetic bipha-sicity cannot be attributed to limited DTNB con-centration. It must, therefore, represent different reactivities of the various accessible SH groups. It is noteworthy that the amplitudes of the fast and

the slow phases are nearly equal; each phase ac-counts for about half of the total absorbance change/activity.

3.3. Effect of SH group modifying reagents on urease acti6_ity

Pigeonpea urease was inactivated on incubation with low concentrations of SH reagents, like p -CMB (100mM), NEM (250mM) or IAM (5 mM). In each case, the reagent concentration was in large excess as compared to that of the enzyme (0.75 mg/ml corresponding to 1.56 mM). A semi-log plot of the data obtained with p-CMB shows biphasic kinetics of the reaction (Fig. 3). The complete time-course of inactivation of pigeonpea urease with excess p-CMB is consistent with (Eq. (2)), similar to that given above for the reaction with DTNB. In the data of Fig. 3,

Afast$Aslow$50% of the initial activity.

A similar pattern of inactivation is observed with NEM and IAM (data not shown). Values of the amplitudes and the rate constants of the two phases are given in Table 1. In each case,

Afast$Aslow$half of the initial activity.

Inactiva-tion of urease with each reagent is consistent with the following scheme.

Fig. 1. Kinetics of reaction of pigeonpea urease with DTNB. The reaction of the enzyme (0.75 mg/ml) and DTNB (0.2 mM), was carried out in 50 mM TEA buffer (pH 8.5) at 37°C and monitored at 412 nm.

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Fig. 2. (A) Semi-log plot of the data of Fig. 1 for first 8 min, after correcting the very slow reaction (see text). (B) Semi-log plot for the inactivation kinetics of pigeonpea urease acivity with DTNB (0.2 mM) at 37°C, pH 8.5.

Active Enzyme

Excess reagent Fast

Half-Active Enzyme

Excess reagent Slow

Inactive Enzyme (3) This is similar to the reaction of accessible SH groups of pigeonpea urease with excess DTNB. Thus, the reactivity of SH groups with a variety of reagents is suggestive of half-site reactivity.

In a separate set of experiments, the enzyme was incubated with p-CMB (100 mM) for 11 min and then treated with a large excess of freshly prepared cysteine (1 mM). Note that the inactivation by

p-CMB is largely reversed on the treatment with cysteine (Fig. 4).

The relationship between the accessible SH groups and catalytic activity of urease has been investigated in a separate experiment. Pigeonpea

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Table 1

Amplitudes and the rate constants for the inactivation of pigeonpea urease with various SH reagents

Fast phase Slow phase

Reagent

Afast(%) kfast(min−1) Aslow(%) kslow(min−1) 50.990.5 0.4390.01 49.190.5

DTNBa _{(pH 7.5, 0.2 mM)}a _0.07₉_0.01

51.990.5 2.8090.1 48.190.5

(pH 8.5, 0.2 mM)a _0.12₉_0.01

(pH 8.5, 0.2 mM)b

DTNBb _51.3₉_0.5 _2.82₉_0.1 _48.7₉_0.5 _0.126₉_0.001

p-CMBb _{(pH 7.3, 0.100 mM)} _51.0₉_0.5 _0.92₉_0.04 _49.0₉_0.5 _0.045₉_0.003 51.890.5 1.1590.1 48.290.5

(pH 7.3, 0.125 mM) 0.06990.005

50.890.5 0.4790.01 49.290.5 0.04690.003 NEMb _{(pH 7.3, 0.250 mM)}

51.790.5 0.5390.03 48.390.5

(pH 7.3, 0.330 mM) 0.06390.004

51.090.5 0.4390.01 49.090.5 0.02290.004 IAMb _{(pH 7.3, 5 mM)}

51.590.5 0.5490.04

(pH 7.3, 8 mM) 48.590.5 0.04790.003

a_{Kinetics of reaction of SH groups based on absorbance change at 412 nm.} b_{Kinetics of inactivation of the enzyme based on activity measurements.}

urease was incubated with excess SH group reagents (p-CMB, NEM and IAM) and aliquots withdrawn at different time intervals were assayed for residual activity as well as for the residual accessible SH groups (titration with DTNB as described earlier). A plot of % residual activity against the number of accessible SH groups blocked is shown forp-CMB in Fig. 5. A linear relationship is observed between the two parameters and all the six ‘accessible’ SH groups need to be blocked for complete inactivation of the urease.

Urea, at 0.2 M concentration, brought about a weak protection against inactivation by either NEM orp-CMB. Finally, when urease was incubated with excess NEM in the presence of different concentra-tions of fluoride ion, the enzyme was protected against time-dependent inactivation (Fig. 6).

4. Discussion

Titration of pigeonpea urease (native and SDS-heat denatured) with excess DTNB has revealed the presence of one accessible and one inaccessible SH groups per monomer of the homohexameric enzyme molecule. The later group is titratable only on SDS-heat denaturation. Jack bean urease contains a total of 17 (cysteine+1

2 cystine) residues per

96,000 Da subunit on the basis of amino acid analysis of the performic acid oxidized protein. However, only 15 cysteine residues per subunit are

titrated by DTNB under denaturing conditions [12,28].K.aerogenesurease, which is inactivated by the formation of a mixed disulfide, can be fully restored by treatment with dithiothreitol [12].

Fig. 3. Kinetics of inactivation of pigeonpea urease with

p-CMB. The enzyme was incubated with 0.1 mM and 0.125 mMp-CMB in 50 mM Tris-acetate buffer pH 7.3 at 37°C and aliquots withdrawn at different time intervals were assayed for the residual activity as described in Materials and meth-ods.

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Fig. 4. Enzyme and 0.1mM p-CMB were incubated for 11 min in 50 mM Tris-acetate buffer pH 7.3 at 37°C, after which excess cysteine (1 mM) was added and the time-dependent reversal of inactivation monitored.

A variety of evidence indicated that urease from jack bean contains a unique cysteine residue per subunit (Cys592_{) and that urease is modified}

cova-lently [12]. Labeling studies of jack bean urease indicated that four cysteines/subunit react rapidly with a disulphide reagent, 2,2%-dithiodipyridine, with no loss of activity whereas modification of a fifth cysteine was correlated to activity loss [8]. More recently, jack bean urease was modified using N-iodoacetyl-N%

(5-sulfo-1-naphthyl)ethyl-enediamine and N-(7-dimethyl amino-4-methyl-3-coumariyl)-maleimide; three thiols were modified with no loss of activity whereas alkylation of two additional thiols (Cys207_{and Cys}592_{) was}

concomi-tant with activity loss. Complete modification of Cys207 _{with the latter reagent resulted in an}

activ-ity loss of only 50%; thus the authors concluded that Cys592 _{(not Cys}207_{) is important for activity}

[14].

Kinetic analysis of the reaction of accessible SH groups in pigeonpea urease with excess DTNB shows that all six accessible groups of the hexam-eric urease molecule are not equally reactive with DTNB at pH 7.5 and 8.5. Three out of six SH group react faster with DTNB than the remaining

Fig. 6. Effect of fluoride ion on the rate of inactivation of pigeonpea urease by NEM. Enzyme was incubated with 0.25 mM NEM in 50 mM Tris-acetate buffer, pH 7.3 at 37°C and in the absence and presence of different concentrations of fluoride ion. Aliquots withdrawn at different time intervals were assayed for the residual activity.

Fig. 5. Relationship between loss of catalytic activity and blocking of ‘accessible’ SH groups of pigeonpea urease with 0.1 mM p-CMB. The aliquots withdrawn were assayed for the residual activity and residual accessible SH groups using DTNB at pH 8.5 (see text).

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as a result of pH change. Time-dependent activity loss of pigeonpea urease with DTNB was also found to be biphasic. This result is consistent with the absorbance change with DTNB at 412 nm for pigeonpea urease. These observations are similar to the site – site heterogeneity, more specifically to the half-site reactivity, observed earlier in thermal inac-tivation of pigeonpea urease [19].

Sulfhydryl specific reagents inactivate pigeonpea urease even at low concentrations. In each case, the concentration of the inactivating agent was kept much larger than the enzyme concentration. If all the SH groups reacting with these reagents had the same reactivity, the inactivation reaction should have exhibited pseudo first-order kinetics (i.e. single exponential decay). However, in each case, the time-course of inactivation was biphasic. Both the phases exhibited pseudo first-order kinetics (Eq. (1)). As already noted, similar behavior was ob-served with the reaction of accessible SH groups with DTNB. The rate constants of the two phases (kfastandkslow) depend on the nature and

concentra-tion of the reagent, but the relative amplitudes are not significantly affected.

Withp-CMB, it has been shown that inactivation of pigeonpea urease is directly proportional to the blocking of its accessible SH groups. A plot of % residual activity versus number of accessible SH groups blocked extrapolated to six accessible SH groups for complete inactivation. Thus, it appears that all the accessible SH groups are necessary for catalytic activity of urease. Presumably IAM, NEM andp-CMB bring about inactivation of this enzyme by a common mechanism, namely blocking of the accessible SH groups. Benini et al. [17] have shown that the X-ray structure ofb-ME-inhibitedBacillus pasteurii urease reveals that inhibition occurs by targeting enzyme sites that are both directly (metal centres) and indirectly (the cysteine side chain) participating in substrate positioning and activation. As the urease active site is conserved among all sources as shown by extensive identity in the protein sequences, it is likely that a similar mechanism of inhibition may be operative in case of pigeonpea urease, where a thiol group is in-volved.

In many cases inactivation of SH enzymes by

p-CMB is reversed on the addition of excess cys-teine. This is believed to be due to deblocking of SH group by mercaptide exchange reaction. Simi-lar results have been obtained with pigeonpea urease (Fig. 4). Since the recovery is not quantita-tive, the blocking of SH groups may be followed by a slow renaturation of the enzyme protein, as has been noted to occur with p-CMB [29].

When urease was incubated with excess NEM in the presence of different concentrations of fluoride ion, it provided a strong protection of the urease activity, in a time-dependent inactivation (Fig. 6). Fluoride ion competitively inhibits pigeonpea ure-ase-catalyzed hydrolysis of urea by binding to the nickel ion at the active site (unpublished results), and may thus protect against inactivation by NEM. The relatively small size of the fluoride ion suggests that it does not sterically inactivate urease [30]. It may decrease the positive charge of nickel ion by coordination and consequently, the reactiv-ity of the cysteine in the vicinreactiv-ity of nickel ion may be decreased. Similar findings have also been re-ported by Takishima and Mamiya [30] for jack bean urease. Dixon et al. [31] have determined dissociation constant of fluoride ion binding to nickel in jack bean urease to be 1.0 mM at pH 7.0 and 38°C. Recently, Todd and Hausinger [32] have suggested that the microbial enzyme has many features similar to the published jack bean urease inhibition results. Prakash and Bhusan [33] have recently shown a time-dependent inactivation of water melon (Citrullus6ulgaris) urease with IA, NEM and p-hydroxy mercuribenzoate in a bipha-sic manner. Mahadevan and Coworkers [34] have reported that the bovine rumen urease was com-pletely inhibited by NEM at 0.1 mM indicating that the thiols groups on the enzyme are required for the activity.

The chemical reactivity of accessible SH groups with DTNB and inactivation of the enzyme with IAM, NEM and p-CMB all suggest that the six accessible SH groups of pigeonpea urease can be divided into two categories of three groups each. The SH groups of one category react faster than those of the other. Varying the nature or the concentration of the reagent and the pH of the reaction solution brings about large changes in the values of rate constants for the reactions of the groups of these categories. However, equal distri-bution of groups in the two categories is not

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affected by changing the reactant or reaction con-ditions. This characteristic distribution is sugges-tive of ‘half-site reactivity’. It is noteworthy that the pigeonpea urease exhibits half-site reactivity of its SH groups as well as its thermal inactivation.

Acknowledgements

Financial assistance of the Council of Scientific and Industrial Research, New Delhi (Junior Re-search Fellowship to P.K.S.) is gratefully acknowl-edged. We are thankful to anonymous referees for helpful suggestions to improve our manuscript.

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-janus cajan L.) urease immobilized on glutaraldehyde activated chitosan beads and its analytical applications, Appl. Biochem. Biotechnol. (2000) in press.

[24] O.H. Lowry, J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265 – 280.

[25] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82 (1959) 70 – 77.

[26] A.M. Kayastha, A.K. Gupta, An easy method to deter-mine the kinetic parameters of biphasic reactions, Biochem. Edu. 15 (1997) 135.

[27] O.P., Malhotra, Srinivasan, L.R., Singh, 1985. Reactiv-ity of active site groups and site heterogeneReactiv-ity in mung bean glyceraldehyde 3-phosphate dehydrogenase: effect of coenzyme and substrate, Indian J. Biochem. Biophys. 22 (1985) 281 – 285.

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[28] R. Blakeley, B. Zerner, Jack bean urease: The first nickel enzyme, J. Mol. Catal. 23 (1984) 263 – 292.

[29] O.P. Malhotra, A.M. Kayastha, Chemical inactivation and active site groups of phosphoenolpyruvate phos-phatase from germinating mung beans (Vigna radiata), Plant Sci. 65 (1989) 161 – 170.

[30] K. Takishima, G. Mamiya, Location of the essential cysteine residue of jack bean urease, Protein Seq. Data Anal. 1 (1987) 103 – 106.

[31] N.E. Dixon, P.W. Riddles, C. Gazzola, R.L. Blakely, B. Zerner, Urease (EC 3.5.1.5.) V. On the mechanism of action on urea, formamide, acetamide, N-methyl urea

and related compounds, Can. J. Biochem. 58 (1980) 1335 – 1344.

[32] M.J. Todd, R.P. Hausinger, Fluoride inhibition ofKleb

-siella aerogenes urease: Mechanistic implications of a pseudo-uncompetitive, slow-binding inhibitor, Biochem-istry 39 (2000) 5389 – 5396.

[33] Om Prakash, G. Bhushan, A study of inhibition of urease from seeds of water melon (Citrullus6_ulgaris_{), J.}

Enz. Inhib. 13 (1998) 69 – 77.

[34] S. Mahadeven, F.D. Sauer, J.D. Erflee, Purification and properties of urease from bovine rumen, Biochem. J. 163 (1977) 495 – 501.

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Active Enzyme

Excess reagent

Fast

Half-Active Enzyme

Excess reagent

Slow

Inactive Enzyme (3)

This is similar to the reaction of accessible SH groups of pigeonpea urease with excess DTNB. Thus, the reactivity of SH groups with a variety of reagents is suggestive of half-site reactivity.

In a separate set of experiments, the enzyme was incubated with p-CMB (100 mM) for 11 min and then treated with a large excess of freshly prepared cysteine (1 mM). Note that the inactivation by

p-CMB is largely reversed on the treatment with cysteine (Fig. 4).

The relationship between the accessible SH groups and catalytic activity of urease has been investigated in a separate experiment. Pigeonpea

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Table 1

Amplitudes and the rate constants for the inactivation of pigeonpea urease with various SH reagents

Fast phase Slow phase

Reagent

Afast(%) kfast(min−1) Aslow(%) kslow(min−1)

50.990.5 0.4390.01 49.190.5

DTNBa _{(pH 7.5, 0.2 mM)}a _0.07₉_0.01

51.990.5 2.8090.1 48.190.5

(pH 8.5, 0.2 mM)a _0.12₉_0.01

(pH 8.5, 0.2 mM)b

DTNBb _51.3₉_0.5 _2.82₉_0.1 _48.7₉_0.5 _0.126₉_0.001

p-CMBb _{(pH 7.3, 0.100 mM)} _51.0₉_0.5 _0.92₉_0.04 _49.0₉_0.5 _0.045₉_0.003

51.890.5 1.1590.1 48.290.5

(pH 7.3, 0.125 mM) 0.06990.005

50.890.5 0.4790.01 49.290.5 0.04690.003

NEMb _{(pH 7.3, 0.250 mM)}

51.790.5 0.5390.03 48.390.5

(pH 7.3, 0.330 mM) 0.06390.004

51.090.5 0.4390.01 49.090.5 0.02290.004

IAMb _{(pH 7.3, 5 mM)}

51.590.5 0.5490.04

(pH 7.3, 8 mM) 48.590.5 0.04790.003

a_{Kinetics of reaction of SH groups based on absorbance change at 412 nm.} b_{Kinetics of inactivation of the enzyme based on activity measurements.}

4. Discussion

2 cystine) residues per 96,000 Da subunit on the basis of amino acid analysis of the performic acid oxidized protein. However, only 15 cysteine residues per subunit are

titrated by DTNB under denaturing conditions [12,28].K.aerogenesurease, which is inactivated by the formation of a mixed disulfide, can be fully restored by treatment with dithiothreitol [12].

Fig. 3. Kinetics of inactivation of pigeonpea urease with p-CMB. The enzyme was incubated with 0.1 mM and 0.125 mMp-CMB in 50 mM Tris-acetate buffer pH 7.3 at 37°C and aliquots withdrawn at different time intervals were assayed for the residual activity as described in Materials and meth-ods.

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A variety of evidence indicated that urease from jack bean contains a unique cysteine residue per subunit (Cys592_{) and that urease is modified} cova-lently [12]. Labeling studies of jack bean urease indicated that four cysteines/subunit react rapidly with a disulphide reagent, 2,2%-dithiodipyridine, with no loss of activity whereas modification of a fifth cysteine was correlated to activity loss [8]. More recently, jack bean urease was modified using N-iodoacetyl-N% (5-sulfo-1-naphthyl)ethyl-enediamine and N-(7-dimethyl amino-4-methyl-3-coumariyl)-maleimide; three thiols were modified with no loss of activity whereas alkylation of two additional thiols (Cys207_{and Cys}592_{) was} concomi-tant with activity loss. Complete modification of Cys207 _{with the latter reagent resulted in an} activ-ity loss of only 50%; thus the authors concluded that Cys592 _{(not Cys}207_{) is important for activity} [14].

(4)

three. This distribution of the ‘accessible’ SH groups (into two sets of three SH groups each) remains the same at pH 7.5 and 8.5, although the magnitudes of the pseudo first-order rate constants for the two sets of groups (kfastandkslow) undergo large changes as a result of pH change. Time-dependent activity loss of pigeonpea urease with DTNB was also found to be biphasic. This result is consistent with the absorbance change with DTNB at 412 nm for pigeonpea urease. These observations are similar to the site – site heterogeneity, more specifically to the half-site reactivity, observed earlier in thermal inac-tivation of pigeonpea urease [19].

In many cases inactivation of SH enzymes by

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Acknowledgements

References

[1] H.L.T. Mobley, M.D. Island, R.P. Hausinger, Molecu-lar biology of microbial urease, Microbiol. Rev. 59 (1995) 451 – 480.

[2] M.J. Krogmeir, G.W. McCarty, J.M. Bremner, Phyto-toxicity of foliar-applied urea, Proc. Natl. Acad. Sci. USA 86 (1989) 8189 – 8191.

enzyme urease, J. Biol. Chem. 69 (1926) 435 – 451. [5] N.E. Dixon, C. Gazzola, R.L. Blakeley, B. Zerner, Jack

bean urease (EC 3.5.1.5). A metalloenzyme. A simple biological role for nickel?, J. Am. Chem. Soc. 97 (1975) 4131 – 4133.

[6] R.K. Andrews, R.L. Blakeley, B. Zerner, Urease a Ni (II) metalloenzyme, in: J.R. Lancaster Jr. (Ed.), Bioinor-ganic Chemistry of Nickel, VCH Publishers, New York, 1988, pp. 141 – 166.

[7] E. Jabri, M.B. Carr, R.P. Hausinger, P.A. Karplus, The crystal structure of Klebsiella aerogenes, Science 268 (1995) 998 – 1004.

[8] R. Norris, K. Brocklehurst, A convenient method of preparation of high activity urease fromCanca6_{alia ensi} -formisby covalent chromatography and an investigation of its thiol group by 2,2%-dithiopyridyl disulphide as a thiol titrant and reactivity probe, Biochem. J. 159 (1976) 245 – 257.

[9] M.J. Todd, R.P. Hausinger, Identification of the essen-tial cysteine residue in Klebsiella aerogenes urease, J. Biol. Chem. 266 (1991) 24327 – 24331.

[10] A.T. Andrews, F.J. Reithel, The thiol groups of jack bean urease, Arch. Biochem. Biophys. 141 (1970) 538 – 546.

[11] M.J. Todd, R.P. Hausinger, Reactivity of essential thiol ofKlebsiella aerogenesurease, J. Biol. Chem. 266 (1991) 10260 – 10267.

[12] P.W. Riddles, R.K. Andrews, R.P. Blakeley, B. Zerner, Determination of thiol and disulphide content, Biochim. Biophys. Acta 743 (1983) 115 – 120.

[13] G. Gorrin, C.C. Chin, Urease IV: Its reaction with N-ethylthaleimide and with silver ion, Biochim. Biophys. Acta 99 (1965) 418 – 426.

[15] J. Jose, U.K. Schaffer, H. Kaltwasser, Threonine is present instead of cysteine at active site of urease from Staphylococcus xylosus, Arch. Microbiol. 161 (1994) 384 – 392.

[17] S. Benini, W.R. Rypniewski, K.S. Wilson, S. Ciurli, S. Mangani, The complex ofBacillus pasteuriiurease with

b-mercaptoethanol from X-ray data at 1.65 A, resolu-tion, J. Biol. Inorg. Chem. 3 (1998) 268 – 273.

[19] A.M. Kayastha, N. Das, Kinetics of thermal inactiva-tion and molecular asymmetry of urease from dehusked pigeonpea (Cajanus cajan L.) seeds, J. Plant Biochem. Biotechnol. 7 (1998) 121 – 124.

[20] N. Das, P. Prabhakar, A.M. Kayastha, R.C. Srivastava, Enzyme entrapped inside the reverse micelle in the fabri-cation of new urea biosensor, Biotechnol. Bioeng. 54 (1997) 329 – 332.

[22] N. Das, A.M. Kayastha, Immobilization of urease from pigeonpea (Cajanus cajan L.) in flannel cloth using polyethyleneimine, World J. Microbiol. Biotechnol. 14 (1998) 927 – 929.

[23] A.M. Kayastha, P.K. Srivastava, 2000. Pigeonpea (Ca -janus cajan L.) urease immobilized on glutaraldehyde activated chitosan beads and its analytical applications, Appl. Biochem. Biotechnol. (2000) in press.

[24] O.H. Lowry, J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265 – 280.

[25] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82 (1959) 70 – 77.

[26] A.M. Kayastha, A.K. Gupta, An easy method to deter-mine the kinetic parameters of biphasic reactions, Biochem. Edu. 15 (1997) 135.

(6)

[28] R. Blakeley, B. Zerner, Jack bean urease: The first nickel enzyme, J. Mol. Catal. 23 (1984) 263 – 292.

[29] O.P. Malhotra, A.M. Kayastha, Chemical inactivation and active site groups of phosphoenolpyruvate phos-phatase from germinating mung beans (Vigna radiata), Plant Sci. 65 (1989) 161 – 170.

[30] K. Takishima, G. Mamiya, Location of the essential cysteine residue of jack bean urease, Protein Seq. Data Anal. 1 (1987) 103 – 106.

[31] N.E. Dixon, P.W. Riddles, C. Gazzola, R.L. Blakely, B. Zerner, Urease (EC 3.5.1.5.) V. On the mechanism of action on urea, formamide, acetamide, N-methyl urea

and related compounds, Can. J. Biochem. 58 (1980) 1335 – 1344.

[32] M.J. Todd, R.P. Hausinger, Fluoride inhibition ofKleb -siella aerogenes urease: Mechanistic implications of a pseudo-uncompetitive, slow-binding inhibitor, Biochem-istry 39 (2000) 5389 – 5396.

[33] Om Prakash, G. Bhushan, A study of inhibition of urease from seeds of water melon (Citrullus6_ulgaris_{), J.}

Enz. Inhib. 13 (1998) 69 – 77.

[34] S. Mahadeven, F.D. Sauer, J.D. Erflee, Purification and properties of urease from bovine rumen, Biochem. J. 163 (1977) 495 – 501.

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Significance of sulfhydryl groups in the activity of urease from

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L.) seeds

Punit K. Srivastava, Arvind M. Kayastha *

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