Jack Bean Urease VI. Determination of thiol and disulfide content

Biochimica et Biophysica Acta, 743 (1983) 115-120

115

Elsevier Biomedical Press BBA31520

JACK BEAN UREASE VI. D E T E R M I N A T I O N OF T H I O L AND DISULFIDE C O N T E N T REVERSIBLE INACTIVATION OF T H E ENZYME BY T H E BLOCKING O F T H E U N I Q U E CYSTEINE RESIDUE * PETER W. RIDDLES a, ROBERT K. ANDREWS b, ROBERT L. BLAKELEY b and BURT ZERNER b.**

aCSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private Bag No. 3, Indooroopilly, Queensland 4068, and bDepartment of Biochemistry, University of Queensland, St. Lucia, Queensland 4067 (Australia) (Received September 27th, 1982)

Key words: Cysteine residue; Urease; Thiol content; Active site," (Jack bean)

The total thiol content of highly pure jack bean urease (urea amidohydrolase, EC 3.5.1.5) has been determined by titration with 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of 6 M guanidinium chloride. Urease contains 15 thioi groups per 96.6-kDa subunit. Coupled with amino acid analysis data, this result establishes that urease contains one cystine disulfide bond per 96.6-kDa subunit. Slow loss of enzymatic activity in the presence of fl-mercaptoethanol and oxygen is due to the formation of a mixed disulfide which involves the unique active-site cysteine residue. Enzymatic activity can be fully restored by treatment of inactive urease with 0.1 M dithiothreitol at pH 7.3. Urease is quite stable when stored in 0.05 M sulfite/0.02 M phosphate buffer (pH 7.2, 1 mM in EDTA).

Introduction The presence of thiol groups in an enzyme appears first to have been described by Sumner and Poland, who titrated crystalline urease from jack beans with nitroprusside in 1933 [1]. Although native urease contains many thiol groups [1-5], absolute quantification has become possible only recently with the secure evaluation of the subunit molecular weight ( M r = 96600) and of other properties of the enzyme [6,7]. In addition,

* Part V is: Dixon, N.E., Riddles, P.W., Gazzola, C., BlakeIcy, R.L. and Zerner, B. (1980) Can. J. Biochem. 58, 1335-1344. ** To whom correspondence should be addressed. Abbreviations: DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); TNB 2-, 5-thio-2-nitrobenzoate dianion. 0167-4838/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

our reassessment of DTNB as a thiol reagent now allows a hitherto unattainable accuracy in titration of thiol groups [8,9]. We report herein the total thiol content of urease as determined by titration with DTNB in the presence of 6 M guanidinium chloride, and adduce evidence for the presence of a single cystine disulfide bond in urease. A variety of evidence indicates that urease contains one unique cysteine residue per subunit [10] and that urease is inactive when this residue is covalently modified. The system is complicated by the presence of five other cysteine residues per 96.6-kDa subunit which are more reactive than the unique residue, but whose covalent modification does not affect enzymatic activity. Titrations have been carried out with N-ethylmaleimide [2,3], 2,2'dipyridyl disulfide [4] and DTNB [3,11], and a pK~ of 9.15 at 25°C has been assigned to the

116

unique cysteine residue [4]. In this paper, we show that the slow inactivation of urease which occurs in the presence of fl-mercaptoethanol and oxygen [6] may be understood in terms of formation of a mixed disulfide between fl-mercaptoethanol and the unique cysteine residue. Materials and Methods

Materials. Buffers were prepared from Analytical Grade reagents, and pH was measured at 25°C with standardization according to Bates [12]. NEthylmorpholine was purified as previously described [13]. Oxygen-free buffers were prepared from deionized distilled water which had been boiled for 20 min and bubbled with nitrogen while cooling. DTNB was prepared and characterized as described elsewhere [8,9], and stock solutions were prepared in 0.0282 M KH2PO4/0.0718 M NaEHPO 4 buffer (pH 7.27, 1 mM in EDTA). Guanidinium chloride (Schwarz/Mann, Ultrapure) was dried in vacuo over P205, and stock solutions were prepared in the 0.1 M phosphate buffer at 25°C with the inclusion of approx. 14 mM NaOH to bring the measured pH to 7.27 _+ 0.05. Manipulations of buffer and reagents were carried out under a gentle stream of nitrogen. Hydroxyethyl disulfide and cystamine dihydrochloride were products of Aldrich Chemical Co. The purification of urease included gel chromatography and ion-exchange chromatography as previously described [7,13]. Enzymatic activity was determined using a pH-stat assay, and protein concentrations were determined spectrophotometrically [7,13]. For DTNB titrations, urease was dialyzed exhaustively at 4°C against oxygen-free water through which nitrogen was bubbling gently. The protein solution was centrifuged at 30 000 × g for 20 min under nitrogen and was then titrated. Thio! titrations. The thermostatted sample compartment of a Cary 17 spectrophotometer was used for DTNB titrations at 25.0°C [8,9]. Aliquots (3 ml) of buffered 6.40 M guanidinium chloride were placed in the sample and reference cuvets, and A412 was adjusted to zero. A 100-/tl aliquot of the 0.1 M phosphate buffer was added to the reference cuvet, and the solution was stirred with a flat-ended glass rod. After addition of a 100-Fl aliquot of DTNB solution to the sample solution,

again recorded ( A D T N B ) . A 100-/~1 aliquot of the final diffusate of the protein dialysis was added to the reference cuvet, and a 100-/~l aliquot of protein solution was added to the sample cuvet (light path 1 cm). Reactivation experiments. Prior to reactivation, urease which had been partially inactivated was dialyzed at 1-2 mg. ml- t at 4°C into oxygen-free 0.05 M N-ethylmorpholinium chloride buffer (pH 7.3, 1 mM in EDTA) and then centrifuged to remove traces of turbidity. After determination of the specific activity, reactivation was initiated by addition of dithiothreitol to a final concentration of 0.1 M. A412 w a s

Results and Discussion

Titration of urease with D T N B in 6 M guanidinium chloride The absorbance due to formation of TNB 2reached a stable value (Aei.al) within 3 min of initiation of the reaction (Fig. 1). The net increase in absorbance at 412 nm (AA412) was calculated from Eqn. 1: Z~A4, 2 = Afina ' -- ( 3 . 1 / 3 . 2 ) ( A D . r N a -

Ab.fr~)

(l)

Urease contributes negligibly to the absorbance at

i "

I!

Afi~

i :

i

-r-

\

-~-t130

--

~0.10

!

t!

J

ii

!°31\ ! 2

it

JL 4

i i-2-

Ti[il

!tFii

TINE {~N )

.....

i

i .

: I

:!

; I 2

I[

I i~

Fig. 1. Speetrophotometric titration of jack bean urease (0.108 m g . m l - ] ) with D T N B (0.217 m M ) in buffered 6 M guanidinium chloride. Absorbance at 412 n m as a function of time: Ab,ffcr, buffer vs. buffer, adjusted to zero absorbance; A OTNa, D T N B vs. buffer; A fi h a l , (protein + D T N B ) vs. buffer. The protein sample w a s a d d e d at zero time. Note the absorbance offset in the presence of protein.

117

412 nm under these conditions. The following reaction takes place between DTNB (ArSSAr) and a thiolate anion ( R S - ) : R S - + ArSSAr ~ RSSAr + A r S -

(2)

It has been established that under the conditions described above, ~'1 412 -- - E412 "riga2- [RSH]0

(3)

where E TNB2"= 13600 M -1 .cm -1 in 6 M guani412 dinium chloride, and [RSH]0 represents the concentration of thiol groups which have reacted with D T N B [8]. Fig. 2 shows that zL44~2 for titration of urease with DTNB in 6 M guanidinium chloride is directly proportional to the concentration of urease. This indicates that the equilibrium in Eqn. 2 lies completely to the right and it establishes the absence of complications related to disaggregation of subunits in this denaturing system. Table I shows that the total thiol titre of urease is reproducible among three different batches of enzyme of high specific activity. Further, the titre

0.~

~0.~

°a-

'

d,

[UREASE] (g.[-1)

'

Fig. 2. Effect of protein concentration on the titration of u r e a s e with 0.20 mM D T N B in buffered 6 M guanldinium chloride. AA412is defined by Eqn. I. Each point represents two to four titrations whose standard deviation from the mean A.4412 is less than +0.7%. See Table I for conditions.

TABLE I T I T R A T I O N O F U R E A S E THIOLS W I T H D T N B IN 6.0 M GUANIDINIUM CHLORIDE Titrations were carried out at 25°C in 0.1 M phosphate buffer (pH 7.27, 1 m M in EDTA, 6.0 M in guanidinium chloride). Each titre is the mean of two to six titrations whose standard deviation from the mean titre was less than +0.6%. The specific activity of urease was 88-93 (mkat/l)/A2s o. Urease batch

[Protein] (g. 1- l )

[DTNB]0 (mM)

[Thiol]/[protein] a (mmol. g - l )

1 1 2 3 3

0.108 0.108 0.109 0.179 0.176

0.217 0.108 0.218 0.200 0.200

0.157 0.158 0.149 0.155 0.154

a [Thiol] in units of mmol. 1 - =; [protein] in units of g. 1- I.

is independent of the initial concentration of DTNB, again establishing that the equilibrium in Eqn. 2 lies completely to the fight. Finally, urease contains 2.0 tightly bound Ni(II) ions per 96 600dalton subunit [6], and these ions are released in 6 M guanidinium chloride [13]. When 3.0 # M Ni(II) ion (in the form of nickelous ammonium sulfate) was added to the sample cuvet at the end of the first titration in Table I, there was absolutely no change in A4~2. Since the solution already contained 2.2 # M Ni(II) ions and 17 # M TNB 2- , this result establishes that the Ni(II) ion released from urease does not affect A412 of TNB 2- . (The absorption spectrum of 4-nitrothiophenolate ion is markedly altered when it binds to nickel ion in Ni(II)-carbonic anhydrase [ 141.) A mean value of 0.154(6) mmol thiol groups per gram protein (Table I) corresponds to 14.9(3) cysteine residues per 96.6-kDa subunit. Cysteic acid analysis of performic acid-oxidized urease shows that the protein contains a total of 17.05 (halfcystine + cysteine) residues per 96.6-kDa subunit [7]. There are accordingly 2.1(2) residues per subunit which are inaccessible to DTNB in the presence of 6 M guanidinium chloride. These experiments therefore establish that urease contains one cystine disulfide bond per subunit. Further, since urease has five reactive, non-essential cysteine residues plus one moderately reactive, unique cysteine residue per 96.6-kDa subunit [2-4,7,11],

118

there are nine thiol groups per subunit which are unreactive unless the enzyme is denatured. It should be noted that the urease used in these titrations had a specific activity very close to the maximum observed value [7,13]. The thiol titre is therefore maximal and is not diminished by the presence of a mixed disulfide between fl-mercaptoethanol and the unique cysteine residue (see below).

5.0

to

Inactivation and reactivation of urease

Concentrated solutions of highly pure urease (10-20 mg-m1-1) have routinely been stored in 0.02 M phosphate buffer (pH 7.14, 20 mM in fl-mercaptoethanol and 1 mM in EDTA) at 4°C [13]. The enzyme generally undergoes a slow decrease in specific activity upon storage, but there is not a corresponding loss of nickel ions [6,13]. We have found that the activity of some samples of relatively low activity urease may be substantially restored over a period of hours at 25°C by treatment with 0.1 M dithionite or 0.2 M sulfite at pH 5.2 or with 0.2 M fl-mercaptoethanol at pH 5.2 or 7.3. However, dithiothreitol is the best reagent found to date for reactivation. When urease which after storage retained only approx. 8% of its original specific activity was treated with 0.1 M dithiothreitol at pH 7.3, reactivation obeyed a first-order rate law (Fig. 3a). The specific activity after seven half-lives was comparable to that of the enzyme as originally prepared. The rate of reactivation was not changed by increasing the concentration of dithiothreitol from 0.1 to 0.2 M or 0.4 M. Since dithiothreitol binds to Ni(II) in urease ( g d < 0.1 mM at pH 7.43 [15]), reactivation presumably takes place in the urease-dithiothreitol complex. A possible explanation for these observations is shown in Eqns. 4 and 5, in which E-SH denotes the unique cysteine residue in urease:

C

A

3.0

c.

1.0

0

I

I 100 TIME

I

I 200

(min)

Fig. 3. First-order plots for reactivation of urease in the presence of 0.1 M dithiothreitol in 0.05 M N-ethylmorpholinium chloride buffer (pH 7.3, 1 mM in EDTA) at 25°C. The leastsquares line corresponds to the equation ln([ELo--[E])= - kobst +C, where [El is the concentration of active enzyme in mkat/1 at any time and [E]~ is evaluated at 7-12 half-lives. (a) Urease whose specific activity had 'spontaneously' decreased progressively from 64 (mkat/l)/A2s o to 5 (mkat/l)/A28 o in 0.02M phosphate buffer (pH 7.14, 20 mM in fl-mercaptoethanol, 1 mM in EDTA) over a period of 3 months at 4°C. kobs=(l.8+0.2)-10 -4 s -x. (b) Urease inactivated by 5 mM dihydroxyethyl disulfide from 64 (mkat/l)/AE8 o to 2 (mkat/l)/A28 o in the N-ethylmorpholinium buffer at 25°C. kobs = ( 1.6 _+0.1). 10- 4 s - I. (c) Urease progressively inactivated from 90 (mkat/l)/AE8 o to 14 (mkat/l)/AEs o over 50 h at 25°C by oxygen (from gentle bubbling of air) in 0.02 M phosphate buffer (pH 7.14, l mM each in CuSO 4 and EDTA, 29 mM in fl-mercaptoethanol), kob s = (1.65 + 0.03). 10 -4 s -1. The ordinate values for line (c) have been displaced upwards by In 2 for graphical clarity.

2 HOCH2CH2SH + -~O2 "-* HOCH2CH2SSCH2CH2OH + H 20

(4) E-SH + HOCH 2CH 2SSCH 2CH 2OH "-~ HOCH 2CH 2SH + E-SSCH 2CH2OH

The

enzymatically

(5)

inactive

product

(E-

SSCH2CH2OH ) contains a mixed disulfide involving the unique thiol group and fl-mercaptoethanol. Analogous mixed disulfides have been identified with other enzymes [ 16-19]. Reactivation of inactive urease in the presence of excess sulfite, flmercaptoethanol or dithiothreitol is readily ex-

119

plained by nucleophilic attack on sulfur with displacement of E-S-. As a partial test of this proposition, urease was treated with 5 mM 2-hydroxyethyl disulfide ((HOCH2CH2S)-2) in the absence of flmercaptoethanol. Loss of specific activity had a half-life of about 6 min at 25°C, and the resulting enzyme retained approx. 3% of its inital specific activity. After treatment with dithiothreitol, the 2-hydroxyethyl disulfide-inactivated urease regained its original specific activity, and the rate constant for reactivation (Fig. 3b) was the same as that for the enzyme which had deteriorated in the presence of fl-mercaptoethanol (Fig. 3a). This reasonably indicates that the inactive form of urease is the same in both cases, and it is also consistent with Eqns. 4 and 5 for the 'spontaneous' inactivation of urease in the presence of fl-mercaptoethanol. Since cupric ions catalyze the oxidation of thiols to disulfides [9,19,20], urease was equilibrated with oxygen and fl-mercaptoethanol in the presence of Cu(II) ion. When only 16% of the original specific activity remained, the inhibitory agents were removed by dialysis and the inactive enzyme was then treated with dithiothreitol. The enzyme regained 84% of its original specific activity, and the rate constant for reactivation (Fig. 3c) was the same as that for previous samples. This experiment establishes that the Cu(II)-promoted inactivation of urease in the presence of fl-mercaptoethanol and oxygen produces the same inactive species that is produced by 'spontaneous' inactivation upon storage and by treatment with 2-hydroxyethyl disulfide. These experiments do not preclude the direct oxidative formation of E-SSCH2CH2OH from E-SH and fl-mercaptoethanol. However, the high concentration of fl-mercaptoethanol in these experiments strongly suggests that 2-hydroxyethyl disulfide is an intermediate in both the 'spontaneous' and the Cu(II)/oxygen-promoted inactivation of urease in the presence of fl-mercaptoethanol. Stable sulfenic acids may be produced by mild oxidation of sterically hindered thiols [19,21-23], and some of the foregoing observations would be accounted for if the unique cysteine residue of urease were oxidized to a sulfenic acid (E-SOH) in

the reversibly inactivated urease. However, reaction of E-SOH with sulfite would be expected to produce an enzymatically inactive, stable Ssulfonate (E-SSO3--) [19,21], whereas it is found that sulfite restores activity. Moreover, the formation of E-SOH by reaction of E-SH with 2hydroxyethyl disulfide is exceedingly unlikely. ESOH must therefore be regarded as a poor candidate for the inactive form of E-SH observed in these experiments. Another possible structure of reversibly inactivated urease would contain one additional disulfide bond per subunit, formed by displacement of fl-mercaptoethanol from E-SSCH2CH2OH by a normally unreactive cysteine residue as in Eqn. 6 [24,251. S E-SSCH2CH2OH ~ Ex/I + HSCH2CH2OH I s SH

(6)

If inactivation of urease obeyed Eqn. 6, the rate constant for reactivation of inactive urease by dithiothreitol would be independent of the structure of the disulfide used to inactivate the enzyme. Urease was treated with 1.4 mM cystamine ((H2NCH2CH2S-)-2) in oxygen-free 0.05 M N-ethylmorpholinium chloride buffer (pH 7.3, 1 mM in EDTA) at 25°C. The half-life of inactivation under these conditions is fortuitously also about 6 min. After removal of excess cystamine by dialysis, the residual specific activity was 4.5% of the initital value. Reactivation in the presence of 0.1 M dithiothreitol was biphasic, with the first 80% of reactivation having a rate constant approx. 30times greater than that for reactivation of 2hydroxyethyl disulfide-inactivated urease. While the system is incompletely defined, this result is fully consistent with E-SSCHECH2N+H3 as the inactive form of the enzyme produced by cystamine treatment, and it provides no support for the formation of an intramolecular disulfide (Eqn. 6) in the reversible inactivation of urease by disulfides. In 1976, Staples and Reithel [5] provided qualitative electrophoretic evidence for the presence of disulfide bonds in urease, in that, after alkylation of urease by N-ethylmaleimide in 8 M guanidium chloride, protein thiol groups are released upon

120 a d d i t i o n of 10 m M f l - m e r c a p t o e t h a n o l (Ref. 5, b u t c o m p a r e Ref. 3). T h e results were i n t e r p r e t e d in terms of two different types of disulfide b o n d in urease: one t y p e was p u r p o r t e d to involve an accessible thiol g r o u p in an e n z y m a t i c a l l y inactive subunit; a n d it was suggested that the o t h e r c o u l d b e r e d u c e d only when the p r o t e i n was d e n a t u r e d . T h e i r urease h a d b e e n p r e p a r e d in the presence of f l - m e r c a p t o e t h a n o l , b u t its specific activity was n o t reported. All of their o b s e r v a t i o n s relating to an accessible disulfide b o n d in inactive subunits are simply e x p l a i n e d b y the existence of a flm e r c a p t o e t h a n o l - u r e a s e m i x e d disulfide at the active site of a s u b s t a n t i a l p r o p o r t i o n of their enz y m e molecules. T h e single cystine disulfide whose presence we have established q u a n t i t a t i v e l y in fully active urease c o r r e s p o n d s to the disulfide of Staples a n d Reithel which can be r e d u c e d o n l y when urease is d e n a t u r e d . Because of slow i n a c t i v a t i o n of urease in the presence of f l - m e r c a p t o e t h a n o l a n d oxygen, we have reinvestigated the use of sulfite at all stages of purification after the initial a c e t o n e - w a t e r extraction a n d crystallization of urease from j a c k b e a n meal in the presence of 1% ( v / v ) flm e r c a p t o e t h a n o l [26,27]. W e have f o u n d that urease is highly stable in the presence of 0.05 M s u l f i t e / 0 . 0 2 M p h o s p h a t e buffer, ( p H 7.2, 1 m M in E D T A ) , a n d this stabilization is being further investigated.

Acknowledgements W e w o u l d like to t h a n k Dr. T o m M a l e f y t for p u r i f i c a t i o n of urease in the presence of sulfite. This w o r k was s u p p o r t e d b y the A u s t r a l i a n Research G r a n t s Scheme a n d b y the U n i v e r s i t y of Queensland.

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