Biochem J. (1980) 191, 533-541Printed in Great Britain

Active-site modification of native and mutant forms of inosine 5'-monophosphate dehydrogenase from Escherichia coli K 12

Harry J. GILBERT* and William T. DRABBLEtDepartment ofBiochemistry, University ofSouthampton, Southampton S09 3TU, U.K.

(Received 8 April 1980/Accepted 25 July 1980)

IMP dehydrogenase of Escherichia coli was irreversibly inactivated by Cl-IMP(6-chloro-9-ff-D-ribofuranosylpurine 5'-phosphate, 6-chloropurine ribotide). The in-activation reaction showed saturation kinetics. 6-Chloropurine riboside did notinactivate the enzyme. Inactivation by Cl-IMP was retarded by ligands that bind at theIMP-binding site. Their effectiveness was IMP > XMP > GMP > AMP. NAD+ did notprotect the enzyme from modification. Inactivation of IMP dehydrogenase wasaccompanied by a change in Amax. of Cl-IMP from 263 to 290nm, indicating formationof a 6-alkylmercaptopurine nucleotide. The spectrum of 6-chloropurine riboside was notchanged by IMP dehydrogenase. With excess Cl-IMP the increase in A290 with time wasfirst-order. Thus it appears that Cl-IMP reacts with only one species of thiol at theIMP-binding site of the enzyme: 2-3mol of Cl-IMP were bound per mol of IMPdehydrogenase tetramer. Of ten mutant enzymes from guaB strains, six reacted withCl-IMP at a rate similar to that for the native enzyme. The interaction was retarded byIMP. None of the mutant enzymes reacted with 6-chloropurine riboside. 5,5'-Dithiobis-(2-nitrobenzoic acid), iodoacetate, iodoacetamide and methyl methanethio-sulphonate also inactivated IMP dehydrogenase. Reduced glutathione re-activated themethanethiolated enzyme, and 2-mercaptoethanol re-activated the enzyme modified byCl-IMP. IMP did not affect the rate of re-activation of methanethiolated enzyme.Protective modification indicates that Cl-IMP, methyl methanethiosulphonate andiodoacetamide react with the same thiol groups in the enzyme. This is also suggested bythe low incorporation of iodo[U4Clacetamide into Cl-IMP-modified enzyme. Hydrolysisof enzyme inactivated by iodo[14C]acetamide revealed radioactivity only in S-carboxymethylcysteine. The use of Cl-IMP as a probe for the IMP-binding site ofenzymes from guaB mutants is discussed, together with the possible function of theessential thiol groups.

IMP dehydrogenase (IMP-NAD+ oxidoreduct-ase, EC 1.2.1.14) of Escherichia coli consists ofidentical subunits, with the active form of theenzyme being predominantly tetrameric (Gilbert etaL, 1979). Mutant (guaB) strains of E. coli, lackingfunctional IMP dehydrogenase, have been isolated;several of these produce material (CRM) cross-

reacting to anti-(IMP dehydrogenase) antiserum.Complementation in vitro between CRM+ guaB

Abbreviations used: Cl-IMP, 6-chloro-9-fl-D-ribofur-anosylpurine 5'-phosphate (6-chloropurine ribotide);GSH, reduced glutathione.

* Present address: Centre for Applied MicrobiologicalResearch, Porton Down, Salisbury SP4 OJG, U.K.

t To whom reprint requests should be addressed.

Vol. 191

strains reveals the presence of active enzyme inmixed cell-free extracts of certain pairs of mutants(Gilbert & Drabble, 1980). Cl-IMP has been shownto inactivate the IMP dehydrogenase from Aero-bacter aerogenes, possibly by interacting with a thiolgroup at the IMP-binding site of the enzyme(Hampton & Nomura, 1967; Brox & Hampton,1968). In the present paper we describe the use ofCl-IMP to investigate the integrity of the IMP-binding site of enzymes isolated from severalcomplementing guaB strains of E. coli.

Other agents known to modify thiols are found toinactivate IMP dehydrogenase. Mutual protectionagainst inactivation by these agents indicates thatCl-IMP, methyl methanethiosulphonate and iodo-acetamide modify the same thiol group at theIMP-binding site. IMP does not retard re-activation

0306-3275/80/110533-09$01.50/1 (© 1980 The Biochemical Society

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H. J. Gilbert and W. T. Drabble

of methanethiolated enzyme, suggesting an import-ant function for this thiol group in the binding ofIMP.

Materials and methods

Bacterial strainsThe guanine auxotrophs used were PL1072

(guaBS2), PL1096 (guaB76), PLi105 (guaB85),PLI138 (guaB105), MW1087 (guaB792), HGI0OO(guaB1001), HG1005 (guaB1005), HG1017(guaB1017), HG1018 (guaB1018), HG1019(guaB1019). Native IMP dehydrogenase was iso-lated from the guaA mutant PL1068 (guaA48). Allthese mutants were derived from E. coli K12 strainW3 110 (Gilbert & Drabble, 1980).

ChemicalsNAD+ was obtained from Boehringer Corp.

(Lewes, Sussex, U.K.) and Cl-IMP from Calbio-chem (Bishop's Stortford, Herts., U.K.). GSH,6-chloropurine riboside (chloroinosine), guaninehydrochloride and IMP were from Sigma (London)Chemical Co. Butyl-PBD [5-(biphenyl-4-yl)-2-(4-t-butylphenyl)- l-oxa-3,4-diazoleI and toluene (sul-phur-free) were obtained from Koch-Light Labora-tories (Colnbrook, Bucks., U.K.) and Hyaminehydroxide (1 M solution in methanol) was fromNuclear Enterprises (Reading, Berks., U.K.) Iodo-[1-14C]acetamide (sp. radioactivity 4OmCi/mmol)was obtained from The Radiochemical Centre(Amersham, Bucks., U.K.) Methyl methanethiosul-phonate was prepared by Dr. D. P. Bloxham of thisDepartment. All other chemicals were supplied byBDH, Poole, Dorset, U.K.

Purification ofIMP dehydrogenaseThe enzyme was purified to homogeneity from

guaA and guaB mutants by affinity chromato-graphy as described by Gilbert et al. (1979).

Assayfor IMP dehydrogenaseThe enzyme-assay system used was as described

by Gilbert et al. (1979) unless otherise stated.

Protein assayThe protein content of preparations was routinely

determined by the method of Lowry et al. (1951),with crystalline bovine serum albumin (fraction V)as standard. The protein content of two samples ofpurified IMP dehydrogenase was also estimated byacid hydrolysis followed by amino acid analysis(Gilbert et al., 1979); 627,ug of enzyme proteinestimated by this method corresponded to 1 mg ofenzyme protein estimated by the Lowry et al. (1951)method with the bovine serum albumin standard.

SpectrophotometryKinetic measurements and absorption spectra

were recorded with a Cary 118 spectrophotometer(Varian Instruments) unless otherwise stated.

Radioactivity measurementsProtein samples, dissolved in 1 M-Hyamine

hydroxide, were added to 15 ml of butyl-PBDscintillation solution (8 g of butyl-PBD in 1 litre oftoluene) together with 0.25 ml of acetic acid (toneutralize the Hyamine hydroxide). Radioactivitywas measured in a Philips liquid-scintillationanalyser (wide 14C channel).

ElectrophoresisProtein hydrolysate (0.1 ml) was spotted on to

Whatman 3MM chromatography paper(460mm x 570mm). e-Carboxymethyl-lysine, S-car-boxymethylcysteine. N1-carboxymethylhistidine andN3-carboxymethylhistidine were also applied (car-boxymethylated standards were used because acidhydrolysis of the modified enzyme converts amidederivatives into free acids). The amino acids wereseparated by electrophoresis in pyridine/acetic acid/water (1: 10:289, by vol.) at pH3.6 and 3000V for1 h by using a Gilson high-voltage Electrophoratormodel D. The paper was dried, cut into 15mmsquares, and counted for radioactivity in scin-tillation solution [8g of butyl-PBD in 1 litre oftoluene/methanol (3:1, v/v)]. The carboxymethyl-amino acid standards were detected by sprayingwith ninhydrin reagent.

Results

Inactivation ofIMP dehydrogenase by Cl-IMPCl-IMP inactivated IMP dehydrogenase with

apparent first-order kinetics (Fig. 1). The inactiva-tion was not dependent on the presence of GSH andwas not reversed by extensive dialysis nor byincreasing the concentration of substrates in theassay system. Unlike the enzyme from A. aerogenes(Hampton & Nomura, 1967), no enzyme activitywas recovered by including GSH in the assay ofenzyme inactivated in the absence of GSH (how-ever, the activity of unmodified enzyme was in-creased by GSH by 40-70%; Table 3). These resultssuggest covalent modification of the enzyme byCl-IMP, even enzyme that is not fully activated(omission of GSH). A linear relationship was givenby plotting 1/kapp. (reciprocal of observed first-orderrate constants for inactivation; Fig. 1) versus1/[Cl-IMPI, indicating a 'rate saturation effect'(Baker et al., 1962) and confirming that inactivationby Cl-IMP is preceded by reversible binding to theenzyme.

1980

534

Covalent modification ofIMP dehydrogenase

0.75

N 0.60

0.aQ45

,0.30.0

o 0.150

0 1 2 3 4 5 6Time (min)

Fig. 1. Inactivation of IMP dehydrogenase at differentconcentrations ofCl-IMP

IMP dehydrogenase (30,ug/ml) was incubated at230C in 20mM-potassium phosphate buffer, pH 7.0,containing 25 mM-KCl and 5 mM-GSH with Cl-IMPat the following concentrations: 11IM (0), 18,pM(A), 24pM (O), 48,UM (0), 60pM (A). Samples(50p1) were removed at the times indicated andassayed for enzyme activity.

Although IMP binds tightly to IMP dehydro-genase, the corresponding nucleoside, inosine, doesnot (Nichol et al., 1967). 6-Chloropurine riboside(200,UM) did not inactivate the enzyme (0.1 mg/ml)within 2 h, whereas Cl-IMP (at one-tenth theconcentration) caused total inactivation after 15 minunder the same conditions. This supports theconclusion that initial binding to the active siteprecedes the covalent inactivation of the enzyme byCl-IMP.

Protection by nucleotides against inactivation byCl-IMPA further indication of chemical modification at

the active site of an enzyme is the protectionafforded by ligands which bind to or affect the activesite. The inactivation of IMP dehydrogenase byCl-IMP was retarded by nucleotides (Fig. 2). IMPprovided the best protection, followed by GMP andXMP, with AMP only slightly retarding the rate ofinactivation. This order of effectiveness is goodevidence that Cl-IMP is acting at the IMP-bindingsite, as the protection provided by the nucleotidescorrelates with their respective Km and K1 values(Powell et al., 1969; Gilbert et al., 1979). NAD+ didnot protect the enzyme; this indicates that Cl-IMP isnot interacting with the NAD+-binding site.

Changes in the absorption spectrum ofCl-IMPAs Cl-IMP reacted with IMP dehydrogenase,

there was a progressive development of a new

.~50

0

E 20

E l oC)N

40 5 10 15 20

Time (min)

Fig. 2. Inactivation of IMP dehydrogenases by Cl-IMPin the presence ofnucleotides

IMP dehydrogenase (lOOpg/ml) was treated at230C with Cl-IMP (65pM) in 20mM-potassiumphosphate buffer, pH 7.4, containing 25 mM-KCland 5 mM-GSH. Samples (20p1) were removed at thetimes indicated and assayed for enzyme activity.Nucleotides (200,uM) present during the incubationwere IMP (0), GMP (A), XMP (5), AMP (A),none (A).

absorption peak at 290nm (Cl-IMP has Amax at263 nm), accompanied by loss of enzyme activity.These changes in the absorption spectrum of Cl-IMPwere similar to those described by Brox & Hampton(1968) for the enzyme from A. aerogenes. Nospectral changes were observed in the absence ofenzyme, and a 25-fold excess of 6-chloropurineriboside produced neither a change in absorbancenor loss of enzyme activity over the same timeperiod. IMP retarded the appearance of the 290nmpeak, half-maximum absorbance at 290nm occur-ring after 5 and 120min respectively in the absenceand presence of IMP (Table 1). However, the samerelationship between A290 and enzyme activity wasdemonstrated in both the absence and presence ofIMP. Measurement of increases in A290 can there-fore be used to follow enzyme inactivation.Enzyme inactivation was pH-dependent (Fig. 3)

in a simple manner that suggests the involvement ofan ionizable group with a PKa of 8.4. As Cl-IMPcontains no group with a pKa of this value, the groupmust be part of the enzyme, being possibly a thiol(these have PKa values around 8-9). In addition, the

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H. J. Gilbert and W. T. Drabble

Table 1. Covalent modification of native and mutantforms ofIMP dehydrogenase

IMP dehydrogenase, purified from gua mutants(Gilbert et al., 1979), was concentrated by (NH4)2SO4precipitation and redissolved in 20mM-potassiumphosphate buffer, pH 7.4, containing 25 mM-KCl and5 mM-GSH. The enzyme solution was then dialysedfor 18h at 40C against lOOvol. of the same buffersolution. The enzyme concentration was adjusted to1 mg/ml. Cl-IMP (32 nmol) was then added to 1 mlof enzyme solution with and without IMP (200 nmol)and the A290 recorded continuously at 10°C. -, Noreaction. The fine-structure map of the guaB gene(shown below) is modified from Gilbert & Drabble(1980). + or - shown above the allele numbersindicates that the IMP dehydrogenase isolated fromthe mutant strain does or does not react withCl-IMP.

1005 76 1001 52 1018 1017 792 85 105

\\\1//'~~~'

-ecx

xqs

0 -7.0 7.5 8.0 8.5 9.0 9.5

pH

guaO 4- aguaB v| guaA

Source of enzymePL1068 guaA48*HG 1005 guaB1005HG1019 guaB1019MW 1087 guaB792PL1072 guaB52PLI 105 guaB85PL1 138 guaBl05HG1001 guaB1001HG1007 guaB1OI7HG1018 guaB1018PL 1096 guaB76

* Native enzyme.

Time (min) to reach half-maximum A290

IMP IMP (0.2 mM)absent present5.0 1204.0 1026.5 917.0 1014.0 527.5 927.5 133

appearance of a new max. at 290nm suggests theformation of a 6-alkylmercaptopurine nucleotidederivative. A molar absorption coefficient of 16800at 290nm and pH7.4 has been given for 6-alkyl-mercaptopurine nucleotides (Brox & Hampton,1968). From this value, the absorbance at 290nmassociated with 97% inactivation of the enzymecorresponds to the formation of about 2 mol of6-alkylmercaptopurine nucleotide/mol of tetramericenzyme. This ratio was established with threeenzyme preparations (Table 4).Enzyme was incubated with a 50-fold excess of

Fig. 3. Rate of inactivation of IMP dehydrogenase byCl-IMP at diferentpH values

IMP dehydrogenase (lOOpg/ml) in 50mM-Tris/HClbuffer, pH 7.1-9.5, containing 25 mM-KCI wasincubated at 230C with Cl-IMP (30,M). Samples(20pl) were removed at intervals and assayed forenzyme activity in the absence of GSH. kapp. at eachpH was calculated from the slopes of the lines(x 2.303/60) obtained by plotting the first-orderkinetic equations (as in Fig. 1).

8x

,o

0--,e6

_C'0DIVse

Time (min)

Fig. 4. Changes in A290 when excess Cl-IMP is incu-bated with IMP dehydrogenase

IMP dehydrogenase (1 mg/ml, 1 7pM) was incu-bated at 10°C with excess Cl-IMP (850pM) in20mM-potassium phosphate buffer, pH 7.4, con-taining 25 mM-KCl and 5 mM-GSH.

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Covalent modification of IMP dehydrogenase

Cl-IMP and the reaction (A290) was followed at10°C with a Unicam SP.1800 spectrophotometer.The linear relationship obtained (Fig. 4) denotes

Table 2. Inactivation ofIMP dehydrogenase by chemicalmodification

IMP dehydrogenase (lOO,ug/ml) was incubated at0°C with the agents listed in 20mM-potassiumphosphate buffer, pH 7.4, containing 25 mM-KCl.At timed intervals, samples (3,ul) were removedand assayed (in the absence of GSH) for enzymeactivity.

Time (min) for 50% decreasein enzyme activity

Modifying agentMethyl methanethio-

sulphonate (1.7,pM)5,5'-Dithiobis-(2-nitro-

benzoate) (10puM)lodoacetate (1 mM)Iodoacetamide (100,UM)Cl-IMP (20,UM)

* Too rapid to determine.

IMPabsent0*

0.5

IMP (0.2mM)present

7

4.03.01.5

201525

first-order kinetics and therefore the modification ofonly one species of thiol group.

Covalent modification by Cl-IMP ofIMP dehydro-genasefrom guaB mutantsIMP dehydrogenase from ten complementing

CRM+ guaB mutants (Gilbert & Drabble, 1980)was treated with Cl-IMP. The enzyme purified fromsix of the mutants and the native enzyme (PL1068)react with Cl-IMP at a similar rate. The times takento reach 50% of maximum absorbance at 290nmare presented in Table 1. Each reaction resulted in ashift of Amax from 263 to 290nm and gave the samemaximum absorbance at 290nm as the nativeenzyme treated identically. These reactions wereconsiderably retarded by IMP (Table 1). Noreaction occurred between 6-chloropurine ribosideand any enzyme. IMP dehydrogenase from theremaining four guaB mutants did not react withCl-IMP. Those mutant enzymes that react withCl-IMP may have an unimpaired IMP-binding site,as modification is preceded by an initial reversiblebinding between reagent and enzyme. The retar-dation of the reactiQn by IMP (competes withCl-IMP at the active site), together with the absenceof any reaction with 6-chloropurine riboside (sug-

Table 3. Protective modification ofIMP dehydrogenaseIMP dehydrogenase (lOOpug), previously inactivated in the absence of a thiol reagent by either Cl-IMP or methylmethanethiosulphonate (MMTS), was treated at 00C in a final volume of 1 ml with 0.1 mM-iodoacetamide (Cl-IMP-and MMTS-treated enzymes) or 0.1 mM-Cl-IMP (MMTS-treated enzyme) in 20mM-potassium phosphate buffer,pH 7.4, containing 25 mM-KCl. Unmodified enzyme was used as a control. When the control enzyme was fullyinactivated, IMP dehydrogenase that had been pre-treated with Cl-IMP or MMTS was assayed for enzyme activity.The enzyme assay was as described by Gilbert et al. (1979), except that the thiol reagent (5mM) was omittedunless stated otherwise. Enzyme activity is expressed relative to that of unmodified enzyme assayed in the presenceof 5 mM-GSH (line 2).

Pretreatment Alkylating agent added Enzyme activityof enzyme to pre-treated enzyme Additions to assay system (%)

12345 Cl-IMP6 Cl-IMP7 Cl-IMP8 Cl-IMP91011 MMTS12 MMTS13 MMTS14 MMTS151617 MMTS18 MMTS1920

GSH2-Mercaptoethanol2-Mercaptoethanol + GSH

IodoacetamideIodoacetamidelodoacetamideIodoacetamide

Cl-IMPCl-IMPCl-IMPCl-IMPIodoacetamidelodoacetamidelodoacetamideIodoacetamide

2-Mercaptoethanol

2-Mercaptoethanol

2-Mercaptoethanol

GSH

GSH

GSH

GSH

GSH

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701001001000

100086000

1000

88000

9400

537

H. J. Gilbert and W. T. Drabble

gests an initial specific binding), are evidence for anintact IMP-binding site in these mutant enzyme.Those mutations producing Cl-IMP-insensitive en-zymes are not closely grouped on the gene map(Table 1), therefore it is not possible to locate anyone region of the guaB gene that codes for theIMP-binding site of the enzyme.

Inactivation of IMP dehydrogenase by other modi-fying agents

The inactivation of IMP dehydrogenase byprotein-modifying agents that are not substrateanalogues was investigated. The times taken for a50% decrease in enzyme activity are given in Table2. Reactivity towards the enzyme is predictable fromthe relative reactivities of the agents towards thiols.The exception is Cl-IMP; this does not react withthiols such as 2-mercaptoethanol and GSH at pH 7.4(Brox & Hampton, 1968). An initial specific bindingof Cl-IMP to the enzyme may be responsible for the

2.0

xcE 1.5

0C-0

0 1.0UU

05

N0_E0.5

on

Time (min)

Fig. 5. Re-activation of methanethiolated IMP dehydro-genase by GSH in the presence and absence ofIMPIMP dehydrogenase (1 mg, 0.5mg/ml) in 50mM-potassium phosphate buffer, pH 7.4, was inacti-vated at 0°C by successive additions of methylmethanethiosulphonate. The enzyme was thendiluted 20-fold into the same buffer solution con-taining 1 mM-GSH. Samples (2jug of protein)removed at timed intervals were assayed in theabsence of GSH for enzyme activity. For thecontrol, unmodified enzyme was treated in the sameway; this enzyme had constant activity over thecourse of the experiment. IMP (200pM) was eitherpresent in (0) or absent from (@) the dilution buffer.

rapid reaction with the enzyme at pH 7.4. IMPprotects the enzyme from inactivation by all theagents except methyl methanethiosulphonate. How-ever, that compound reacts so rapidly (Table 2) thatan accurate assessment of protection by IMP wasnot possible. Nevertheless methyl methanethiosul-phonate and Cl-IMP probably modify the same thiolgroups, because both protect the enzyme againstiodoacetamide (see the next section).

Protective modification ofIMP dehydrogenaseIMP dehydrogenase, after complete inactivation

by C1-IMP, was assayed in either the presence orabsence of 5 mM-2-mercaptoethanol (Tables 3, lines5 and 6). The modified enzyme was fully re-activatedby 2-mercaptoethanol after approx. 5 min. Additionof 5 mM-2-mercaptoethanol did not affect the ac-tivity of native enzyme assayed in the presence of5 mM-GSH (Table 3, lines 2 and 4). Similarly,enzyme that had been fully inactivated by methylmethanethiosulphonate (methanethiolated) could bere-activated, with full recovery of activity, by5 mM-GSH (Table 3, lines 11 and 12).The protective effect of one agent against in-

activation of IMP dehydrogenase by a second agentwas investigated. Treatment of Cl-IMP-modifiedenzyme with iodoacetamide, under conditions thatinactivate native enzyme, did not prevent re-activa-tion by 2-mercaptoethanol (Table 3, lines 7-10).Methanethiolated enzyme, after further treatmentwith Cl-IMP under conditions that completelyinactivate native enzyme, was re-activated by GSH(Table 3, lines 13-16). Finally methanethiolatedenzyme was active after treatment with iodoacet-amide provided that the assay mixture containedGSH (Table 3, lines 17-20). Therefore Cl-IMPprotects the enzyme from alkylation by iodoacet-amide, and methyl methanethiosulphonate protectsthe enzyme from alkylation by iodoacetamide andCl-IMP. This indicates that all three agents modifythe same thiol groups ofIMP dehydrogenase.

The rate of re-activation by GSH of methane-thiolated IMP dehydrogenase was determined in thepresence and absence of IMP (Fig. 5). The enzymewas fully re-activated after 40 min, with no ob-servable retardation by IMP. These data suggestmodification of an active-site thiol group by methylmethanethiosulphonate such that binding of IMP isprecluded. The thiol group modified by methylmethanethiosulphonate may therefore be importantin the binding of IMP to the active site of theenzyme.

Incorporation of iodoacetamide into native andCl-IMP-modified IMP dehydrogenaseIMP dehydrogenase was treated with iodo[ l-14C1-

acetamide (Table 4). The alkylation was accom-

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Covalent modification ofIMP dehydrogenase

Table 4. Incorporation of iodo[1-14C]acetamide intonative and Cl-IMP-modified IMP dehydrogenase

Purified IMP dehydrogenase (4mg/ml, 68pM) in20mM-potassium phosphate buffer, pH 7.4, contain-ing 25mM-KCI was fully inactivated at 230C byCl-IMP. The modified enzyme (4mg), and nativeenzyme used as a control, were treated with 0.5,umolof iodo[1-14C]acetamide (sp. radioactivity 75pCi/mmol). Samples (0.2ml) were placed at intervalsinto 1 ml of ice-cold 10% (w/v) trichloroacetic acid.As a control, 0.2ml of iodo[ 1-14c]acetamide(0.1,umol) in the same buffer was placed in 1mlof 10% trichloroacetic acid. After 30min, cell-freeextract (Lambden & Drabble, 1973) of strain PL1068(10mg of protein) was added to each acidifiedsample to act as a carrier. After a further 20 min.the precipitated protein was washed five times with5% trichloroacetic acid, twice with acetone, andthen dissolved in 0.5 ml of 1 M-Hyamine hydroxideby heating at 60°C for 1 h. Radioactivity wasmeasured as described in the Materials and methodssection. Samples (2,g of protein) were also removedfrom the reaction and assayed for enzyme activity.Results from three separate experiments are shown.Protein concentration was determined by the methodof Lowry et al. (1951), and molarity of tetramericenzyme was calculated by using 232000 for themol.wt. (Gilbert et al., 1979). Values shown for molof modifying agent incorporated per mol of tetra-meric enzyme are increased by a factor of 1.6 whenthe protein concentration estimated from amino acidanalysis is substituted (see the Materials andmethods section).

7500-

0

250

0180 150 120 90 60 30 0 30 60 90

(D Distance from origin (mm) 0

Fig. 6. Electrophoresis of acid-hydrolysed IMP dehydro-genase after treatment with iodo[14Clacetamide

Purified IMP dehydrogenase (2mg) that had beenfully inactivated at 230C by iodo[l1-14C]acetamide(sp. radioactivity 75,pCi/mmol) was hydrolysed-under nitrogen with 6 M-HCI at 1 100C for 24 h. Thehydrolysate was dissolved in 0.1 ml of water beforeelectrophoresis as described in the Materials andmethods section. Carboxymethylated amino acidsused as markers were e-carboxymethyl-lysine (CM-Lys), S-carboxymethylcysteine (CM-Cys), N1-car-boxymethylhistidine (N1-CM-His) and N3-carboxy-methylhistidine (N3-CM-His).

Incorporation (mol/mol of tetramericenzyme) required for complete

inactivation-A. --

Cl-IMP2.05, 1.92, 2.13

Iodoacetamide2.13, 2.30, 2.200.25, 0.16,0.27

origin probably corresponded to precipitated pro-tein. No radioactivity moved to the cathode,therefore cysteine is the only amino acid modified byiodoacetamide.

panied by a progressive decrease in enzyme activity.For each of three preparations, the amount ofiodoacetamide bound per tetramer that caused totalinactivation of the enzyme was similar to the amountof bound Cl-IMP required for total inactivation(Table 4). IMP dehydrogenase, previously modifiedwith Cl-IMP, incorporated only about 10% of theiodoacetamide incorporated into native enzyme.These results give further support to the proposalthat iodoacetamide and Cl-IMP modify the samegroups on the enzyme.

The amino acid modified by iodoacetamide wasidentified as described in the Materials and methodssection. After hydrolysis of the enzyme, most of theradioactivity coincided with S-carboxymethylcys-teine (Fig. 6). A small peak of radioactivity at the

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Discussion

The inactivation of IMP dehydrogenase of E. coliby Cl-IMP is consistent with covalent modificationat the active site of the enzyme. Thus the kinetics ofthe reaction display the rate-saturation effect of atwo-stage inactivation (Baker et al., 1962), andpurine nucleotides protect against modification to anextent predictable from their affinities for theenzyme. NAD+ does not protect, however, even atthe high concentration of 2mM; this implies a

specific action of Cl-IMP at the IMP-binding site.NAD+ binds to the K+-enzyme complex -in a non-

compulsory order with respect to IMP (Heyde &Morrison, 1976), so presumably NAD+ binds underthe experimental conditions described in the presentpaper. Additional evidence for initial specific andreversible binding of Cl-IMP to the enzyme is theinertness of 6-chloropurine riboside. It should be

EnzymepreparationNativeModified byCl-IMP

539

540 H. J. Gilbert and W. T. Drabble

noted that inosine does not bind to IMP de-hydrogenase (Nichol et al., 1967).

Apparently 2mol of Cl-IMP are required to in-activate 1 mol of IMP dehydrogenase tetramer, anindication of half-site reactivity (Levitzki & Kosh-land, 1976). However, this calculation is based on adetermination of protein concentration by themethod of Lowry et al. (1951), with a bovine serumalbumin standard, and is therefore not whollyreliable. In addition, only half the enzyme units wererecovered from the affinity column during puri-fication (Gilbert et al., 1979), so contaminatinginactive enzyme may be present in the preparation.Inactivation of 1 mol of tetrameric enzyme byapprox. 3 mol of Cl-IMP is indicated by using anestimate of enzyme concentration based on the totalquantity of amino acids released by completehydrolysis of the protein. The linear increase in logA290 with time also argues against half-site reac-tivity; a non-linear relationship would be expectedfor groups with different reactivities (unless twosubunits of the tetramer are modified very rapidly toinactivate the enzyme before the other two subunitshave reacted). Sites with different degrees of cata-lytic activity and reactivity towards Cl-IMP havebeen proposed for IMP dehydrogenase of A.aerogenes (Brox & Hampton, 1968), an enzymecontaining non-identical subunits, albeit of the samemolecular weight. The enzyme of E. coli hasidentical subunits (Gilbert et al, 1979; Gilbert &Drabble, 1980). No enzyme activity was detectableafter denaturing and renaturing enzyme modified byCl-IMP (Gilbert & Drabble, 1980), but someactivity would be expected from the combination offour unmodified subunits from half-reacted enzyme.However, the preferred conformation of tetramericenzyme could be two modified plus two nativesubunits. In summary, the data do not permitunambiguous assignment of stoichiometry to thereaction, but the results are consistent with a specificmodification, be it full or half-site, at the active siteof the enzyme.

Cl-IMP provides a useful probe for the IMP-binding site of IMP dehydrogenase from guaBmutants because the interaction can be easilyfollowed spectrophotometrically. A mutant enzymewas considered to have an intact IMP-binding sitewhen it (a) reacted with Cl-IMP at a rate com-parable with that for native enzyme, (b) wasprotected from modification by IMP, and (c) wasnot modified by 6-chloropurine riboside. On thisbasis, six of the ten CRM+ guaB strains withmutations of known map position produce enzymeswith intact IMP-binding sites. The positions of thefour mutations that abolish IMP binding are widelyspaced on the gene map, therefore several regions ofthe protein, well separated in -the primary sequence,are involved in binding IMP. It should be possible to

extend this experimental approach to the NAD+-binding site by using affinity-labelling analogues ofthat substrate.

The shift in the Amax of Cl-IMP from 263 to290nm on addition of IMP dehydrogenase indicatesformation of a 6-alkylmercaptopurine nucleotidederivative (Brox & Hampton, 1968) by reaction ofC-6 of the purine with a thiol group at theIMP-binding site. As 6-alkylaminopurines and 6-alkoxypurines have absorption maxima at 248nmand 266-267 nm respectively (Johnson et al., 1958),an amino or hydroxy group is not modified byCl-IMP. However, the spectra of modifying agentsbound to protein can be quite different from spectrain free solution, because of the influence of the localenvironment around the protein-bound ligand.Nevertheless, the following considerations suggestmodification of a thiol by Cl-IMP. Variations in therate of modification by Cl-IMP at different pHvalues suggest the presence of a reactive group inIMP dehydrogenase with a PKa of 8.4, i.e. within therange expected for thiols. In addition to Cl-IMP,agents that are known to modify thiol groups inenzymes also inactivate IMP dehydrogenase, andlabelling with iodo( 4C]acetamide gives rise only toradioactive carboxymethylcysteine on hydrolysis ofthe enzyme. The mutual protection against in-activation provided by these agents indicates themodification of the same thiol groups. This con-clusion is supported by the observations that similarmolar amounts of iodoacetamide and Cl-IMP arerequired to abolish enzyme activity and that pre-treatment of the enzyme with Cl-IMP decreasesiodoacetamide binding by 10-fold.

The recovery of activity when methanethiolatedenzyme is treated with GSH is unaffected by IMP.Therefore even a small modification of the active-sitethiol group results in an enzyme unable to bind IMP.GSH, a bulky charged molecule, would be unable tore-activate a thiol group protected by a boundnucleotide. It should be noted that IMP protects theenzyme against inactivation by iodoacetate andiodoacetamide, both smaller molecules than GSH,and Cl-IMP, although removed from the enzyme by2-mercaptoethanol, is not by GSH. Several de-hydrogenases have a cysteine residue at the activesite. As modification of this amino acid inactivatesthese enzymes, an essential function in catalysis hasbeen proposed for this residue. However, methane-thiolated lactate dehydrogenase retains full catalyticactivity, but with a large increase in the Km forpyruvate (Bloxham & Wilton, 1977; Bloxham et al.,1979). The thiol is not involved, therefore, in anyessential covalent step in the catalytic mechanism,but in the binding of substrate, possibly by stabiliz-ing an ionic interaction. The reactive thiol groups ofIMP dehydrogenase may have a similar function inbinding an ionic species ofIMP (Hampton, 1963).

1980

Covalent modification of IMP dehydrogenase 541

H. J. G. thanks the Scientific Research Council for aresearch training studentship.

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