Purification and some properties of uracil phosphoribosyltransferase from Escherichia coli K12

Biochimica et BiophvsicaActa 881 (1986) 268 275

268

Elsevier BBA 22295

P u r i f i c a t i o n a n d s o m e p r o p e r t i e s o f uracil p h o s p h o r i b o s y i t r a n s f e r a s e

from

Escherichia coli K 1 2

U l l a B. R a s m u s s e n *, B e n t e M y g i n d a n d P e r N y g a a r d ** Enzyme Division, University Institute of Biological Chemistry B, Solvgade 83, D K-1307 Copenhagen K (Denmark) (Received December 31st, 1985)

Key words: Uracil phosphoribosyltransferase; Pyrimidine metabolism: Gene amplification; ( E. coli)

Uracil phosphoribosyltransferase from Escherichia coil KI2 was purified to homogeneity as determined by polyacrylamide gel electrophoresis. For this purpose a pyrimidine-requiring strain harboring the upp gene on a ColEI plasmid was used, which showed 15-times higher uracil phosphoribosyitransferase activity in a crude extract. When this strain was grown under conditions of uracil starvation, an additional 10-times elevation of the enzyme activity was obtained. The molecular weight of uracil phosphoribosyltransferase was determined to be 75 000; the enzyme consists of three subunits with a molecular weight of 23 500. Uracil phosphoribosyltransferase is specific for uracil and some uracil analogues. The apparent K m values for uracil and PRib-PP were 7 p M and 300 pM, respectively. As an effector of enzyme activity, GTP lowered the K m for PRib-PP to 90 p M and increased the V,.ax value 2-fold, but had no effect on the K,, for uracil. The effect of GTP was found to be pH-dependent. The enzymatic characterization of uracil phosphoribosyltransferase and the observed regulation of its synthesis emphasizes the role of the enzyme in pyrimidine salvage.

Introduction

Uracil phosphoribosyltransferase (EC 2.4.2.9) catalyzes the conversion of uracil and 5-phosphoribosyl c~-l-pyrophosphate ( P R J b - P P ) to UMP and PPi- UMP is the precursor for all pyrimidine nucleoside triphosphates, and in the cell it is synthesized via the de novo pathway and the salvage pathway [1]. The role of uracil phosphoribosyltransferase in the salvage of endogenously formed uracil and in the utilization of exogenous uracil and cytosine has been demonstrated in several microorganisms including E. coli [1-3]. Mutants of E. coli lacking the enzyme (upp) but with an intact uracil transport system fail to grow on uracil as a pyrimidine source and they * Present address: Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114, U.S.A. ** To whom correspondence should be addressed.

excrete uracil into the culture medium [1,2]. Furthermore they are resistant to 20 ~M 5-fluorouracil, this being a phenotype which has been used in the selection of upp mutants [1]. Uracil phosphoribosyltransferase from E. coli is activated by GTP and inhibited by uridine nucleotides [4]. The activation by GTP is abolished by ppGpp as shown in a partially purified extract of uracil phosphoribosyltransferase from E. coli B [3]. Fast [3] proposed that the allosteric behaviour of the enzyme is the mechanism behind the regulation of uracil uptake. The incorporation of uracil into the pyrimidine ribonucleotide pool in E. coli is strongly restricted under stringent conditions, a situation in which the GTP pool decreases and ppGpp accumulates. In accordance with this is the observation that a mutant of Salmonella typhimurium with a low GTP pool is resistant to 5-fluorouracil [5]. Some properties are known of partially purified uracil phosphoribosyltransferase

0304-4165/86/$03.50 ¢2 1986 Elsevier Science Publishers B.V. (Biomedical Division)

269 from other organisms: yeast [6], Tetrahymena pyriformis [7], and Acholeplasma laidlawii [8]. The objective of the present study was to purify uracil phosphoribosyltransferase in order to improve our understanding of the effect of G T P on the regulation of uracil utilization, and to determine the specificity of the enzyme towards other pyrimidine bases and towards some uracil analogues. This report describes the first purification of uracil phosphoribosyltransferase to apparent homogeneity and some properties of the enzyme from E. coli K12. The procedure combines the advantages of gene amplification and derepression and the use of Blue Sepharose affinity chromatography. Materials and Methods Bacterial strains

The strains used and their genotypes are listed in Table I. The ColEI: p u r M ÷, upp ÷ plasmid was obtained from the pooled gene bank Of E. coli constructed by Clarke and Carbon [10]. By mating their collection with F - phenocopies [11] of SO1342, a derivative of RW599 harbouring a deletion covering purM-upp (Table I), and selecting for purine prototrophy, SO1344 was obtained. The presence of the upp gene on the plasmid was verified by testing for sensitivity to 5-fluorouracil (20 /~M) and by enzyme assay. The F-mediated transfer of the plasmid from SO1344 to PC0631, a

pyrimidine and purine auxotroph, was performed in the same manner, again selecting for purine prototrophy. Materials

Pyrimidine nucleobases and phosphoribosylpyrophosphate were obtained from the Sigma Chemical Co., St. Louis, MO, U.S.A. Radiolabelled compounds were from Amersham International plc, Amersham, U.K. Growth conditions

If not otherwise stated, bacteria were grown with shaking in minimal medium at 37°C with 0.2% glucose as carbon source. When required, the concentration of the nutrients were, per ml: 1 /~g biotin, 1 /~g thiamin, 15 /~g hypoxanthine, 10 ~tg uracil, 40/~g methionine and 50 #g arginine. Cell growth was monitored in an Eppendorf photometer at 436 nm. An absorbance of 1 corresponds to about 3 . 1 0 s cells/ml [9]. Preparation of extracts

For the determination of enzyme levels, cells were harvested by centrifugation at 6000 × g for 5 min. The pellet was washed in 0.9% NaCI and resuspended in 20 mM Tris-HCl (pH 7.5)/1 mM E D T A / 5 mM fl-mercaptoethanol. After sonication for 1 min and centrifugation at 8000 × g for 15 rain the supernatant was used for enzyme assay.

TABLE I BACTERIAL STRAINS USED Strain

Genotype

RW599 SO1342

HfrH galE, A(att-bio), thi HfrH galE, A(att-bio), thi

SO1344 PC0631 SO1346

A ( purM, upp) HfrH galE, Zl(att-bio), thi A ( purM, upp ) ColE1 : purM +, upp ÷ HfrR4, carAB35, purM48 a, m e t B 1 rel-1 HfrR4, carAB35, purM48, metB1, rel-1 ColE1 : purM ÷, upp +

a FormerlypurG [9]. h Obtained through the E. coli Genetic Stock Center

Nutritional requirements biotin, thiamin biotin, thiamin, hypoxanthine biotin, thiamin

Source

hypoxanthine, uracil, methionine, arginine uracil, methionine, arginine

P.G. de Haan b

M. Gottesmann I. Quinto and B. Mygind this work

this work

270

Assay for uracil phosphoribosyltransferase One enzyme unit (U) is defined as the amount of enzyme that converts 1 nmol substrate per rain at 37°C. Specific activities are given as units per mg protein. The uracil phosphoribosyltransferase assay, initially developed by Crawford et al. [12], is based on conversion of [2-14C]uracil and PRib-PP to [2-14C]UMP. When necessary the enzyme was diluted in 10 mM Tris-HC1 (pH 7.6)/2 mM flmercaptoethanol/20% ethylene glycol. The standard assay mix contained 50 mM Tris-HCl/20 mM sodium phosphate (pH 7.5)/5 mM M g C l J 0 , 8 mM fl-mercaptoethanol/1 mM G T P / 0 . 1 mM [214C]uracil (1 ~ C i / / ~ m o l ) / 1 . 2 m M P R i b PP/0.1-20 U uracil phosphoribosyltransferase, usually in a total volume of 50 #1. After 2 min of preincubation the reaction was started by adding PRib-PP. 10 /~1 aliquots were withdrawn and spotted on polyethylenimine-impregnated Whatman paper strips (20 cm x 1.5 cm x 0.3 mm) or polyethylenimine-impregnated cellulose thin layers [9]. The paper strips were dried in cold air, and the remaining uracil was washed out in a tray under running ion-exchanged water for 10 min. The thin-layer plates were developed in water, which allows the separation of uracil from UMP. After drying, the UMP spots were marked under ultraviolet light, cut out and counted in a Packard Tri-Carb liquid scintillation spectrometer. Usually, different dilutions of the enzyme preparation were assayed simultaneously and the average was calculated. Protein concentrations were determined by the method of Lowry et al. [13], using bovine serum albumin as standard.

Purification of uracil phosphoribosyltransferase All purification steps were carried out at 0-4°C. SO 1346 was grown in a fermentor in minimal medium with the required nutrients and under stepwise uracil starvation. 2 ~g uracil/ml was added from the beginning, and increments of 1 p,g/ml were added later after starvation periods (lag in growth), each of 1-2 h. This was repeated four times at the initial growth stage, during which the absorbance at 436 nm increased from 0.1 to 1.5. At the later growth stage, when the absorbance increased to 10 the same procedure was repeated three to five times but now with the

stepwise addition of 10 fig uracil/ml. The cells were harvested by centrifugation and kept frozen at - 70°C. Cell lysis. 72 g cells were thawed, washed and resuspended in a total volume of 370 ml 20 mM Yris-HCl (pH 7.5)/1 mM E D T A / 5 mM flmercaptoethanol. The cells were disrupted in a French pressure cell twice (10-13 MPa) and sonicared for 1 min. The cell debris was removed by centrifugation at 12000 X g for 15 rain (fraction I). Anion exchange chromatography. To remove nucleic acids, streptomycin sulphate was added to the supernatant to a final concentration of 45 mg per ml, and stirring was continued for one h. After centrifugation (20000 x g for 20 min) the supernatant was collected and dialysed overnight against 10 vol. buffer 1 (10 mM Tris-HCl (pH 7.5)/10 mM MgCI2/2 mM fl-mercaptoethanol). After centrifugation (20000 x g for 20 min) the supernatant was again dialysed against buffer 1 for 1.5 h. The extract was then applied to a (3 x 45 c m ) DEAE-cellulose (Pharmacia) column (185 ml bed volume) equilibrated with buffer 1 and eluted with a linear 0-500 mM NaC1 gradient in buffer 1. Uracil phosphoribosyltransferase is eluted between 100 and 200 mM NaC1, with the peak fraction at 140 mM. The fractions containing more than 30% uracil phosphoribosyltransferase activity, relative to the peak fraction, were pooled and dialysed against 10 vol. buffer 1 (fraction II). Concentration. Fraction II was concentrated by applying the fraction to a DEAE-cellulose column (42 ml bed volume) equilibrated in buffer 1. Uracil phosphoribosyltransferase activity was eluted as a sharp peak with 300 mM NaCI in buffer 1 and dialysed against 10 vol. buffer 1 (fraction III). Affinity chromatograph)'. Fraction III was applied to a (2.5 x 30 cm) Blue Sepharose CL-6B (Pharmacia) column (66 ml bed volume) equilibrated with buffer 1. The column was washed with buffer 1 containing 150 mM NaCI, which did not elute any uracil phosphoribosyltransferase activity. Uracil phosphoribosyltransferase was eluted in buffer 1 containing 10 mM PRib-PP, and the fractions containing enzyme activity were pooled (fraction IV). This fraction was concentrated as described above (fraction V).

271

P olyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis was performed in cylindrical gels (0.5 x 9 cm) containing 7.5% (w/v) polyacrylamide/300 mM Tris-HCl (pH 9.1). The upper buffer contained 42.6 mM TrisHC1/46.4 mM glycine (pH 8.9)/0.2 mM EDTA, and the lower buffer contained 120 mM Tris-HC1 (pH 9.1)/0.2 mM EDTA. To the samples were added 40% (w/v) sucrose and 0.001% (w/v) bromophenol blue. A constant current of 2.5 mA was applied per gel. All gels were cut through the tracking dye after the run. Denaturing SDS-polyacrylamide (10% w/v) gel electrophoresis was performed as described by Weber et al. [14]. The gels were stained with Coomassie brilliant blue. Results

Construction of a strain with elevated uracil phosphoribosyltransferase activity The ColE1 plasmid, harbouring the uracil phosphoribosyltransferase gene (upp), was transferred to a strain (PC0631) impaired in both the pyrimidine and purine biosynthetic pathways, as described under Materials and Methods. The resulting mutant, designated SO1346 (Table I), was resistant to colicin, indicating the presence of the ColE1 plasmid. The strains PC0631 and SO1346 were grown in minimal medium either with an excess of uracil or under stepwise uracil starvation, a condition which increased the level of uracil phosphoribosyltransferase activity [1]. Enzyme activity was assayed in crude extracts of the cells. The results in Table II show that the presence of

the plasmid results in an approx. 15-fold increase in uracil phosphoribosyltransferase synthesis (compare a and c in Table II). This was also found when uracil phosphoribosyltransferase activity of RW599 and SO1344 was compared (data not shown). Uracil starvation results in a 10-fold derepression of the enzyme (compare a with b and c with d in Table II). The uracil phosphoribosyltransferase activity of SO1346 grown under uracil starvation is about 150-fold higher than the wildtype level. SO1346 grown under conditions of uracil starvation thus served as source for the purification of uracil phosphoribosyltransferase.

Elution of uracil phosphoribosyltransferase from Blue Sepharose A summary of the purification of uracil phosphoribosyltransferase is given in Table III. The Blue Sepharose column was washed with 150 mM NaCI in buffer 1 before the enzyme was eluted with PRib-PP. This washing step elutes about 12% of the total protein applied, including orotate phosphoribosyltransferase which also can be eluted with PRib-PP [15]. Other specific ligands were tested for their ability to elute the enzyme. UMP (10 mM) or uracil (10 mM) does not elute uracil phosphoribosyltransferase activity. GTP (10 mM) also elutes the enzyme, but the recovery is lower than with PRib-PP. The enzyme is nonspecifically eluted from Blue Sepharose by ATP (10 mM) and by 200 mM NaCI in buffer 1. The recovery of activity in the Blue Sepharose purification step is about 20% (Table III). If uracil phosphoribosyltransferase was eluted with NaCI (500 mM), the

TABLE II URACIL PHOSPHORIBOSYLTRANSFERASE ACTIVITY IN P Y R I M I D I N E - R E Q U I R I N G STRAINS G R O W N EITHER IN EXCESS OF, OR STARVED OF, URACIL Cells were grown in minimal medium. In (a) and (c) all the uracil was added from the beginning. In (b) and (d) 2 #g uracil/ml were added from the beginning, and increments of 1 /.tg/ml were added later, after starvation periods (lag in growth) each of 1-2 h. Strain

upp gene on plasmid

Uracil added (total) (#g/ml)

Cell density at harvest A 436

Specific activity (U/mg)

(a) PC0631 (b) PC0631 (c) SO1346 (d) SOl 346

+ +

10 6 10 5

2.3 1.5 2.0 1.8

1.5 18.4 21.7 192.1

272 TABLE Ill PURIFICATION OF URACIL PHOSPHORIBOSYLTRANSFERASE Fraction

Purification step

Volume (ml)

Total activity (103 U)

Total protein (mg)

Specific activity (U/rag)

Purification (fold}

Yield (%)

1 II Ill lV V

Crude extract DEAE-cellulose Concentration Blue Sepharose Concentration

325 195 38 49 8

1 268 544 532 110 73

6468 917 809 20 11

196 605 658 5540 6602

1 3 3 28 34

100 44 42 9 6

recovered activity was also about 20%. If the activity was reapplied to the column and eluted again with NaCI (500 mM), only about 25% of the applied activity was recovered.

Gel electrophoresis When 30 /~g protein of fraction V (Table III) was electrophoresed through a polyacrylamide gel and stained with Coomassie brilliant blue (Fig. 1, lane 1) a single band with a mobility of 0.6 was obtained. No other bands became visible when the amount of applied protein was increased to 180 ~g (lane 2). Two gels (30 ~g protein each) were run in parallel; one was stained and the other was sliced and extracted for uracil phosphoribosyltransferase activity. Activity coincided with the stained band (data not shown).

Stability of uracil phosphoribosyltransferase When stored at 4°C in buffer 1, 56% activity remains after 8 days. Omission of MgClz in the storage buffer lowers the stability by about 50%. fl-Mercaptoethanol is not essential for stability in long-term storage. 5 mM G T P in buffer 1 labilizes uracil phosphoribosyltransferase, with only 15% activity remaining after 13 days, compared to a control without GTP, while 5 mM PRib-PP seems to have a slightly stabilizing effect, the labilizing effect of G T P being abolished in the presence of PRib-PP. Storage in buffer 1 adjusted to pH 8.3 also labilizes the enzyme. 20% ( v / v ) ethylene glycol significantly increases the stability, with virtually no loss of activity after storage at 4°C for 13 days and after a year at - 7 0 ° C . Generally, after purification the enzyme was immediately frozen in

aliquots and stored in buffer 1 containing 20% ( v / v ) ethylene glycol.

pH optimum Uracil phosphoribosyltransferase activity was determined using the standard assay with different p H values. Optimum activity for the purified enzyme and in crude extracts is between p H 7.5 and 8.5 and is influenced by GTP. A 2-3-fold stimulation by G T P of the purified enzyme and a 7-fold stimulation in crude extracts was observed at p H 7.5, while G T P had no effect at p H 8.5 on either of the two preparations.

Molecular weight and subunit composition To determine the subunit composition of uracil phosphoribosyltransferase, SDS-polyacrylamide gel electrophoresis of fraction V was performed. Examination of the gel (Fig. 1, lane 6) showed one single band. By coelectrophoresis of fraction V with combinations of six different molecular weight marker proteins as reference (Fig. 1, lanes 3-5), the molecular weight was determined to be 23 500. By elution of uracil p h o s p h o r i b o syltransferase from Sephadex G-200 with molecular weight marker proteins (catalase, alcohol dehydrogenase, bovine serum albumin, and cytochrome C) [16], an average molecular weight of 75 000 was obtained.

Dilution effect A characteristic of uracil p h o s p h o r i b o syltransferase is that the rate of reaction at greater dilutions is not proportional to the amount of protein in the incubation assay, due to inactivation

273 authentic unlabelled nucleotide on polyethylenimine-impregnated cellulose thin layers.

Kinetic properties

Fig. 1. Analytical polyacrylamide gel electrophoresis of purified uracil phosphoribosyltransferase (fraction V). Lane 1, 30 ~g protein; lane 2, 180 /xg protein. Lanes 3-6 (SDS gels) contained 7 p,g protein (fraction V) and 10 /~g standard proteins. Lane 3, phosphorylase a (Mr 94000), cytochrome c (Mr 11700); lane 4, glyceraldehyde-3-phosphatedehydrogenase(Mr 36000), serum albumin (Mr 68000); lane 5, ovalbumin (Mr 43000); lane 6, catalase (Mr 60000). Protein was stained with Coomassie brilliant blue.

of the enzyme [3,4]. This effect of dilution on enzyme activity was observed at all stages of the purification, and is observed whether the enzymatic reaction is started by the addition of R RJb-PP, uracil, or a combination of both. However, addition of ethylene glycol protected against enzyme inactivation. No loss of activity is seen with the purified enzyme (fraction V) within 1 h at 4°C when diluted up to 100-fold.

Substrate specificity The specificity of uracil p h o s p h o r i b o syltransferase (fraction V) towards various nucleobases (200 /zM, 0.8 /~Ci/ml) was investigated. Activity was determined essentially as described with uracil. If activity with uracil was set to 100%, 6-azauracil showed 97% and 5-fluorouracil showed 216% activity, while orotate, cytosine, thymine and hypoxanthine all showed less than 1% activity. The product of the enzymatic reaction with uracil as substrate was found to be 5'-UMP, as identified by chromatography with the

The initial velocity of the uracil phosphoribosyltransferase-catalyzed reaction was measured as a function of both the uracil and the PRib-PP concentration, in the presence and in the absence of GTP; under all conditions, hyperbolic saturation curves were obtained. The apparent K m and V,,,~ were determined from an Eadie-Hofstee plot. The K,, value for uracil is not influenced by GTP. while GTP lowers the K m value about 3-fold for PRib-PP. A 2-fold increase in V,.... was observed when GTP was present (Table IV). It was found that the inhibition by 5-fluorouracil was competitive with respect to uracil; the K~ was determined to be 15 #M. Pyrophosphate (0.1 mM) had no effect on the uracil phosphoribosyltransferase activity. The actual concentration of PRib-PP in the used solutions was determined by its capacity to convert [14C]uracil to [14C]UMP through the reaction catalyzed by uracil phosphoribosyltransferase using limiting concentrations of P Pdb-PP.

Regulation of the synthesis of uracil phosphoribosyltransferase To test whether uracil phosphoribosyltransferase synthesis is regulated together with other pyrimidine salvage enzymes, cells have been grown under conditions which influence the synthesis of cytosine deaminase encoded for by the codA gene [1]. Expression of the codA gene is derepressed under conditions of pyrimidine starvation [1,17] TABLE IV KINETIC PARAMETERS FOR URACIL PHOSPHORIBOSYLTRANSFERASE Nucleotide present (1 mM)

K m

(#M) for uracil a PRib-PPb

limax (U/mg)

GTP None

7 7

1800 810

90 300

a Concentration range 2-18 #M at a fixed PRib-PP concentration of 200/,tM. b Concentration range 6-1100 p,M at a fixed uracil concentration of 120 #M.

274 a n d we also o b s e r v e d this for the upp gene (Table II). While a d d i t i o n of p u r i n e a n d p y r i m i d i n e bases to the growth m e d i u m reduces the level of cytosine d e a m i n a s e [1,17], we f o u n d that the level of uracil p h o s p h o r i b o s y l t r a n s f e r a s e was not affected ( d a t a not given).

Discussion U r a c i l p h o s p h o r i b o s y i t r a n ~ f e r a s e has been purified to a p p a r e n t h o m o g e n e i t y from a strain of E. coli h a r b o r i n g the cloned upp gene. T h e enzyme ( M r 75 000) consists of three subunits of similar size ( M r 23 500) a n d differs from the enzyme of yeast ( M r 80000) which seems to c o n t a i n two n o n i d e n t i c a l subunits [6]. T h e e n z y m e from Tetrahymena has a m o l e c u l a r weight of 36 000 [7] a n d that from Acholeplasma of 80000 [8], as d e t e r m i n e d b y gel filtration. T h e enzyme specifically reacts with uracil a n d some uracil analogues. T h e high activity of the E. coli enzyme with 5fluorouracil as s u b s t r a t e m a y explain why this c o m p o u n d is toxic to E. coli at low c o n c e n t r a tions. T h e M i c h a e l i s - M e n t e n c o n s t a n t s o b s e r v e d in this study (Table IV) are similar to those r e p o r t e d for p r e p a r a t i o n s of Mycoplasma, uracil K m = 2.6 /~M a n d 51 /~M for P R i b - P P [18] a n d Acholeplasma, uracil K m = 4 . 2 / I M a n d 6 6 / I M for P R i b PP [8], while g m values of 0 . 4 / ~ M for uracil a n d 6.9/~M for P R i b - P P of the e n z y m e from Tetrahymena [7] a n d 21 /~M for uracil a n d 26 /IM for PRJb-PP have been o b s e r v e d for the yeast enz y m e [7]. C r u d e p r e p a r a t i o n s of uracil p h o s p h o r i b o s y l t r a n s f e r a s e from Neisseria [19] a n d Mycoplasma [18] r e s e m b l e the E. coli e n z y m e in that they are activated b y G T P . G T P has no effect on the purified e n z y m e from Tetrahymena [7] a n d yeast [6]. W e now p r e s e n t evidence that in E. coli the effect of G T P a p p e a r s to be m e d i a t e d t h r o u g h lowering of the K m value for P Pdb-PP a n d an increase in Vmax (Table IV). A t present we can offer no e x p l a n a t i o n for the m o r e p r o n o u n c e d G T P effect in c r u d e extracts [3,4]. T h e r e m o v a l of the reaction p r o d u c t s from the e n z y m e which occurs in c r u d e extracts m a y be of i m p o r t a n c e ; however, we find no i n h i b i t i o n b y 0.1 m M PPi, a n d U M P o n l y p a r t i a l l y i n h i b i t e d the enzyme

(23% i n h i b i t i o n at 0.1 m M [4]). W e f o u n d that the s t i m u l a t o r y effect of G T P was p H d e p e n d e n t , an o b s e r v a t i o n which has also been m a d e for the effect of G T P on a d e n i n e p h o s p h o r i b o s y l t r a n s ferase of Mycoplasma [20]. T o g e t h e r with cytosine d e a m i n a s e , uracil p h o s p h o r i b o s y l t r a n s f e r a s e is involved in cytosine salvage [1,17,21]. T h e o b s e r v e d p a t t e r n of regulation of the genes coda a n d upp which respectively e n c o d e the two enzymes is different, i n d i c a t i n g s e p a r a t e r e g u l a t o r y control mechanisms.

Acknowledgements This investigation was s u p p o r t e d b y grants from the D a n i s h N a t u r a l Science R e s e a r c h Council. T h e a u t h o r s t h a n k Drs. K . F . Jensen a n d L.R. F i n c h for helpful advice d u r i n g the study.

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19 Jyssum, S. and Jyssum, K. (1979) J. Bacteriol. 138, 320-323 20 Sin, J.L. and Finch, L.R. (1972) J. Bacteriol. 112, 439-444 21 West, T.P.. Shanley, M.S. and O'Donovan, G.A. (1982) Biochim. Biophys. Acta 719, 251-258