THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 17, Issue of June 15, pp. S204-8209,1988 Printed in U.S.A.

Conservative Replacement of Methionine by Norleucine in Escherichia coli Adenylate Kinase*

(Received for publication, October 22, 1987)

Anne-Marie GillesS, Philippe Marliereg, Thierry Rosel, Robert Sarfati(1, Robert Longin**, Alain Meier**, Serge FermandjianSS, Monique MonnotSS, Georges N. Coheng, and Octavian BiirzulQQ From the $Unite de Chimie des Proteines, §Unite de Bwchimie Cellulaire, 7Unite de Biochimie des Regulations Cellulaires, II Unite de Chimie Organbe, and **Unite de Phvswlonie Cellulaire, Znstitut Pasteur, 75724 Paris and the StLuboratoire de Biochimie et EnzymoLgie; Zktitut Gustaue Rowiy, 94805

Escherichia coli grown in limited methionine and excess norleucine media accumulate cyanogen bro- mide-resistant species of proteins after the methionine supply is exhausted. Bacteria, transformed by recom- binant plasmid pIPD37 carrying the adk gene and grown under limiting methionine and excess norleu- cine, synthesize 16-20% of adenylate kinase molecules having all 6 methionine residues replaced by norleu- cine. Species showing only partial replacement of me- thionine residues by norleucine are identified by so- dium dodecyl sulfate-polyacrylamide gel electropho- resis after cyanogen bromide treatment of pure en- zyme.

Norleucine-substituted adenylate kinase shows structural and catalytic properties similar to the wild- type protein as indicated by circular dichroism spec- troscopy and kinetic experiments but exhibits a much higher resistance to hydrogen peroxide inactivation under denaturing conditions.

In vivo replacement of naturally occurring amino acids by their analogs offers a convenient and powerful tool for probing the effect of molecular alterations on the biological activity of proteins (1). The methionine analog norleucine is known to increase the culture yield of methionine auxotroph strains of Escherichia coli (2,3), although it cannot alone sustain the growth of such auxotrophs (3), as does selenomethionine (4). Sterically superimposable to methionine, norleucine lacks a sulfur atom and is thus unable to ensure the metabolic func- tions of methionine, via the formation of S-adenosylmethio- nine. On the other hand, norleucine-containing proteins re- semble their wild-type counterparts, as demonstrated for E. coli @-galactosidase (5) and for staphylococcal nuclease (6).

In search of new methods for obtaining complementary fragments of E. coli adenylate kinase able to reconstitute catalytically active species (7, €9, we investigated the effect of in vivo substitution of methionine residues by norleucine. Since norleucine is incorporated randomly in place of methi- onine (3), we expected a large variety of adenylate kinase species containing from 1 to 6 methionine residues replaced

* This work was supported by Grants UA 1129 and ATP 955 492 from the Centre National de la Recherche Scientifique. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed Unit6 de Bio- chimie de RBgulations Cellulaires, Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France.

Villejuif,. France

by norleucine. Some of these species might be easily identified by resistance to CNBr’ cleavage and tested for their catalytic properties or their ability to interact with nucleotides.

In this paper we show that E. coli transformed by recom- binant plasmid pIPD37 carrying the adk gene and grown in the presence of an excess of DL-norleucine (1.5 X M) over L-methionine (2 X lo-’ M) synthesizes a large proportion (between 16 and 20%) of active adenylate kinase which is insensitive to CNBr cleavage. We also examined the conse- quences of this amino acid replacement on the stability of adenylate kinase in the presence of chemical reagents such as oxidants.

EXPERIMENTAL PROCEDURES

Chemicak-Adenine nucleotides, substrates, and coupling enzymes were from Boehringer Mannheim (a generous gift of Professor F. H. Schmidt (University of Heidelberg)). TPCK-trypsin and soybean trypsin inhibitor were from Sigma. Blue-Sepharose was obtained from Pharmacia LKB Biotechnologies Inc. Urea (fluorimetrically pure) was from Schwarz/Mann. [azP]H3POl (carrier-free) and ~ - [ ~ ~ S ] m e - thionine (1300 Ci/mmol) were purchased from the Radiochemical Centre (Amersham, United Kingdom). BQATP was synthesized as described by Williams and Coleman (9). [ya2P]BzATP was prepared according to Glynn and Chappell(l0). The identity of the compound was checked by thin layer chromatography on cellulose or polyethyl- ene imine-cellulose, from its absorption spectrum (6% = 41) (11) and substrate capacity for yeast phosphoglycerate kinase (good) and hex- okinase (poor). Photoreactivity was ascertained as described by Wil- liams and Coleman (9).

Bacterial Strains and Culture Conditwns-The strain POPX used in this work is a derivative of Hfr P4X8 bearing a metB mutation from the Pasteur Institute collection (12). The methionine auxotro- phy conferred by this mutation does not revert in standard genetic assays (13) and was not analyzed further. Following conventional procedures (14), strain POPX was transformed with plasmid pIPD37 (kindly provided by Dr. Isabelle Saint Girons (Pasteur Institute, Paris)), a derivative of pBR322 carrying the E. coli adk gene (7) yielding strain PMX.

The strain PMX was grown in a 20-liter Chemap fermenter filled with 15 liters of synthetic medium (pH 7) having the following composition (g/liter): KHzPOI, 6; KzHPOI, 18.3; (NHI)zSOI, 4; MgS04. 7H20, 0.4; FeSO+. 7H20, 5 X 1O-I; glycerol, 8; ampicillin, 0.1; L-methionine, 3 X (2 X M); DL-norleucine, 0.2 (1.5 X M). The culture was heated at 37 “C and oxygenated with an air flow of 10 liters/min under vigorous agitation. When a predetermined value of turbidity was attained, a fraction (0.1-3 liters) of the culture was withdrawn and immediately centrifuged at 5,000 X g for 30 min at 4 “C. The resulting pellet was washed and resuspended either with 50 mM Tris-HC1 (pH 7.4) or with 50 mM ammonium bicarbonate (pH 7.8) and disrupted by sonication at 20 KHz and 100 watts (3 X 4

The abbreviations used are: CNBr, cyanogen bromide; BzATP, 3’-0-(4-benzoyl)benzoyl adenosine 5”triphosphate; SDS, sodium do- decyl sulfate; TPCK, L-1-tosylamido-2-phenyl-ethylchloromethyl ke- tone; Blue-Sepharose, Cibacron Blue 3G-A Sepharose CL-GB.

8204

Norleucine-containing Adenylate Kinase of E. coli 8205 min). Cell debris were removed by centrifugation at 13,000 X g for 20 min at 4 ‘C.

Purification of Adenylate Kinuse and Assay of Enzymatic Actiuity- Adenylate kinase from the overproducing strain was purified as described previously (7, 15), by a two-step procedure involving chro- matography on Blue-Sepharose and gel permeation chromatography on Ultrogel AcA 54. After extensive dialysis against several changes of 50 mM ammonium bicarbonate, the enzyme solution was lyophi- lized and was cyanylated according to Jacobson et al. (16). The cyanylated protein was cleaved and the resulting peptides (C1, resi- dues 1-76; Cf, residues 77-214) purified under denaturing conditions as described previously (7). Thermosensitive adenylate kinase from E. coli strain CR341T28 was purified according to Gilles et al. (17). Adenylate kinase activity was determined a t 27 “C in a final volume of 1 ml using a spectrophotometric assay system both in the direction of ATP (forward reaction) and of ADP (reverse reaction) formation (7). One unit of enzyme activity corresponds to 1 pmol of product formed/min.

Chemical Cleavage of Adenylate Kinase with Cyanogen Bromide- Pure adenylate kinase or E. coli extracts in ammonium bicarbonate were lyophilized, then dissolved in a solution of 2.5% CNBr in 70% formic acid (200 pl for each milligram of protein). After 24 h of incubation at room temperature in the dark, samples were diluted 5- fold with twice distilled water and lyophilized.

Circular Dichroism Measurements-The CD spectra were recorded with a Jobin Yvon Mark IV dichrograph connected to a Digital microcomputer. Enzyme was solubilized in 5 mM ammonium bicar- bonate (pH 7.8). Measurements were performed in quartz optical cylindrical cells with 0.1-cm path length, for the spectral range from 260 to 185 nm. Results are expressed in mean residue molar ellipticity, ( O ) , in degrees.cm2.dmol”. For estimation of secondary structure, CD curves in the 190-260 nm range were processed by the method of Chen et al. (18).

Analytical Procedures-Proteins were measured according to Brad- ford (19) with purified adenylate kinase (A:% = 5.0) as the calibration standard. Amino acid analysis after hydrolysis of proteins a t 110 “C with 6 N HCI for 24 h was performed on a Biotronik LC 5001 amino acid analyzer. Equilibrium dialysis experiments and SDS-polyacryl- amide gel electrophoresis were performed as described in previous publications (7, 15, 17, 20).

RESULTS

Cyanogen Bromide-resistant Species of Adenylate Kinase Accumulate after Methionine Depletion in the Growth Me- dium-Preliminary experiments with methionine auxotrophs grown on limiting L-methionine (3 to 5 X M) showed that growth ceased when the methionine supply was exhausted. With 30-50-fold molar excess DL-norleucine over L-methio- nine, there was an additional growth of bacteria corresponding to about a doubling in mass, in agreement with data of Cowie et al. (3) and Barker and Bruton (2) obtained’ under similar experimental conditions.

Adenylate kinase specific activity, determined in cell ex- tracts of E. coli harvested a t different phases of growth in limited methionine and excess norleucine media, shows little variation within the limit of experimental errors. However, the species resistant to CNBr accumulate only in the late phase of growth. In bacteria from the stationary phase, be- tween 16 and 20% of total adenylate kinase activity was found resistant to CNBr cleavage (Fig. hi). Since enzyme expressed by the pIPD37 plasmid represents 12-15% of total E. coli proteins, SDS-polyacrylamide gel electrophoresis of crude extracts before and after treatment with CNBr showed accu- mulation of those species having presumably all methionine residues replaced by norleucine (Fig. 1B). Gel analysis was in good agreement with adenylate kinase activity assays.

Amino Acid Composition of Adenylute Kinase Purified a t Different Stages of Growth in Excess Norleucine Medium- During the early phase of growth, E. coli accumulated mainly methionine-containing species of proteins, so that only 5% of the methionine residues were substituted by norleucine in purified preparations of adenylate kinase (Table I), At the

r A B 45 116 R U

Y 52’3.

S A B C D F I G ““1 ” - L 2

0 1 2 3 4 5 6 7 T I M E (HOURS)

FIG. 1. Synthesis of CNBr-resistant species of adenylate kinase during growth of E. coli in restricted methionine and excess norleucine medium. A, bacteria grown in minimal medium supplemented with glycerol ampicillin, L-methionine, and DL-norleu- cine as described under “Experimental Procedures” were harvested at different phases of growth (indicated by the arrow), disrupted by sonication in 50 mM ammonium bicarbonate, then assayed for ade- nylate kinase activity. Numbers on the left side of arrows indicate the adenylate kinase specific activity in sonicated extracts (pmol of ATP formed/min/mg of protein). Duplicate samples from each extract corresponding to 2 mg of protein were treated with CNBr in formic acid, lyophilized, then treated with 1 ml of 6 M urea in 50 mM Tris. HCI (pH 7.4). Samples of 1-5 pl were used for measurement of enzyme activity in the direction of ATP formation. The renaturation yield of adenylate kinase from urea treated samples varied between 70 and 95%. Numbers on the right side of arrows indicate the per- centage of adenylate kinase activity recovered after CNBr treatment taking controls (samples treated with formic acid in the absence of CNBr) as 100%. B, extracts (about 100 pg of protein) in 50 mM ammonium bicarbonate from bacteria harvested at different phases of growth and corresponding to an optical density a t 570 nm of 0.18 (lanes A and C) , 0.3 (lane D) , 0.4 (lane E ) , 0.6 (lane F ) , and 0.7 (lanes B and C) were treated (lanes C-G) or not (lanes A and B ) with CNBr, then examined on a 12.5% SDS-polyacrylamide gel and stained with Coomassie Blue. Lane S, standard proteins, from top to bottom: a, phosphorylase a (94,000); b, bovine serum albumin (67,000); c, oval- bumin (43,000); d, carbonic anhydrase (30,000); e, soybean trypsin inhibitor (20,100); f , lysozyme (14,000). Arrows situated on the right side of the figure indicate the position of adenylate kinase and of peptide corresponding to residues 97-214 (resistant to CNBr cleavage since its Met”‘ is followed by a Thr residue).

stationary phase, half of the methionine residues of adenylate kinase were found replaced by norleucine. This suggests that only adenylate kinase species having all of the 6 Met residues replaced by norleucine (with the possible exception of the N- terminal Met) remain active after CNBr treatment. Fig. 2A shows the electrophoretic pattern of adenylate kinase purified from bacteria grown in presence of an excess of norleucine and cleaved with CNBr. Adenylate kinase treated with CNBr yielded several polypeptide bands in SDS-polyacrylamide gel electrophoresis. The dominant bands corresponded to the uncleaved enzyme and to a polypeptide fragment 97-214. The other peptide fragments, 22-214, 35-214, and 54-214, could be identified according to their molecular weights. Under identical experimental conditions, methionine-containing ad- enylate kinase is completely cleaved, with no detectable resid- ual enzymatic activity. The 97-214 peptide formed again the major cleavage product. This peptide results from an incom- plete cleavage since it contains a methionine residue (Metli4) followed by a threonine residue, which makes this particular peptide bond resistant to CNBr digestion (21).

Susceptibility to Trypsin and BzATP Photolabeling of E. coli Adenylate Kinase and of Peptides Resulting from Incom- plete Cleavage with CNBr-Adenylate kinase isolated from bacteria grown in excess norleucine was first treated with CNBr, then subjected to trypsin proteolysis. CNBr-resistant

8206 Norleucine-containing Adenylate Kinase of E. coli TABLE I

Amino acid composition of E. coli adenylate kinase purified at different phases of growth in restricted methionine and excess

norleucine medium Adenylate kinase was purified from bacteria harvested a t different

phases of growth in media containing 2 X M L-methionine and 1.5 x lo-' M DL-norleucine, and corresponding to optical densities of 0.3 (A), 0.6 (B), and 0.7 (C, stationary phase). Numbers in column D correspond to adenylate kinase purified from bacteria in the station- ary phase of growth, treated with CNBr then with trypsin under protection with ATP. Uncleaved protein was separated from frag- ments by chromatography on Blue-Sepharose and elution with ATP + AMP. Column E indicates the amino acid composition of wild-type enzyme as deduced from the nucleotide sequence of the adk gene.

Amino Residues/molecule acid A B C D E

cys 1.1 1.4 1.3 1.2 1 Asx 21.6 21.8 21.7 22.0 21 Thr 9.8 9.7 10.5 9.7 11 Ser 4.6 4.6 4.8 4.5 5 Glx 26.0 25.6 25.9 25.8 26 Pro 8.9 8.7 10.5 8.1 10

Ala 19.0" 19.0 19.0 19.0 19 Val 17.9 18.1 19.4 18.0 19 Met 4.7 3.1 3.1 0.0 6 Ile 12.5 12.8 14.2 13.8 14 Leu 14.9 15.9 17.3 16.8 16 TY r 5.6 5.1 6.9 5.5 7 Phe 4.8 4.6 5.4 4.8 5 His 3.3 3.1 3.5 3.8 3 LY s 15.6 17.1 18.9 17.4 18 -4% 11.5 12.6 15.3 13.0 13 Trp NDb ND ND ND 0

G ~ Y 20.1 20.0 19.4 21.5 20

Norleucine 0.3 2.5 2.9 5.9 0 ' Arbitrarily taken as reference value for M, = 23,500.

Not determined.

species of adenylate kinase were also resistant to proteolysis by trypsin, especially when ATP was present in the incubation medium. All polypeptides resulting from partial cleavage of adenylate kinase with CNBr were rapidly proteolyzed by trypsin, irrespective of the presence or absence of ATP in the incubation medium (Fig. 2B). It has been observed previously that isolated C1 and CP peptides are completely digested by trypsin in less than 10 min at 30 "C, whereas reconstituted CICz protein shows a much higher resistance toward trypsin proteolysis (7).

Bz2ATP which acts as a substrate for E. coli adenylate kinase (7) is a suitable photoaffinity label for the enzyme.' Upon irradiation with long wave UV light, BzzATP effectively inactivates the adenylate kinase. ATP protects adenylate kinase against photoinactivation by Bz2ATP (not shown). The irreversible binding of [y-3'P]Bz2ATP to adenylate ki- nase was shown by SDS-polyacrylamide gel electrophoresis and autoradiography (Fig. 3). We wanted to know if the polypeptides resulting from incomplete CNBr cleavage of the norleucine-enriched adenylate kinase could be photolabeled by Bz2ATP. As shown in Fig. 3 (lane A), only the uncleaved protein incorporated [y-3'P]Bz2ATP, thus indicating that the peptide fragments do not adopt a conformation recognizing ATP or its analog. CICn-reconstituted adenylate kinase, which shows binding properties for adenine nucleotides and catalytic activity (7), incorporates radioactivity (more than 85% located on CP fragment) upon photoirradiation with [y-32P]Bz2ATP.

Isolation of Adenylate Kinase Species Containing Norleucine in Place of All Methionine Residues-As norleucine is ran-

' R. Sarfati, A.-M. Gilles, T. Rose, and 0. Birzu, manuscript in preparation.

A

a b

C

d adenvlate kinase

A B C D E S A B

FIG. 2. SDS-polyacrylamide gel (12.5%) analysis of CNBr peptides of adenylate kinase from E. coli grown in limiting methionine and excess norleucine medium. A, adenylate kinase, from bacteria grown until the stationary phase in norleucine-rich media, was purified according to standard procedures. 20 mg of pure protein were cleaved with CNBr (100 mg in 4 ml of 70% formic acid) for 24 h a t room temperature. A 25-pgsample of protein was examined electrophoretically (lane B ) in parallel with methionine-containing adenylate kinase previously cleaved with CNBr (lane A ) . S, the same standard proteins as those in Fig. lA. Arrows on the right side indicate the fragments of adenylate kinase resulting from incomplete replace- ment of methionine residues with norleucine. B, CNBr-treated ade- nylate kinase (1 mg/ml in 50 mM Tris.HC1 (pH 7.4)) purified from bacteria grown in medium with excess norleucine over methionine (lane A ) was digested with TPCK-trypsin (2 gg/ml) for 10 min (lanes B and D) or 20 min (lanes C and E ) a t 30 "C, in the absence (lones B and C) or in the presence (lanes D and E ) of 4 mM ATP. Further proteolysis was stopped with 5 pg/ml of soybean trypsin inhibitor then 25-pg samples of protein were examined by electrophoresis and stained with Coomassie Blue.

domly incorporated at methionine sites (3), it might seem difficult to isolate a homogenous population of molecules. However, in the particular case of adenylate kinase, all species having even a single methionine residue gave fragments highly sensitive to proteolysis by trypsin after an initial CNBr treat- ment (Fig. 2%). Intact molecules of adenylate kinase, pre- sumed to contain all 6 methionine residues replaced by nor- leucine and resistant to trypsin digestion, were thus purified by rechromatography on Blue-Sepharose. Amino acid analysis of adenylate kinase repurified after CNBr cleavage and tryp- sin treatment (column D in Table I) confirmed the total replacement of methionine by norleucine.

Structural and Catalytic Characteristics of Norleucine-con- taining Species of Adenylate Kinase from E. coli-The CD spectra of methionine-containing and norleucine-containing adenylate kinase are very similar (Fig. 4) showing that nor- leucine plays the same role as methionine in maintaining the intact secondary structure of the protein. The composition in the secondary structure of norleucine-containing adenylate kinase obtained by processing CD curves in the near UV gave 46% a-helix, 14% p-sheet, and 40% remainder, as compared to 50% a-helix, 15% &sheet, and 35% remainder for methio- nine-containing adenylate kinase.

Determination of the kinetic parameters of norleucine- containing adenylate kinase showed a 20% decrease in maxi- mal catalytic activity, (326 pmol/min. mg of protein in the forward reaction) whereas affinity for nucleotides was prac- tically unaffected. Thus, the apparent KiDP was 105 pM for

Norleucine-containing Adenylate Kinase of E. coli 8207

A B C

r)- "-aden late kinase . - 22 -!&4

-54-214 -35- 214

. a+Cc,(77-214) "97-214

c C 1 (1-76)

"front

FIG. 3. Identification of peptides labeled by photoactivation of [y-32P]Bz2ATP. Adenylate kinase purified from bacteria grown in norleucine-rich media was cleaved ( l a n e A ) or not ( l a n e B ) with CNBr. Then 50 p1 of enzyme solution a t 1 mg/ml in 50 mM Tris-HCI (pH 7.4), 50 mM KC1, 2 mM MgCI2, and 1 mM [y3'P]Bz2ATP (approximately 5000 cpm/nmol) were photolyzed for 30 min a t 0 "C employing the long wave UV emission from a UVSL 58 Mineralight at a distance of 5 cm from the sample. Following photolysis proteins were precipitated with trichloroacetic acid then analyzed on a 12.5% polyacrylamide gel under denaturing conditions. After Coomassie Blue staining for peptide identification, the gels were dried and exposed to autoradiography to Kodak X-Omat AR films for 6 h at -80 "C. Lane C corresponds to C1C2 reconstituted adenylate kinase photolabeled with [-p3'P]Bz2ATP in identical conditions.

190 21 0 2 3 0 A t n m l

2 5 0

FIG. 4. Mean residue molar ellipticity of wild-type (WT) and norleucine-containing (NL) adenylate kinase of E. coli in 5 mM ammonium bicarbonate (pH 7.8) at 20 "C. Spectra were recorded at a protein concentration of 1.3 p ~ .

norleucine-containing adenylate kinase, whereas methionine- containing enzyme had an apparent KhDp of 92 PM.

A characteristic of E. coli adenylate kinase is that AMP above 0.3 mM, but not Z'dAMP or other nucleoside mono-

phosphates, inhibits enzyme activity (22). This intriguing property can be interpreted as the result of competition be- tween excess AMP and ATP for the nucleotide donor site of adenylate kinase. However, the apparent KhTp determined at optimal (0.3 mM) and inhibitory concentrations (4 mM) of AMP does not reveal significant differences (48 and 54 p ~ , respectively). Norleucine-containing adenylate kinase shows the same sensitivity to inhibition by excess AMP (more than 50% inhibition a t 4 mM, taking the activity a t 0.3 mM AMP as 100%) (Fig. 5) . It is interesting to note that thermosensitive adenylate kinase (Pros7 + Ser) (17) is not inhibited by excess AMP, in agreement with data reported using partially purified enzyme preparations (22).

Hydrogen peroxide is known as a powerful methionine- oxidizing agent (23, 24). We thus examined its effect on methionine- and norleucine-containing adenylate kinase. In the presence of 0.1 M H202, under nondenaturing conditions, adenylate kinase activity remained unchanged for several hours at room temperature. In the presence of 6 M urea and 0.05 M H202, methionine-containing adenylate kinase was

A M P ( m M )

FIG. 5. Effect of AMP concentration on the reverse reaction catalyzed by E. coli adenylate kinase. The reaction medium contained at 1 ml final volume 50 mM Tris-HC1 (pH 7.4), 100 mM KC1,2 mM MgC12,l mM phosphoenolpyruvate, 0.1 mM NADH, 1 mM ATP, 5 units each of pyruvate kinase and lactate dehydrogenase and different concentrations of AMP. The reaction was started with wild- type (.), thermosensitive (V), or norleucine-containing (W) adenylate kinase.

I I I I I 5 10 15 20

TIME (MINI

FIG. 6. Time course of the effect of 0.06 M H202 on the activity of purified wild-type and norleucine-containing ad- enylate kinase of E. coli. Norleucine-containing (0) and wild-type (W) enzymes (20 pg/ml) were incubated a t 19 'C in the presence of fresh H202 solution, 0.1 M sodium borate (pH 8.8) and 6 M urea. A t the indicated times, reactions were quenched by dilution of enzyme into assay mixture containing 50 mM Tris-HCI (pH 7.41, 100 mM KCI, 1 mM glucose, 0.4 mM NADP+, 2 mM MgCl2, 1 mM ADP, and 3 units each of hexokinase and glucose-6-phosphate dehydrogenase. Residual activity is expressed as percent of nontreated enzyme con- trol.

8208 Norleucine-containing Adenylate Kinase of E. coli

90% inactivated within 20 min at 19 "C (Fig. 6). The kinetics of inactivation showed two distinct steps. The rapid phase of inactivation corresponded to methionine oxidation to its sulf- oxide, whereas the slow phase probably corresponded to oxi- dation of the cysteine residue. Under identical experimental conditions, norleucine-containing adenylate kinase was inac- tivated at a rate which corresponded to the slow phase inac- tivation of methionine-containing enzyme. When excess 8- mercaptoethanol was added to protein in Hz02-urea medium after complete inactivation, full enzyme activity was restored within several minutes both in the case of methionine- or norleucine-containing adenylate kinase.

DISCUSSION

Evidence has been accumulating from molecular genetics and protein engineering that proteins are largely impervious to point changes. Thus, the vast majority of @-galactosidase missense mutants show only a small loss of activity (25), and many residues at invariant sites of homologous proteins can be changed without causing much damage (26, 27). This robustness has been interpreted as a built-in anticipation of close sequence variants of a protein arising either by trans- lation errors (28) or by point mutations (27). When incorpo- rated into the bulk of cellular proteins amino acid analogs afford the phenotypic equivalent of many simultaneous mu- tations of a unique type. The fact that bacteria built with an artificial, albeit structurally conservative analog, remain via- ble and their proteins functional widens these current hy- potheses of protein robustness (27, 28).

Thus, norleucine-substituted adenylate kinase of E. coli shows catalytic and structural properties close or identical to the wild-type protein. Our results are consistent with earlier observations on E. coli 6-galactosidase and Staphylococcus aureus nuclease substituted with norleucine (5, 6), as well as on a-amylase of Bacillus subtills with ethionine (29).

When a methionine auxotroph strain of E. coli is grown in the presence of a restricted amount of methionine and an excess of norleucine, a final substitution rate of about 50% is attained. About one further doubling of the bacterial popula- tion takes place after methionine has been exhausted. The protein species synthesized during the last bacterial doubling following methionine exhaustion contains almost exclusively norleucyl residues at methionine sites. On the other hand, during the early phase of growth, norleucine does not enter the translation pathway because of the large bias of the Met- tRNA ligase toward methionine (30). In contrast, the methi- onine permease shows no marked preference for the natural amino acid (31).

After the growth completion of strain PMX in limited methionine and excess norleucine, a population representing about 20% of active adenylate kinase survives to CNBr treat- ment. Knowing that the overall substitution rate of methio- nine with norleucine is 50% (compare norleucine and methi- onine contents in Table I, column C), even if one (wrongly) assumes that the remaining 30% (i.e. 50-20%) of norleucine residues are randomly scattered within 80% (Le. 100-20%) of the protein chains, a substantial proportion of at least 12% (i.e. 100 x (1 - (0.5-0.2))6) of the chains should be devoid of norleucine. Therefore, one might envision each protein in the cells of such stationary cultures as an entire distribution of molecular species with defined sequences of methionines and norleucines, both ends of the spectrum, one free of the natural amino acid and the other free of the analog, being grossly over-represented.

The thioether group of methionine makes this amino acid susceptible to oxidizing agents, in particular to hydrogen

peroxide. In vivo incorporation of norleucine might thus help overcome the susceptibility inherent to some proteins that is due to the presence of a critical methionyl residue. In order to assess the validity of this notion, we subjected adenylate kinase to hydrogen peroxide treatment under denaturing con- ditions and showed that its stability toward this reagent increases dramatically when the enzyme contains norleucine instead of methionine. These results are consistent with a purely structural role for the 6 methionine residues of E. coli adenylate kinase. Kress and Noda (32) showed that the mod- ification of 2 to 3 methionyl residues of rabbit muscle adenyl- ate kinase with iodoacetic acid (the thiol groups being previ- ously blocked and later unblocked) was accompanied by com- plete loss of activity. However, a conclusion was not drawn as to whether derivatization had directly affected the catalytic mechanism or had modified the conformation.

In .light of the data presented above, we favor the idea that phenotypic substitution of methionine by norleucine might conveniently overcome the need to engineer this particular residue in oxidation-prone proteins (24, 33). Indeed, the in- sertion of a single leucine or valine (taken as conservative choices) at one methionine site of a protein can result in a decreased activity (24, 33) whereas the replacement of all methionine residues by norleucine seems to maintain the catalytic properties of enzymes (5, 6). Amino acid analogs have hitherto mainly been used for selecting mutants with enhanced defense mechanisms toward them (34). In the future they will perhaps serve more constructive goals like tuning the activity or improving the stability of certain proteins, without resorting to directed mutagenesis.

Acknowledgments-We thank S. Michelson for carefully reading this manuscript and M. Ferrand for expert secretarial help.

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