Selective chemical modification of Escherichia coli elongation factor G. N-Ethylmaleimide modification of a cysteine essential for nucleotide binding.

Escherichia coli Elongation Factor G is inhibited ireversibly by the chemical modification of 1 cysteine residue with N-ethylmaleimide. At pH 5.2, this cysteine is approximately 130 times more reactive than beta-mercaptoethanol toward N-ethylmaleimide. Inhibition is not prevented by either the ribosome or GTP alone at concentrations approximately equal to that of Elongation Factor G, but in combination they reduce the inhibition by 50%. Increasing the stability of the Elongation Factor G-ribosome-GDP complex by the addition of fusidec acid, completely protects against N-ethylmaleimide inhibition. The modified protein cannot form either the Elongation Factor G-ribosome-GMP-P(CH2)P or the Elongation Factor G-ribosome-GDP-fusidic acidcomplex. However, the modification had no effect on its ability to form the Elongation Factor G-ribosome complex. These results suggest that the cysteine residue modified by N-ethylmaleimide is at or near the nucleotide binding site.

cysteine residue modified by N-ethylmaleimide is at or near the nucleotide binding site.
Elongation Factor G interacts with the ribosome and GTP to catalyze the translocation step of the protein synthesis elongation cycle (2). While the mechanism of action of this protein has been investigated in a number of laboratories, the chemical features of the protein which are essential for its function remain undefined.
Since shortly after its discovery, EF-G' has been assumed to contain 1 or more essential cysteine residues. This conclusion was based on the observations that its participation data. Division of the P-mercaptoethanol/EF-G ratio at which 50% maximal inhibition occurs by the molar excess of MalNEt yields the relative reactivity. In this way, at pH 5.2, EF-G was calculated to be 130 times more reactive toward MalNEt than was B-mercaptoethanol. Identification of Modified Residue-In order to identify the amino acid residue in EF-G modified by MalNEt a sample of [2,3-"C]MalNEt-inactivated protein was hydrolyzed in uacuo at 110" for 72 hours with 6 N HCl. The hydrolysate was chromatographed on a Beckman model 119 amino acid analyzer. The effluent was collected and fractions were monitored for radioactivity.
A standard sample of S-(ethyl[2,3-"C]succinimido)cysteine prepared by the method of Fruton et al. (9) was hydrolyzed and chromatographed in the same manner. Seventy-two hours of hydrolysis were employed to ensure complete conversion of the S-(N-ethylsuccinimido)cysteine to S-(succinyl)cysteine (10). The radioactivity derived from the "C-labeled MalNEtinactivated protein eluted in a single peak at 42 ml. No radioactivity was observed in any other position of the chromatography. The "C-labeled S(succinyl)cysteine also eluted at 42 ml, indicating that cysteine is the amino acid modified in EF-G.
Protection against MalNEt Inhibition-The ability of the ribosome, GTP, and fusidic acid, either individually or in combination, to protect EF-G against MalNEt inhibition was examined as described under "Methods." As shown in Table I, significant protection against inhibition was observed under these conditions only when both the ribosome and GTP were present. In the absence of fusidic acid the EF-G 'ribosome. GDP complex is relatively labile and only 50% protection was observed. Fusidic acid is known to increase the stability of this complex (ll), and addition of the antibiotic resulted in essentially complete protection against inhibition.
These results indicate that the modification occurred at or near one of the binding sites of one of these molecules. The site containing the modified cysteine was identified as described below.
Site of MalNEt Modification-The activity measurement employed in this investigation was based on the formation and quantification of the EF-G.ribosome.
[3H]GDP.fusidic acid complex. This complex can be formed by EF-G and the ribosome in the presence of fusidic acid by a single round of hydrolysis of [$H]GTP (12). Modification of EF-G which would prevent either the binding of ribosomes, nucleotide, or fusidic acid, or the hydrolysis of GTP to GDP would result in the observed inhibition.
In order to determine which of these sites contained the modified cysteine, the ability of the MalNEtinhibited EF-G to participate in the complexes listed in Table  II was examined. The substitution of [$H]GDP for [SH]GTP in the assay obviates the requirement for hydrolysis prior to complex formation. As shown in the table, the same degree of inhibition was observed when either [9H]GTP.or [SH]GDP was employed. This would suggest but not prove that inhibition was not due to the modification of a catalytically essential residue.
The ability of the modified enzyme to form the EF-G.ribosome.
[SH]GMP-P(CH,)P and [SH]EF-G.ribosome complexes can be used to define which of the three binding sites contains the modified residue. The formation of the ['H]EF-G.ribosome complex requires neither nucleotide nor fusidic acid, while the EF-G.ribosome.
[SH]GMP-P(CH,)P complex does not require fusidic acid. Only the formation of the ['HI EF-G.ribosome complex is unaffected by MalNEt modification suggesting that inhibition is due to the inability of the modified enzyme to participate in the binding of nucleotide.

DISCUSSION
The modification of EF-G with MalNEt was complicated by the required presence of @mercaptoethanol in at least a 1500.fold excess over EF-G. In spite of this large excess of fl-mercaptoethanol, inhibition of EF-G was observed at low molar excesses of N-ethylmaleimide, indicating that the protein cysteine was more reactive toward N-ethylmaleimide than was fi-mercaptoethanol.
Although rapid inhibition was observed under these reaction conditions, approximately 40% of the EF-G appeared to be refractory to MalNEt inhibition. The subsequent observation that preincubation of the protein in high concentrations of /3-mercaptoethanol converted the MalNEt-insensitive fraction to an MalNEt-sensitive form parallels the results of a previous study of the effects of air oxidation on activity in which we reported that air oxidation led to inactivation which could be reversed by the addition of fl-mercaptoethanol (4). In fact, reactivation was observed during activity measurements due to the high P-mercaptoethanol/EF-G ratio in the assay. It is likely that the MalNEt-insensitive EF-G is protein which had oxidized upon storage, thereby making the active cysteine unavailable for modification.
During the assay for activity, this oxidized EF-G is reduced giving rise to the observation that a fraction of the EF-G appears to be both fully active and MalNEt-insensitive.
This hypothesis was substantiated further by the observation that the per cent of MalNEt-insensitive EF-G is a function of the length of storage. Preparations of EF-G which were stored for a shorter time then that used in The presence of the unreactive fraction of EF-G had no effect on either the outcome or the interpretation of experiments reported here other than limiting the maximal level of inhibition of 60 to 70%. However, the observation that EF-G tends to undergo a reversible oxidation upon storage may have significant effects on the selective modification of other residues at the active site. It is possible that due to this oxidation, normally accessible residues near the cysteine may become inaccessible to reagent. This limitation will have to be born in mind in subsequent modifications of the active site (or sites) of EF-G.
The relative reactivity of the cysteine residue modified by MalNEt exhibited an unusual pH dependence; its relative reactivity increased as the pH was lowered from neutrality of pH 6.5. Since the reaction with MalNEt occurs via nucleophilic substitution, the unprotonated form of cysteine is the more reactive species. This pH dependence suggests that the reactive cysteine has a lowered pK,.
In order to draw meaningful conclusions about the nature of the active center of a protein from chemical modification studies, two criteria have to be fulfilled: (a) the modification must be selective, e.g. a linear relationship must exist between inhibition and incorporation of the modifying reagent; (b) modification must occur at or near the active center. The modification of EF-G with MalNEt described here fulfills both of these requirements. Incorporation of radiolabeled MalNEt parallels the observed inhibition. Extrapolation to 100% inactivation indicates that 0.97 mol of MalNEt would be incorporated per mol of EF-G.
Protection against inhibition by equal molar concentrations of GTP and ribosomes indicate that the reactive cysteine is located at or near the active center of the protein. In its broadest terms, this active center contains sites for the sM. S. Rohrbach and J. W. Bodley, unpubl i shed observation.
interaction with the ribosome, nucleotide, and fusidic acid. The identification of the site containing the modified cysteine was elucidated from the ability of the modified protein to form the EF-G.ribosome.GDP.fusidic acid, EF-G.ribosome.GMP-P(CH,)P, and EF-G.ribosome complexes. The observation that the MalNEt-modified EF-G could not form the first two complexes but was fully competent in the formation of the third indicated that the modified cysteine was located at the nucleotide binding site.
These results represent the first functional assignment of an amino acid residue in EF-G. It is interesting to note that EF-Tu also contains a reactive cysteine at its nucleotide binding site (14). It may be that this is a general characteristic of the nucleotide binding sites of the protein synthesis factors.