The specificity of carboxyl group modification during the inactivation of the Escherichia coli F1-ATPase with dicyclohexyl[14C]carbodiimide.

When the Escherichia coli F1-ATPase (EFI) is inactivated with dicy~lohexyl[~~C]carbodiirnide and then gelfiltered, about 1 g atom of I4C is bound per mol of enzyme. However, only 45% of this radioactivity remained bound to the protein when the subunits were isolated in the presence of 8 M urea. The majority of the radioactivity which remained bound to the protein during subunit isolation was associated with the p subunit that contained 0.13 g atom of I4C per mol. A tryptic peptide derived from the labeled p subunit was isolated which contained  the  majority of the  radioactivity bound to it. Based on the amino acid composition of this peptide, the amino acid compositions of radioactive fragments derived from it, and the amino acid sequence of the /3 subunit of EFl translated from the gene (Saraste, M., Gay, N. J., Eberle, A., Runswick, M. J., and Walker, J. E. (1981) Nucleic Acids Res. 9,5287-5296 and Kanazawa, H., Kayano, T., Kiyasu, T., and Futai, M. (1982) Biochem. Biophys. Res. Commun. 105,1257-1264), it is concluded that the glutamic acid residue marked with an asterisk in the sequence Glu-Gly-Asn-Asp-PheTyr-His-Glu*-Met-Thr-Asp-Ser-Asn-Val-Ile-Asp-Lys is labeled when the enzyme is inactivated with dicyclohe~yl['~C]carbodiimide. The major radioactive cyanogen bromide peptide derived from the p subunit after inactivating the FI-ATPase from the thermophilic bacterium, PS3 (TFI), with dicyclohexyl['4C]carbodiimide, has been isolated. The amino acid sequences of the COOH-terminal tryptic fragments derived from this cyanogen bromide peptide have been determined. From this analysis it is now known that the amino acid sequence of the /3 subunit of TFI in the vicinity of the glutamic acid residue that reacts with dicyclohexylcarbodiimide is Ala-Gly-ValGly-m-Arg-Thr-Arg-Glu-Gly -Am-Asp-Leu-Tyr-His&-Met where Glu represents the N-y-glutamyl derivative of dicyclohexyl['4C]urea. Except for the fact that Glu is labeled in TFI rather than the glutamic acid


Tyr-His-Glu*-Met-Thr-Asp-Ser-Asn-Val-Ile-Asp-Lys
The soluble F,-ATPases from a variety of species are inactivated by DCCDI in reactions which are slowed in the presence of Mg" (1-5). Inactivat,ion of TFI, the F,-ATPase from the thermophilic bacterium, PS3, by ["CIDCCD has been correlated with the modification of a specific glutamic acid residues in the p subunit residing in the sequence Ala-Gly-Val-Gly-G-Arg where t.he underlined clu designates the labeled residue (4). Surprisingly, a different glutamic acid residue was labeled when MF,, the FI-ATPase from bovine heart mitochondria, was inactivated with ["CIDCCD (5). The The last three residues in the amino acid sequence of the i4Clabeled CNBr peptide from the / 3 subunit after inactivating MFI with ["C]DCCD were previously reported by us to be: . . . Glu*-His-Hse, which was based on the release of "C neai the end of the automatic Edman degradation of the peptide (5). Our conclusion was in error.
This was kindly pointed out to us by Dr. John E. Walker of the MRC Laboratory of Molecular Biology, Cambridge, England, who has completed the entire amino acid sequence of the / I subunit of MF, which will be published soon (personal communication). The sequence deduced in Dr. Walker's laboratory for these three amino acid residues is: Glu-His-Met. Moreover, his laboratory has also shown that the amino acid sequence, translated from the DNA sequence, in the homologous region of the / 3 subunit of EFI, is also Glu-His-Met (17). in the two ATPases, and the observed protection against DCCD inactivation provided by Mg2+ to both ATPases, led to the suggestion that the two Glu's highlighted in the bovine sequence shown above might be essential residues in all F1-ATPases. Both glutamic acid residues might function to bind the Mg" moiety of magnesium complexes of adenine nucleotides at the catalytic site. The amino acid sequence analyses presented here for the Fi-ATPases from Escherichia coli, and the thermophilic bacterium, PS3, support this contention. in a boiling water bath and then digested with trypsin known to be contaminated with chymotrypsin at a weight ratio of 509 for 20 h at 37 "C. The digests were then subjected to HPLC using the triethylamine:phosphoric acid:acetonitrile elution program described in detail previously (4). The radioactive profiles of the collected fractions were identical, showing a major, sharp peak with a retention time of 80 min corresponding to the peptide Ala-Gly-Val-Gly-Glu*-Arg where Glu* represents the labeled residue. To compare the effect of temperature on the specificity of labeling, TF1 was inactivated with 0. sharp radioactive peak appeared at 80 min with some small, less defined radioactive peaks appearing later in the elution profiles. Thus temperature variation and the presence and absence of ADP do not affect the specificity of labeling when TF, is inactivated with ['4C]DCCD. The results presented show that the radioactivity incorporated into EFI as a covalent label which survives the purification procedures is associated with the glutamic acid residue marked with an asterisk in Fig. 1. However, the yield of the radioactive tryptic fragment derived from the /3 subunit on which this assignment is made is surprisingly low when compared to the yields of labeled peptides obtained after inactivating TFI and MF1 with ['4C]DCCD.
Extrapolating the values for 14C incorporation in Table I of the miniprint to 100% inactivation, 1.12 g atoms of 14C per mol of EF, were incorporated which is similar to the value reported by Satre et al.
On the other hand, TFi and MF, were labeled to a greater extent with 2.15and 2.70-g atoms of 'Y! per mol being incorporated into the two enzymes, respectively, after removing excess radioactive reagent by gel filtration under nondenaturing conditions. Structural analyses of TF, and MF, after inactivation showed that most of the radioactivity incorporated into these ATPases is due primarily to the reaction of single glutamic acid residue in the /3 subunit of each with [14C]DCCD (4,5). While none of the '*C bound initially was removed from TF1 and MFi during the subsequent isolation of subunits in the presence of 8 M urea, only 45% of the '*C remained bound to EF1 under denaturing conditions. These results suggest that the O-acylisourea formed initially during the inactivation of EFI rearranges more slowly than do the 0-acylisoureas formed initially during the inactivation of TF, and MFI. If this is indeed the case, then about 50% of the 14C which remains bound to inactivated EF, after gel filtration under nondenaturing conditions might exist as an 0-acylisourea which decomposes on subsequent denaturation.
The low incorporation of 14C which is observed during the inactivation of EF1 when compared to that observed during the inactivations of TFI and MF1 might be due to the attack of the more stable 0-acylisourea purported to be present in EF, by a neighboring nucleophile. Such a reaction would produce an inactive derivative of the enzyme that would not be radioactive.
The mechanism by which 0-acylisoureas rearrange to form N-acylureas has not been examined directly. However, the rearrangement of isoimides (21) to the corresponding imides and the rearrangement of S-acylisothioureas to N-acylthioureas (22,23) have been examined in detail. By analogy with these rearrangements, Bruice and his colleagues have suggested that 0-acylisoureas rearrange intramolecularly by the attack of the lone pair of the imino nitrogen on the carbonyl carbon, as illustrated in Fig. 2 (22-24) The amino acid sequences shown in Fig. 1 contain invariant glutamic acid residues in the position labeled by [14C]DCCD in TF1 and the position labeled by ['4C]DCCD in MF1 and EFl. Since the rate of inactivation of aLl three F1-ATPases by DCCD is inhibited by MgZ+, it seems appropriate to assume that both carboxyl groups in each ATPase might function to bind the Mg' moiety of magnesium complexes of adenine nucleotides. However, it must be pointed out that Senior and his colleagues have provided strong evidence which indicates that sites other than the catalytic sites in the F,-ATPases also bind Mg2+ (25-27). Moreover, one of these sites, which has high affinity for Mg"+, may have a structural role. Therefore, a more detailed analysis will be necessary to determine the function of the glutamic acid side chains which, when modified by DCCD, lead to inactivation of the F1-ATPases.