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  • Given that inactivation of GLO by BAB is an active

    2021-09-14

    Given that inactivation of GLO1 by 4BAB is an active site directed mechanism, as the transition state analogue CHG protects GLO1 against inactivation by 4BAB (Figs. 2D and S6b), we next set out to determine the amino Pramipexole that is covalently modified by 4BAB. From the X-ray crystallographic structures1, 2 Cys60 is located in the hydrophobic binding pocket and is the most likely candidate to act as the nucleophile to displace the bromine on 4BAB. To test this hypothesis, the GLO1:4BAB complex was digested with trypsin and analyzed by LC–MS/MS and based on the in silico tryptic peptides predicted for GLO1, Cys60 would lie on a 7 amino acid long peptide (C60-D-F-P-I-M-K66, C60-K66). Complete disappearance of the 853.4 m/z ion (corresponding to the singly charged C60-K66 peptide) was not observed, consistent with incomplete inactivation of the enzyme, leaving a fraction of the Cys60 residues unmodified. There should also be a new peak present in the chromatograms for the GLO1:4BAB trypsin solution, corresponding to a modified C60-K66 peptide. Modification of the C60-K66 peptide by 4BAB should produce a singly charged m/z value of 1330.5. However, no new peaks corresponding to the singly or multiply charged ions consistent with this modification were present in the GLO1:4BAB trypsin digests. Unexpectedly, the intact inactivator was not observed on the C60-K66 peptide, and therefore we set out to explore the possible reasons for the absence of the modified C60-K66 peptide in the chromatograms. One possible explanation could be a missed cleavage at Lys59 resulting from increased steric hindrance around this residue, possibly making it inaccessible to trypsin, but this does not seem to be the case since there is no ion present in the chromatographs consistent with modification of the V51-K66 peptide. Another possible explanation could be that the ester functions of the bound 4BAB are hydrolyzed during trypsin digestion, as trypsin has been shown to possess esterase activity towards benzoyl-l-arginine ethyl ester (BAEE) and other l-arginine esters.48, 49 We predicted the possible species (Table S2) that would arise from hydrolysis of these esters, assuming that C60-K66 is the peptide modified, and checked the chromatograms for the existence of these ions. Monitoring the extracted ion currents of these ions showed the presence of an additional peak in the GLO1:4BAB tryptic digest with an elution time of 57min that was not present in the control mixture (Fig. 3C), and this new peak was due to the presence of a singly charged ion with m/z of 911.4 (Fig. 3D). This mass is consistent with the C60-K66 peptide plus the addition of a carboxymethyl group (58massunits), one of the predicted species resulting from the hydrolysis of the ester bonds of 4BAB (C60-K66CM, Table S2). LC–MS/MS analysis confirmed that the modification was localized to either Cys60 or Asp61, both of which could act as nucleophiles to displace the bromine. First, the mass increase of 58 units of the b4 ion in Fig. 4B indicates that the modification must be somewhere on peptide fragment C60-D-F-P63. Second, the fact that the y5 fragment is unchanged in Figure 4B means that residues F62-P-I-M-K66 are excluded as possibilities. This leaves only residues Cys60 and Asp61 as the possible sites of modification. Since low mass fragments in the MS2 spectra were not detected, localization of the modification site had to be achieved through alternative methods. The presence of a carboxymethyl group on either Cys60 or Asp61 would yield two structurally different peptides with similar but different chemical properties (referred to as C60-K66CMCys if modification is at Cys60 and C60-K66CMAsp if modification is at Asp61, Fig. S8). Carboxymethylation of Cys60 results in a peptide with three free carboxylic acid functions and carboxymethylation of Asp61 results in only two free carboxylic acid functions. Therefore, treatment of the purified C60-K66CM peptide with ethanol and propanol in the presence of concentrated H2SO4 should result in esterification of the free carboxylic acid residues on the peptide through a traditional Fisher esterification mechanism. The m/z values of the mono- and di-esterified C60-K66CM peptide would be the same if either Cys60 or Asp61 were carboxymethylated (Table S3). The m/z values for the peaks eluting at 87 and 95min (Fig. 5, Table S3) for the ethanol and propanol esterification, respectively, are consistent with the tri-esterified C60-K66CM peptide, which could only arise if the carboxymethylation occurs on Cys60. The MS/MS data presented in Figure 4 and the esterification data presented in Figure 5 confirms that Cys60 is the site of covalent modification between GLO1 and 4BAB.