PubMed · 8065655
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S L Carter. 1994. Screening tests for cervical abnormalities.. https://doi.org/10.1097/00006205-199406000-00004
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The specific interaction of the upstream element-containing promoter of the Escherichia coli acetate operon with either the RNA polymerase holoenzyme or its alpha subunit has been analyzed by the base removal method. Our results indicate that: (i) direct and specific base contacts can be detected in the acetate promoter-alpha subunit complex; (ii) base elimination in the upstream element of the acetate promoter enhances the binding of RNA polymerase. A similar effect is observed when studying the interactions between RNA polymerase and the rrnB ribosomal operon P1 promoter.
Asp10 of L-2-haloacid dehalogenase from Pseudomonas sp. YL was proposed to act as a nucleophile to attack the alpha-carbon of L-2-haloalkanoic acids to form an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon (Liu, J.-Q, Kurihara, T., Miyagi, M., Esaki, N., and Soda, K. (1995) J. Biol. Chem. 270, 18309-18312). We have found that the enzyme is paracatalytically inactivated by hydroxylamine in the presence of the substrates monochloroacetate and L-2-chloropropionate. Ion spray mass spectrometry demonstrated that the molecular mass of the enzyme inactivated by hydroxylamine during the dechlorination of monochloroacetate is about 74 Da greater than that of the native enzyme. To determine the increase of the molecular mass more precisely, we digested the inactivated enzyme with lysyl endopeptidase and measured the molecular masses of the peptide fragments. The molecular mass of the hexapeptide Gly6-Lys11 was shown to increase by 73 Da. Tandem mass spectrometric analysis of this peptide revealed that the increase is due to a modification of Asp10. When the enzyme was paracatalytically inactivated by hydroxylamine during the dechlorination of L-2-chloropropionate, the molecular mass of the hexapeptide was 87 Da higher. Hydroxylamine is proposed to attack the carbonyl carbon of the ester intermediate and form a stable aspartate beta-hydroxamate carboxyalkyl ester residue in the inactivated enzyme.
Specific non-covalent interactions between aldose reductase (AR), its NADP+ cofactor and five inhibitors have been characterized by electrospray mass spectrometry (ES-MS). These results indicated that the protein could be desorbed and maintained in the gas phase in a form very close to its native conformation. Collisionally induced dissociation (CID)-MS and CID-MS-MS showed that the adenosine diphosphate part of the cofactor interacts strongly with AR. The relative stability of the ternary AR x NADP+ x inhibitor complexes was established and successfully correlated with the IC50 values. All inhibitors were shown to only bind to AR holoenzyme. These results are important for the field of drug development insofar as ES-MS might provide a rapid and very sensitive method for the screening of potential drugs or for the identification of compounds displaying high binding affinity to a target biomolecule.