Journal
JOURNAL OF PROTEIN CHEMISTRY
Volume 22, Issue 7-8, Pages 643-654Publisher
SPRINGER/PLENUM PUBLISHERS
DOI: 10.1023/B:JOPC.0000008729.20730.59
Keywords
chemical modification; differential scanning calorimetry; H-1 NMR; histidyl residues; RNase A; isoelectric pH gradient
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Funding
- NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES [R01AR045599] Funding Source: NIH RePORTER
- NIAMS NIH HHS [AR45599] Funding Source: Medline
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The histidyl residues of bovine pancreatic ribonuclease A (RNase A) play a crucial role in enzymatic activity. Diethylpyrocarbonate (DEPC) is a potent inhibitor of RNase A, and its precise sites of action on the imidazole rings of the four histidyl residues of RNase A are not clearly defined. We have used a multidisciplinary approach including enzyme assay, calculation of accessible surface area (ASA), isoelectric pH gradient technique, fluorescence investigations, circular dichroism spectroscopy, differential scanning calorimetry, and H-1 NMR analysis to study the sites of DEPC interaction with the imidazole rings of the four histidyl residues. Our results demonstrate that among the histidyl residues of RNase A, His48 is not accessible to react with DEPC. However, the sequential carbethoxylation of the imidazole rings of His119, His105, and His12 occurs on the nitrogen atoms of N-delta, N-epsilon, and N-epsilon, respectively. Carbethoxylation of His119 was followed by conversion of the A conformation to the B conformation in the active site. However, the carbethoxylation of His12 was accompanied by a second spatial rotation of the corresponding imidazole ring in the active site to adopt a new conformation. These conformation changes are accompanied by subsequent decrements in the thermal stability of the protein. Therefore, these findings reinforce the important structural roles of the spatial positions for His119 and His12 in the active site of RNase A.
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