The structural and functional studies of His119 and His12 in RNase A via chemical modification.
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 1H 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 Ndelta, Nepsilon, and Nepsilon, 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.
Safarian
Shahrokh
S
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
Moosavi-Movahedi
Ali Akbar
AA
Hosseinkhani
Saman
S
Xia
ZhiCheng
Z
Habibi-Rezaei
Mehran
M
Hosseini
Ghasem
G
Sorenson
Christine
C
Sheibani
Nader
N
D000818:Animals; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry; D007525:Isoelectric Focusing; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature
D004791:Enzyme Inhibitors; D007093:Imidazoles; D011994:Recombinant Proteins; D006639:Histidine; D012259:Ribonuclease, Pancreatic; D004047:Diethyl Pyrocarbonate