期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 279, 期 18, 页码 18711-18716出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M314271200
关键词
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资金
- NCI NIH HHS [R01 CA075954-07, CA75954, R01 CA075954] Funding Source: Medline
Hint, histidine triad nucleotide-binding protein, is a universally conserved enzyme that hydrolyzes AMP linked to lysine and, in yeast, functions as a positive regulator of the RNA polymerase II C-terminal domain kinase, Kin28. To explore the biochemical and structural bases for the adenosine phosphoramidate hydrolase activity of rabbit Hint, we synthesized novel substrates linking a p-nitroaniline group to adenylate (AMP-pNA) and inhibitors that consist of an adenosine group and 5'-sulfamoyl (AdoOSO(2)NH(2)) or N-ethylsulfamoyl (AdoOSO(2)NHCH(2)CH(3)) group. AMP-pNA is a suitable substrate for Hint that allowed characterization of the inhibitors; titration of each inhibitor into AMP-pNA assays revealed their K-i values. The N-ethylsulfamoyl derivative has a 13-fold binding advantage over the sulfamoyl adenosine. The 1.8-Angstrom cocrystal structure of rabbit Hint with N-ethylsulfamoyl adenosine revealed a binding site for the ethyl group against Trp-123, a residue that reaches across the Hint dimer interface to interact with the alkyl portion of the inhibitor and, presumably, the alkyl portion of a lysyl substrate. Ser-107 is positioned to donate a hydrogen bond to the leaving group nitrogen. Consistent with a role in acid-base catalysis, the Hint S107A mutant protein displayed depressed catalytic activity.
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