1RZY

Crystal structure of rabbit Hint complexed with N-ethylsulfamoyladenosine

Summary for 1RZY

• Entry DOI: 10.2210/pdb1rzy/pdb
• Related: 5RHN 6RHN
• Descriptor: Histidine triad nucleotide-binding protein 1, 5'-O-(N-ETHYL-SULFAMOYL)ADENOSINE (3 entities in total)
• Functional Keywords: hit protein; protein-inhibitor complex, hydrolase
• Biological source: Oryctolagus cuniculus (rabbit)
• Cellular location: Cytoplasm: P80912
• Total number of polymer chains: 1
• Total formula weight: 14088.21

Authors

Krakowiak, A.K.,Pace, H.C.,Blackburn, G.M.,Adams, M.,Mekhalfia, A.,Kaczmarek, R.,Baraniak, J.,Stec, W.J.,Brenner, C. (deposition date: 2003-12-29, release date: 2004-03-09, Last modification date: 2024-02-14)

Primary citation

Krakowiak, A.K.,Pace, H.C.,Blackburn, G.M.,Adams, M.,Mekhalfia, A.,Kaczmarek, R.,Baraniak, J.,Stec, W.J.,Brenner, C.
Biochemical, crystallographic, and mutagenic characterization of hint, the AMP-lysine hydrolase, with novel substrates and inhibitors
J.Biol.Chem., 279:18711-18716, 2004

PubMed Abstract: 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-A 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.

PubMed: 14982931
DOI: 10.1074/jbc.M314271200

Experimental method

X-RAY DIFFRACTION (1.8 Å)