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J Biol Chem. 2004 Apr 30;279(18):18711-6. Epub 2004 Feb 24.

Biochemical, crystallographic, and mutagenic characterization of hint, the AMP-lysine hydrolase, with novel substrates and inhibitors.

Author information

1
Structural Biology and Bioinformatics Program, Kimmel Cancer Center, Philadelphia, Pennsylvania 19107, USA.

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.

PMID:
14982931
PMCID:
PMC2556070
DOI:
10.1074/jbc.M314271200
[Indexed for MEDLINE]
Free PMC Article

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