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.
Highlights
Hint Characterization with Novel Substrates and InhibitorsHint substrates were only identified (3) and a catalytic mechanism proposed (1) in 2002
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, Kin[28]
The 1.8-Å 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
Summary
Hint substrates were only identified (3) and a catalytic mechanism proposed (1) in 2002. Using methods we established for analysis of Fhit with the fluorogenic substrate GpppBODIPY (12), we titrated nonlabeled inhibitors into assays of wild-type and mutant Hint enzymes to determine their equilibrium inhibitory binding constants. Biochemical and crystallographic analysis of wild-type Hint with newly synthesized adenosine sulfamoyl inhibitors indicated and located a binding site for an alkylamino leaving group. The inhibited Hint crystal structure provides information about hydrogen bonding that suggests that the carbonyl oxygen of Gly-105 may assist the side chain hydroxyl of Ser-107 as the acid-base catalyst, in contrast to the earlier suggestion of His-114 as the acid-base catalyst (1). Mutant indicates that the Ser hydroxyl plays a facilitative role in catalysis, in its absence, the Gly-105 carbonyl may assist a water molecule bound by the S107A enzyme to provide residual activity for protonation of the leaving group and activation of the hydrolytic water
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