Biochemical, crystallographic, and mutagenic characterization of hint, the AMP-lysine hydrolase, with novel substrates and inhibitors.
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.
Structural Biology and Bioinformatics Program, Kimmel Cancer Center, Philadelphia, Pennsylvania 19107, USA.
Agnieszka
A
Krakowiak
Helen C
HC
Pace
G Michael
GM
Blackburn
Martina
M
Adams
Abdelaziz
A
Mekhalfia
Renata
R
Kaczmarek
Janina
J
Baraniak
Wojciech J
WJ
Stec
Charles
C
Brenner
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D011817:Rabbits; D013379:Substrate Specificity
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; C486517:histidine triad nucleotide-binding protein, mammals; D000249:Adenosine Monophosphate; D006867:Hydrolases; C087067:adenosine 5'-phosphoramidate hydrolase