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

Agnieszka Krakowiak; Helen C Pace; G Michael Blackburn; Martina Adams; Abdelaziz Mekhalfia; Renata Kaczmarek; Janina Baraniak; Wojciech J Stec; Charles Brenner

doi:10.1074/jbc.M314271200

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

Journal article

Open access

Peer reviewed

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

Agnieszka Krakowiak, Helen C Pace, G Michael Blackburn, Martina Adams, Abdelaziz Mekhalfia, Renata Kaczmarek, Janina Baraniak, Wojciech J Stec and Charles Brenner

The Journal of biological chemistry, Vol.279(18), pp.18711-18716

04/30/2004

DOI: 10.1074/jbc.M314271200

PMCID: PMC2556070

PMID: 14982931

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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.

Rabbits

Hydrolases - genetics

Adenosine Monophosphate - chemistry

Carrier Proteins - antagonists & inhibitors

Enzyme Inhibitors - pharmacology

Substrate Specificity

Crystallography, X-Ray

Enzyme Inhibitors - chemical synthesis

Carrier Proteins - genetics

Animals

Adenosine Monophosphate - analogs & derivatives

Carrier Proteins - chemistry

Hydrolases - chemistry

Kinetics

Binding Sites

Amino Acid Substitution

Hydrolases - antagonists & inhibitors

Details

Title: Subtitle: Biochemical, crystallographic, and mutagenic characterization of hint, the AMP-lysine hydrolase, with novel substrates and inhibitors
Creators: Agnieszka Krakowiak - Structural Biology and Bioinformatics Program, Kimmel Cancer Center, Philadelphia, Pennsylvania 19107, USA
Helen C Pace
G Michael Blackburn
Martina Adams
Abdelaziz Mekhalfia
Renata Kaczmarek
Janina Baraniak
Wojciech J Stec
Charles Brenner
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.279(18), pp.18711-18716
DOI: 10.1074/jbc.M314271200
PMID: 14982931
PMCID: PMC2556070
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: R01 CA075954 / NCI NIH HHS R01 CA075954-07 / NCI NIH HHS CA75954 / NCI NIH HHS
Language: English
Date published: 04/30/2004
Academic Unit: Biochemistry and Molecular Biology; Internal Medicine
Record Identifier: 9983788432602771

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