Substrate preference of the HIF-prolyl hydroxylase-2 (PHD2) and substrate-induced conformational change.
J Inorg Biochem
; 126: 55-60, 2013 Sep.
Article
en En
| MEDLINE
| ID: mdl-23787140
HIF prolyl-4-hydroxylase 2 (PHD2) is a non-heme Fe, 2-oxoglutarate (2OG) dependent dioxygenase that regulates the hypoxia inducible transcription factor (HIF) by hydroxylating two conserved prolyl residues in N-terminal oxygen degradation domain (NODD) and C-terminal oxygen degradation domain (CODD) of HIF-1α. Prior studies have suggested that the substrate preference of PHD2 arises from binding contacts with the ß2ß3 loop of PHD2. In this study we tested the substrate selectivity of PHD2 by kinetic competition assays, varied ionic strength, and global protein flexibility using amide H/D exchange (HDX). Our results revealed that PHD2 preferred CODD by 20-fold over NODD and that electrostatics influenced this effect. Global HDX monitored by mass spectrometry indicated that binding of Fe(II) and 2OG stabilized the overall protein structure but the saturating concentrations of either NODD or CODD caused an identical change in protein flexibility. These observations imply that both substrates stabilize the ß2ß3 loop to the same extent. Under unsaturated substrate conditions NODD led to a higher HDX rate than CODD due to its lower binding affinity to PHD2. Our results suggest that loop closure is the dominant contributor to substrate selectivity in PHD2.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Modelos Moleculares
/
Subunidad alfa del Factor 1 Inducible por Hipoxia
/
Prolina Dioxigenasas del Factor Inducible por Hipoxia
/
Hierro
/
Ácidos Cetoglutáricos
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
J Inorg Biochem
Año:
2013
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos