Analysis of the Structural Determinants Underlying Discrimination Between Substrate and Solvent in β-Phosphoglucomutase Catalysis

Journal Article · Thu Jan 01 04:00:00 EST 2009 · Biochemistry

DOI:https://doi.org/10.1021/bi801653r· OSTI ID:980016

Dai, J; Finci, L; Zhang, C; Lahiri, S; Zhang, G; Peisach, E; Allen, K; Dunaway-Mariano, D

The {Beta}-phosphoglucomutase ({Beta}-PGM) of the haloacid dehalogenase enzyme superfamily (HADSF) catalyzes the conversion of {Beta}-glucose 1-phosphate ({Beta}G1P) to glucose 6-phosphate (G6P) using Asp8 of the core domain active site to mediate phosphoryl transfer from {Beta}-glucose 1,6-(bis)phosphate ({Beta}G1,6bisP) to {Beta}G1P. Herein, we explore the mechanism by which hydrolysis of the {Beta}-PGM phospho-Asp8 is avoided during the time that the active site must remain open to solvent to allow the exchange of the bound product G6P with the substrate {Beta}G1P. On the basis of structural information, a model of catalysis is proposed in which the general acid/base (Asp10) side chain moves from a position where it forms a hydrogen bond to the Thr16-Ala17 portion of the domain-domain linker to a functional position where it forms a hydrogen bond to the substrate leaving group O and a His20-Lys76 pair of the cap domain. This repositioning of the general acid/base within the core domain active site is coordinated with substrate-induced closure of the cap domain over the core domain. The model predicts that Asp10 is required for general acid/base catalysis and for stabilization of the enzyme in the cap-closed conformation. It also predicts that hinge residue Thr16 plays a key role in productive domain-domain association, that hydrogen bond interaction with the Thr16 backbone amide NH group is required to prevent phospho-Asp8 hydrolysis in the cap-open conformation, and that the His20-Lys76 pair plays an important role in substrate-induced cap closure. The model is examined via kinetic analyses of Asp10, Thr16, His20, and Lys76 site-directed mutants. Replacement of Asp10 with Ala, Ser, Cys, Asn, or Glu resulted in no observable activity. The kinetic consequences of the replacement of linker residue Thr16 with Pro include a reduced rate of Asp8 phosphorylation by {Beta}G1,6bisP, a reduced rate of cycling of the phosphorylated enzyme to convert {Beta}G1P to G6P, and an enhanced rate of phosphoryl transfer from phospho-Asp8 to water. The X-ray crystal structure of the T16P mutant at 2.7 {angstrom} resolution provides a snapshot of the enzyme in an unnatural cap-open conformation where the Asp10 side chain is located in the core domain active site. The His20 and Lys76 site-directed mutants exhibit reduced activity in catalysis of the Asp8-mediated phosphoryl transfer between {Beta}G1,6bisP and {Beta}G1P but no reduction in the rate of phospho-Asp8 hydrolysis. Taken together, the results support a substrate induced-fit model of catalysis in which {Beta}G1P binding to the core domain facilitates recruitment of the general acid/base Asp10 to the catalytic site and induces cap closure.

• Research Organization: Brookhaven National Laboratory (BNL) National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: AC02-98CH10886
• OSTI ID: 980016
• Report Number(s): BNL--92934-2010-JA
• Journal Information: Biochemistry, Journal Name: Biochemistry Journal Issue: 9 Vol. 48
• Country of Publication: United States
• Language: English

Related Subjects

08 HYDROGEN
36 MATERIALS SCIENCE
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
AMIDES
CATALYSIS
CHAINS
CONVERSION
CRYSTAL STRUCTURE
ENZYMES
FUNCTIONALS
GLUCOSE
HYDROGEN
HYDROLYSIS
INTERACTIONS
KINETICS
MUTANTS
PHOSPHORYLATION
REDUCTION
RESIDUES
RESOLUTION
SOLVENTS
STABILIZATION
SUBSTRATES
SUPPORTS
WATER
national synchrotron light source