Conserved active site aspartates and domain-domain interactions in regulatory properties of the sugar kinase superfamily

Archives of Biochemistry and Biophysics
D W PettigrewD C Dodds

Abstract

The structures of the sugar kinase/heat shock 70/actin superfamily of enzymes show that the active site is located in a deep cleft between two domains whose relative movement defines a domain closure conformational change thought to be involved in the catalytic and regulatory properties of members of the superfamily. To investigate the role of the domain closure in the regulatory behavior, site-directed mutagenesis is used to alter specific domain-domain interactions in Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase), a member of this superfamily. Two active site aspartate residues are conserved throughout the superfamily, one (Asp245 in glycerol kinase) which is proposed to act as a general base during catalysis and one (Asp10 in glycerol kinase) which interacts with the Mg(II) ion of the bound Mg(II)-nucleotide complex. Each of these residues participates in domain-domain interactions that are mediated by the bound substrates. The enzymes containing the substitutions Asp245 to Asn (D245N) or Asp10 to Asn (D10N) were purified by affinity chromatography, and the effects of the substitutions on the catalytic properties and regulation by the allosteric effectors, fructose 1,6-bisphosphate (FBP), ...Continue Reading

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Citations

Aug 31, 2000·Applied and Environmental Microbiology·K A ErlandsonC A Batt
Sep 25, 2007·Journal of Bacteriology·Jason S Richardson, Ivan J Oresnik
Feb 7, 2001·Journal of Bacteriology·C K HoltmanD W Pettigrew
Mar 19, 2005·Journal of Bacteriology·Cheryl Ingram-SmithJames G Ferry
Dec 13, 2006·Microbiology and Molecular Biology Reviews : MMBR·Josef DeutscherPieter W Postma
Aug 6, 2002·Proceedings of the National Academy of Sciences of the United States of America·Aaron C Pawlyk, Donald W Pettigrew
Nov 25, 2006·Journal of Molecular Biology·Eric Di LuccioDavid K Wilson
Jun 5, 2001·Protein Expression and Purification·A C Pawlyk, D W Pettigrew
Apr 9, 2009·Applied Microbiology and Biotechnology·Kouta Hatayama, Tatsuo Yagishita
Dec 7, 2013·Acta Crystallographica. Section D, Biological Crystallography·Magdalena SchacherlUlrich Baumann
Nov 3, 2001·Biochimica Et Biophysica Acta·H Schüler
Sep 18, 2002·Archives of Biochemistry and Biophysics·Joan H F Drosopoulos

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