Conserved active site aspartates and domain-domain interactions in regulatory properties of the sugar kinase superfamily.
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), and the glucose-specific phosphocarrier protein, IIIGlc (also known as IIAGlc), were determined. Each of the residues participates in catalysis; kcat/Katp is decreased 300-fold by the D245N substitution and 100-fold by the D10N substitution. Affinity labeling with the glycerol analog 1,3-dichloroacetone shows that the level of activity seen for the D245N mutant enzyme is not due to deamidation of the substituted asparagine. Each of the substitutions has little effect on regulation by FBP and the apparent affinity for IIIGlc, and the D245N substitution does not affect the extent of inhibition by IIIGlc. However, the D10N substitution decreases the maximum extent of inhibition by IIIGlc from 100 to 60%, thus changing the action of IIIGlc to that of a partial inhibitor. The different sensitivities of the extents of FBP and IIIGlc inhibition to perturbation of a domain-domain interaction mediated by Asp10 suggest that the relations of the actions of these allosteric effectors to the domain closure conformational change are different.
Pettigrew
D W
DW
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA. DPETTIGREW@TAMU.EDU
Smith
G B
GB
Thomas
K P
KP
Dodds
D C
DC
D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001216:Asparagine; D001224:Aspartic Acid; D001665:Binding Sites; D017124:Conserved Sequence; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005635:Fructosediphosphates / Q000378:metabolism; D005991:Glycerol Kinase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D029968:Escherichia coli Proteins; D005635:Fructosediphosphates; D011994:Recombinant Proteins; C032242:crr protein, E coli; D001224:Aspartic Acid; D001216:Asparagine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D005991:Glycerol Kinase; C029063:fructose-1,6-diphosphate