Characterization of ADP-glucose pyrophosphorylase from Rhodobacter sphaeroides 2.4.1: evidence for the involvement of arginine in allosteric regulation.
ADP-glucose pyrophosphorylase (ADPGlc PPase, EC 2.7.7.27) from Rhodobacter sphaeroides 2.4.1 has been purified to near homogeneity. The enzyme reacted in Western blots to polyclonal antibodies raised against other bacterial ADPGlc PPases. The purified enzyme was found to be activated by fructose 6-phosphate, fructose 1,6-bisphosphate, and pyruvate and inhibited by phosphate, phosphoenolpyruvate, ADP, and pyridoxal phosphate. Kinetic studies indicate that AMP, while having little effect on kinetic parameters at pH 8 in the absence of effectors, is a specific ligand for an allosteric site(s). Treatment of the purified enzyme with the arginyl reagents 2,3-butanedione and phenylglyoxal resulted in desensitization of the enzyme to both activation and inhibition by metabolites. Phosphate, fructose 6-phosphate, and AMP were found to protect the enzyme against allosteric desensitization supportive of these metabolites interacting at common site(s) or with a common enzyme form. As a first step in cloning the gene coding for this enzyme, a polymerase chain reaction fragment was generated from genomic DNA using primers based on amino terminal sequencing data and a highly conserved region in known ADPGlc PPases. The sequence of this fragment and position of amino terminal arginines in comparison to other known ADPGlc PPases is discussed in relation to the kinetic and chemical modification data.
Meyer
C R
CR
Department of Chemistry and Biochemistry, California State University at Fullerton, California, 92834, USA. cmeyer@fullerton.edu
Borra
M
M
Igarashi
R
R
Lin
Y S
YS
Springsteel
M
M
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D003931:Diacetyl; D005798:Genes, Bacterial; D051599:Glucose-1-Phosphate Adenylyltransferase; D007202:Indicators and Reagents; D007700:Kinetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D007202:Indicators and Reagents; D001120:Arginine; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase; D003931:Diacetyl