Catalytic implications of the higher plant ADP-glucose pyrophosphorylase large subunit.
ADP-glucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, is composed of a pair of catalytic small subunits (SSs) and a pair of catalytically disabled large subunits (LSs). The N-terminal region of the LS has been known to be essential for the allosteric regulatory properties of the heterotetrameric enzyme. To gain further insight on the role of this region and the LS itself in enzyme function, the six proline residues found in the N-terminal region of the potato tuber AGPase were subjected to scanning mutagenesis. The wildtype and various mutant heterotetramers were expressed using our newly developed host-vector system, purified, and their kinetic parameters assessed. While P(17)L, P(26)L, and P(55)L mutations only moderately affected the kinetic properties, P(52)L and P(66)L gave rise to significant and contrasting changes in allosteric properties: P(66)L enzyme displayed up-regulatory properties toward 3-PGA while the P(52)L enzyme had down-regulatory properties. Unlike the other mutants, however, various mutations at P(44) led to only moderate changes in regulatory properties, but had severely impaired catalytic rates, apparent substrate affinities, and responsiveness to metabolic effectors, indicating Pro-44 or the LS is essential for optimal catalysis and activation of the AGPase heterotetramer. The catalytic importance of the LS is further supported by photoaffinity labeling studies, which revealed that the LS binds ATP at the same efficiency as the SS. These results indicate that the LS, although considered having no catalytic activity, may mimic many of the catalytic events undertaken by the SS and, thereby, influences net catalysis of the heterotetrameric enzyme.
Hwang
Seon-Kap
SK
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA.
Hamada
Shigeki
S
Okita
Thomas W
TW
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D011392:Proline / Q000032:analysis; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D021122:Protein Subunits; D011392:Proline; D051599:Glucose-1-Phosphate Adenylyltransferase