Substrate binding domains in pyruvate phosphate dikinase.
Proteolysis of Clostridium symbiosum pyruvate phosphate dikinase (PPDK) in its free or phosphorylated state with subtilisin Carlsberg followed two different cleavage pathways. The major pathway involved initial cleavage of the holoenzyme (93 kDa) into a stable 25-kDa N-terminal fragment and transiently stable 67-kDa C-terminal fragment. The 67-kDa fragment was cleaved to generate a stable 35-kDa fragment and an unstable 30-kDa fragment (containing the catalytic histidine). Proteolytic cleavage via the minor pathway divided the holoenzyme into an unstable 37-kDa N-terminal piece (which was further cleaved to the stable 25-kDa fragment produced in the major pathway) and a transiently stable 55-kDa C-terminal fragment. The 55-kDa fragment was then cleaved to produce the stable 35-kDa fragment produced by the major pathway. The cleavage pattern of PPDK complexed with the ATP analog adenyl imidodiphosphate was identical to that of the free enzyme, only the rate of cleavage as slower. In contrast, proteolysis of the phosphorylenzyme-oxalate complex generated the 55-kDa fragment indicating that oxalate binding induces a change in protein conformation. Treatment of PPDK with [1-14C]bromopyruvate followed by proteolysis revealed selective radiolabeling of the stable 35-kDa fragment while similar experiments with [14C]2',3'-dialdehyde adenosine 5'-monophosphate resulted in selective radiolabeling of the stable 25-kDa fragment. These results were interpreted to suggest that PPDK contains several structural domains and that the catalytic histidine, the pyruvate binding site, and the ATP binding site may be located on different domains.
Carroll
L J
LJ
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Xu
Y
Y
Thrall
S H
SH
Martin
B M
BM
Dunaway-Mariano
D
D
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology; D006868:Hydrolysis; D008024:Ligands; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D011772:Pyruvate, Orthophosphate Dikinase / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism
D008024:Ligands; D011773:Pyruvates; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D011772:Pyruvate, Orthophosphate Dikinase; D013381:Subtilisins