Kinetic and regulatory properties of rat liver phosphoribosylpyrophosphate synthetase complex are partly distinct from those of isolated recombinant component catalytic subunits.
Rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of two catalytic subunits (PRS I and II, in a ratio of approximately 4:1) and two catalytically inactive PRPP synthetase-associated proteins. To better understand the significance of the complex structure, the properties of the native liver enzyme were compared with those of homologous aggregates of recombinant PRS I and PRS II (rPRS I and rPRS II). (1) The specific activity per catalytic subunits of the liver enzyme was about 2.5 times lower than that of rPRS I over a wide pH range. Km values for substrates and Ka values for Pi and Mg2+ of the three enzymes were similar. (2) Specific activity of the liver enzyme for the reverse reaction was about 2 times lower than those of rPRSs. Km values for substrates of the three enzymes were comparable. (3) The liver enzyme was more stable than were rPRSs when incubated at a high temperature or in the absence of stabilizing agents. (4) The liver enzyme was markedly less sensitive to inhibition by nucleotides compared to rPRS I. GDP at 1 mM inhibited the liver enzyme and rPRS I by 32 and 93%, respectively. This effect is not ascribable to molecular interaction between rPRS I and II, as reconstitution of the two did not alter the sensitivity to nucleotide inhibition. (5) Our observations suggest that complex aggregation states of the native enzyme not only suppress the activities but also stabilize the catalytic subunits and the associated proteins and remarkably reduce the sensitivity to inhibition by nucleotides.
Sonoda
T
T
Department of Biochemistry, Chiba University School of Medicine. cum121j@med.m.chiba-u.ac.jp
Kita
K
K
Ishijima
S
S
Ishizuka
T
T
Ahmad
I
I
Tatibana
M
M
D000818:Animals; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D009711:Nucleotides / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D012268:Ribose-Phosphate Pyrophosphokinase / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology*
D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D009711:Nucleotides; D011994:Recombinant Proteins; D012268:Ribose-Phosphate Pyrophosphokinase