Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure.
Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts ATP, dATP, GTP, CTP, and UTP as diphosphoryl donors. All of these properties are characteristic for class II PRPP synthases. K(m) values for ATP and ribose 5-phosphate are 77 and 48 microM, respectively. Gel filtration reveals a molecular mass of the native enzyme of approximately 110 kD, which is consistent with a homotrimer. Secondary structure prediction shows that spinach PRPP synthase isozyme 4 has a general folding similar to that of Bacillus subtilis class I PRPP synthase, for which the three-dimensional structure has been solved, as the position and extent of helices and beta-sheets of the two enzymes are essentially conserved. Amino acid sequence comparison reveals that residues of class I PRPP synthases interacting with allosteric inhibitors are not conserved in class II PRPP synthases. Similarly, residues important for oligomerization of the B. subtilis enzyme show little conservation in the spinach enzyme. In contrast, residues of the active site of B. subtilis PRPP synthase show extensive conservation in spinach PRPP synthase isozyme 4.
Krath
B N
BN
Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark.
Hove-Jensen
B
B
D000595:Amino Acid Sequence; D007527:Isoenzymes / Q000737:chemistry / Q000145:classification; D007700:Kinetics; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012268:Ribose-Phosphate Pyrophosphokinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000145:classification; D016415:Sequence Alignment; D018724:Spinacia oleracea / Q000201:enzymology*
D007527:Isoenzymes; D011994:Recombinant Proteins; D012268:Ribose-Phosphate Pyrophosphokinase