Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase.
The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms possesses very different thermal properties. The B. caldolyticus enzyme has optimal activity at 60-65 degrees C and a half-life of 26 min at 65 degrees C, compared to values of 46 degrees C and 60 s at 65 degrees C, respectively, for the B. subtilis enzyme. Chemical cross-linking shows that both enzymes are hexamers. Vmax is determined as 440 micromol.min(-1).mg protein(-1) and Km values for ATP and ribose 5-phosphate are determined as 310 and 530 microM, respectively, for the B. caldolyticus enzyme. The enzyme requires 50 mM Pi as well as free Mg2+ for maximal activity. Manganese ion substitutes for Mg2+, but only at 30% of the activity obtained with Mg2+. ADP and GDP inhibit the B. caldolyticus enzyme in a cooperative fashion with Hill coefficients of 2.9 for ADP and 2.6 for GDP. Ki values are determined as 113 and 490 microm for ADP and GDP, respectively. At low concentrations ADP inhibition is linearly competitive with respect to ATP. A predicted structure of the B. caldolyticus enzyme based on homology modelling with the structure of B. subtilis 5-phospho-alpha-D-ribosyl 1-diphosphate synthase shows 92% of the amino acid differences to be on solvent exposed surfaces in the hexameric structure.
Hove-Jensen
Bjarne
B
Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Denmark. hove@mermaid.molbio.ku.dk
McGuire
James N
JN
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012268:Ribose-Phosphate Pyrophosphokinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012267:Ribosemonophosphates / Q000378:metabolism; D016415:Sequence Alignment
D000596:Amino Acids; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012267:Ribosemonophosphates; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; D012268:Ribose-Phosphate Pyrophosphokinase