TAILIEUCHUNG - Báo cáo khoa học: Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase

The amino acid sequence of 5-phospho-a-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticusis 81% identical to the amino acid sequence of 5-phospho-a-D-ribosyl 1-diphosphate synthase from the mesophileBacillus the enzyme fromthe two organisms possesses very different thermal properties. TheB. caldolyticusenzyme has optimal activity at 60–65 C and a half-life of 26 min at 65 C, compared to values of 46 Cand60sat65 C, respectively, for theB. subtilis enzyme. . | Eur. J. Biochem. 271 4526-4533 2004 FEBS 2004 doi Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase Bjarne Hove-Jensen1 and James N. McGuire2 1 Department of Biological Chemistry and 2Center for Enzyme Research Institute of Molecular Biology University of Copenhagen Denmark The amino acid sequence of 5-phospho-a-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81 identical to the amino acid sequence of 5-phospho-a-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 C and a half-life of 26 min at 65 C compared to values of 46 C and 60 s at 65 C respectively for the B. subtilis enzyme. Chemical cross-linking shows that both enzymes are hexamers. Vmax is determined as 440 mol-min-1-mg protein-1 and Km values for ATP and ribose 5-phosphate are determined as 310 and 530 M 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. caldo-lyticus enzyme in a cooperative fashion with Hill coefficients of for ADP and for GDP. Ki values are determined as 113 and 490 IM 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-a-D-ribosyl 1-diphosphate synthase shows 92 of the amino acid differences to be on solvent exposed surfaces in the hexameric structure. Keywords kinetics mesophile nucleotide metabolism PRPP thermophile. The compound 5-phospho-a-D-ribosyl 1-diphosphate PRibPP is a central intermediate in the de novo and salvage .

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