Functional studies of ATP sulfurylase from Penicillium chrysogenum
ATP sulfurylase from Penicillium chrysogenum has a specific activity (V/sub max/) of 6-7 units x mg protein/sup -1/ determined with the physiological substrates of MgATP and SO/sub 4//sup 2 -/ and assayed by (A) initial velocity measurements with APS kinase and inorganic pyrophosphatase present and (B) analysis of nonlinear reaction progress curves. The fact both assays give the same results show the intrinsic activity of ATP sulfurylase is much higher than previously reported. In initial velocity dead-end inhibition studies, the sulfate analog S/sub 2/O/sub 3//sup 2 -/ is a competitive inhibitor of SO/sub 42/..sqrt.. and a noncompetitive inhibitor of MgATP. Monovalent oxyanions such as NO/sub 3//sup -/, ClO/sub 3//sup -/, ClO/sub 4//sup -/, and FSO/sub 3//sup -/ behave as uncompetitive inhibitors of MgATP and thus seem not to be true sulfate analogs. The reverse reaction was assayed by the pyrophosphate dependent release of /sup 35/SO/sub 4//sup 2 -/ from AP/sup 35/S. Product inhibition by MgATP or SO/sub 4//sup 2 -/ is competitive with APS and mixed-type with PP/sub i/. Imidodiphosphate can serve as an alternative substrate for PP/sub i/. ATP sulfurylase binds (but does not hydrolyze) APS. A Scatchard plot of the APS binding is nonlinear, suggesting at least two types of sites. The cumulative results are qualitatively consistent with the random addition of MgATP and SO/sub 4//sup 2 -/ and the ordered release of first MgPP/sub i/ then APS, with APS release being partially rate limiting. Certain quantitative discrepancies suggest either an unknown variable (e.g. enzyme concentration) complicates the analysis or, in light of binding studies that the actual mechanism is more complicated (e.g. alternating sites) than any of the conventional models examined.
- Research Organization:
- California Univ., Davis (USA)
- OSTI ID:
- 5623348
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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HYDROLASES
BIOCHEMISTRY
PENICILLIUM
BIOCHEMICAL REACTION KINETICS
ATP
ENZYME ACTIVITY
SULFATES
SULFUR 35
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMISTRY
DAYS LIVING RADIOISOTOPES
ENZYMES
EVEN-ODD NUCLEI
FUNGI
ISOTOPES
KINETICS
LIGHT NUCLEI
NUCLEI
NUCLEOTIDES
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PLANTS
RADIOISOTOPES
REACTION KINETICS
SULFUR COMPOUNDS
SULFUR ISOTOPES
550201* - Biochemistry- Tracer Techniques