Abstract
The kinetic mechanism of ATP sulfurylase was established from initial velocity, product inhibition, and dead-end inhibition studies. In the forward direction, the reaction is steady state ordered, with MgATP=A, sulfate=B, MgPP1=P, and APS=Q.KmA=0.38 mM, Kia=0.71 mM, KmB=0.50 mM. Nitrate and chlorate are competive with sulfate and uncompetitive with MgATP. KiNO3-=0.25 mM; KiC1O3-= 0.15 mM. AMP and various MgATP analogs are competitive with MgATP and mixed-type inhibitors with respect to SO42-. The Ki for AMP is 0.55 mM. The reaction is rapid equilibrium ordered in the reverse direction with Kiq=0.3 to 1.0 muM and Kmp=0.65 muM. Adenosine 5'-phosphosulfate (APS) exhibits competitive substrate inhibition (KIQ=0.3 mM). The ratio Vmaxf/Vmaxr is 0.018. In the forward direction the ratio VmaxMoO42-/VmaxSO42- is 20. The Keq at pH 8.0 and 30 degrees calculated from the Haldane equation is 6 X 10(-9) to 3.3 X 10(-8) (depending on the Kiq value chosen). The experimental Keq is about 2.5 X 10(-9). The fact that Vmax/Vmaxr is about 1 million times greater than Keq is consistent with the assumed physiological role of the enzyme (APS synthesis). The mechanistic basis of the ordered binding sequence was probed by multiple inhibition analysis. Dead-end inhibitors competitive with MgATP (such as free ATP, Mg alpha,beta-methylene ATP, CrATP, and CaATP) do not induce substrate inhibition by sulfate or alter the inhibition patterns displayed by nitrate. This result suggests (but does not prove) that catalytic action on MgATP must precede the formation of the sulfate binding site.
Highlights
ATP sulfurylase (EC 2.7.7.4, ATP-sulfate adenylyltransfer- praduct of the reaction, APMo, spontaneously hydrolyzes to ase), the first enzyme involved in inorganic sulfate metabolism, ( AMP and MOO,‘, thereby insuring a continuous unidirecis widely distributed in nature and has been studied in a tional flux. (MgPP, is removed continuously by inorganic variety of organisms (l-13)
In this paper we describe the kinetics of the ATP sulfurylase
Kinetics of Forward Reaction-Initial velocity studies of the forward reaction are shown in Figs. 1 and 2
Summary
The kinetic mechanism of ATP sulfurylase was established from initial velocity, product inhibitipn, and dead-end inhibition studies. Nitrate and chlorate are competitive with sulfate and uncompetitive with MgATP. CaATP) do not induce substrate inhibition by sulfate or alter the inhibition patterns displayed by nitrate. This result suggests (but does not prove) that catalytic action on MgATP must precede the formation of the sulfate binding site. ATP sulfurylase (EC 2.7.7.4, ATP-sulfate adenylyltransfer- praduct of the reaction, APMo, spontaneously hydrolyzes to ase), the first enzyme involved in inorganic sulfate metabolism, In spite of the ubiquity of the pyrophosphatase.) Studies with molybdate have yielded valuaenzyme, relatively little is known about its physical, chemical ble information [1,2,3, 12, 13] but the fact remains that MOO,*
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