Farley J R, Cryns D F, Yang Y H, Segel I H
J Biol Chem. 1976 Jul 25;251(14):4389-97.
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.
通过初速度、产物抑制和终产物抑制研究，确定了ATP硫酸化酶的动力学机制。在正向反应中，该反应是稳态有序的，其中MgATP = A，硫酸盐 = B，MgPP1 = P，APS = Q。Km A = 0.38 mM，Ki a = 0.71 mM，Km B = 0.50 mM。硝酸盐和氯酸盐与硫酸盐竞争，与MgATP非竞争性。Ki NO3- = 0.25 mM；Ki ClO3- = 0.15 mM。AMP和各种MgATP类似物与MgATP竞争，对SO42-而言是混合型抑制剂。AMP的Ki为0.55 mM。该反应在反向是快速平衡有序的，Ki q = 0.3至1.0 μM，Kmp = 0.65 μM。腺苷5'-磷酸硫酸酯（APS）表现出竞争性底物抑制（KI Q = 0.3 mM）。Vmax f/Vmax r的比值为0.018。在正向反应中，Vmax MoO42-/Vmax SO42-的比值为20。根据Haldane方程在pH 8.0和30℃计算的Keq为6×10（-9）至3.3×10（-8）（取决于所选的Ki q值）。实验Keq约为2.5×10（-9）。Vmax/Vmax r约比Keq大100万倍这一事实与该酶假定的生理作用（APS合成）一致。通过多重抑制分析探究了有序结合序列的机制基础。与MgATP竞争的终产物抑制剂（如游离ATP、Mgα,β-亚甲基ATP、CrATP和CaATP）不会诱导硫酸盐的底物抑制，也不会改变硝酸盐显示的抑制模式。该结果表明（但未证明）对MgATP的催化作用必须先于硫酸盐结合位点的形成。
