Katz Faith E H, Shi Xinying, Owens Cedric P, Joseph Simpson, Tezcan F Akif
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093, United States.
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093, United States.
Anal Biochem. 2017 Mar 1;520:62-67. doi: 10.1016/j.ab.2016.12.012. Epub 2016 Dec 23.
One of the most common assays for nucleoside triphosphatase (NTPase) activity entails the quantification of inorganic phosphate (P) as a colored phosphomolybdate complex at low pH. While this assay is very sensitive, it is not selective for P in the presence of labile organic phosphate compounds (OPCs). Since NTPase activity assays typically require a large excess of OPCs, such as nucleotides, selectivity for P in the presence of OPCs is often critical in evaluating enzyme activity. Here we present an improved method for the measurement of enzymatic nucleotide hydrolysis as P released, which achieves selectivity for P in the presence of OPCs while also avoiding the costs and hazards inherent in other methods for measuring nucleotide hydrolysis. We apply this method to the measurement of ATP hydrolysis by nitrogenase and GTP hydrolysis by elongation factor G (EF-G) in order to demonstrate the broad applicability of our method for the determination of nucleotide hydrolysis in the presence of interfering OPCs.
用于测定核苷三磷酸酶（NTPase）活性的最常见检测方法之一，是在低pH值下将无机磷酸盐（P）定量为有色磷钼酸盐复合物。虽然这种检测方法非常灵敏，但在存在不稳定有机磷酸盐化合物（OPC）的情况下，它对P没有选择性。由于NTPase活性检测通常需要大量过量的OPC，如核苷酸，因此在存在OPC的情况下对P的选择性在评估酶活性时往往至关重要。在此，我们提出一种改进的方法，用于测量作为释放的P的酶促核苷酸水解，该方法在存在OPC的情况下实现了对P的选择性，同时还避免了其他测量核苷酸水解方法所固有的成本和风险。我们将此方法应用于测定固氮酶催化的ATP水解以及延伸因子G（EF-G）催化的GTP水解，以证明我们的方法在存在干扰性OPC的情况下测定核苷酸水解的广泛适用性。
