Regulating Energy Transfer in the ATP Sulfurylase−GTPase System

Abstract

ATP sulfurylase, isolated from Escherichia coli K-12, is a GTPase-target complex that catalyzes and links the energetics of GTP hydrolysis to the synthesis of activated sulfate (APS). When the GTP concentration is saturating and held fixed with a regenerating system, the APS reaction reaches a steady state in which its mass ratio is shifted (5.4 x 10(6))-fold toward the product by the hydrolysis of GTP. If GTP is not regenerated, the shift toward the product is transient, producing a pulse-shaped progress curve. The mechanistic basis of this transience is the subject of this paper. The product transient is caused by the binding of GDP to the enzyme which establishes a catalytic pathway that allows the chemical potential that had been transferred to the APS reaction to "leak" into the chemical milieu. The system leaks because the E.GDP complex catalyzes the uncoupled APS reaction. The addition of phosphate to the leaky GDP.E.APS.PPi complex converts it into the central Pi.GDP.E.APS.PPi complex which catalyzes the energy-transfer reaction. Thus, Pi binding directs the system through the coupled mechanism, "plugging" the leak. GMPPNP, which also causes a leak, is used to demonstrate that the mass ratio of the APS reaction can be "tuned" by adjusting flux through the coupled and uncoupled pathways. This energy-coupling mechanism provides a means for controlling the quantity of chemical potential transferred to the APS reaction. This versatile linkage might well be used to the cell's advantage to avoid the toxicity associated with an excess of activated sulfate.

Roles of elusive translational GTPases come to light and inform on the process of ribosome biogenesis in bacteria

“…CysN is a component of the enzyme ATP sulfurylase, which forms activated sulfate (adenosine 5′-phosphosulfate; APS) from ATP and sulfate, an obligate step in the metabolic assimilation of sulfur (Leyh and Suo, 1992). GTP hydrolysis by the CysN subunit regulates APS formation in many bacteria (Liu et al , 1998; Margus et al , 2007). Structures of IF2, EF-Tu, SelB, EF-G, RF3, TetM, BipA, and LepA on the ribosome have been determined by cryo-electron microscopy and/or X-ray crystallography (Fischer et al , 2016; Gagnon et al ., 2014; Gao et al ., 2009; Kumar et al ., 2015; Li et al ., 2013; Schmeing et al ., 2009; Sprink et al ., 2016; Zhou et al ., 2012).…”

Reduction of Adenosine-5′-Phosphosulfate Instead of 3′-Phosphoadenosine-5′-Phosphosulfate in Cysteine Biosynthesis by Rhizobium meliloti and Other Members of the Family Rhizobiaceae

“…APS kinase PAPS ϩ ADP GTP hydrolysis shifts the mass ratio of the sulfation reaction toward APS by (5.4 ϫ 10 6 )-fold (20), and the free energy of APS phosphorylation, which is highly negative, further drives the two-step reaction forward. In most eukaryotes, PAPS is used as a sulfate donor in reactions in which the sulfate is directly transferred to a second molecule.…”

Colorimetric Determination of Sulfate via an Enzyme Cascade for High-Throughput Detection of Sulfatase Activity

“…Ideally, a GTP:ATP molar ratio of 1:2 is expected to generate a steady flowing cascade as 2 ATP molecules are needed in the first reaction: one producing APS and the other for APS phosphorylation to yield PAPS with GDP as the byproduct. However, the accumulation of GDP in the system decreases the ATPs efficiency for coupling GTP hydrolysis to APS synthesis and PP i release, as competitive inhibition of the guanine nucleotide binding site occurs between GDP and GTP . Accordingly, the concentration of GTP needed to direct the reaction toward SO 4 2– consumption must be increased and was, therefore, set to at least a 1:1 GTP:ATP molar ratio.…”

“…Too-short times may not be adequate for the complete reaction of sulfate, especially at the higher concentrations in the calibration curve. On the other hand, too-long times might shift the equilibrium back to the production of ATP and SO 4 2– , as previously described for the ATPs reaction . Thus, a reaction time of 45 min was considered optimal and employed.…”