The Role of Enzyme Isomerization in the Native Catalytic Cycle of the ATP Sulfurylase−GTPase System

Abstract

ATP sulfurylase, from E. coli Kappa-12, is a GTPase.target complex that conformationally couples the free energies of GTP hydrolysis and activated sulfate (adenosine 5'-phosphosulfate, or APS) synthesis. Energy coupling is achieved by an allosterically driven isomerization that switches on and off chemistry at specific points in the catalytic cycle. This coupling mechanism is derived from the results of model studies using analogue complexes that mimic different stages of the native catalytic cycle. The current investigation extends the analogue studies to the native catalytic cycle. Isomerization is monitored using the fluorescent, guanine nucleotide analogues mGMPPNP (3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-[beta, gamma-imido]triphosphate) and mGTP [3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-triphosphate]. The isomerization is shown to be initiated by an allosteric interaction that requires the simultaneous occupancy of all three substrate-binding sites. Stopped-flow fluorescence and single-turnover studies were used to define and quantitate the isomerization mechanism, and to show that the isomerization precedes and rate-limits both GTP hydrolysis and APS synthesis. These findings are incorporated into a model of the energy-coupling mechanism.

The Trifunctional Sulfate-activating Complex (SAC) of Mycobacterium tuberculosis

“…The SAC APS kinase domain couples APS synthesis to PAPS formation, and PAPS synthesis is sufficiently favorable (K eq ϭ 2 ϫ 10 3 , 50 mM Hepes, pH/K ϩ ϭ 8.0, T ϭ 25°C) (22) to produce detectable, albeit low, quantities of [ 35 S]PAPS from ATP and 35 SO 4 in the absence of GTP; reactions initiated at 4.0 mM ATP and 0.50 mM 35 SO 4 produce a maximum of ϳ2% conversion of SO 4 to PAPS, and APS levels under these conditions are too low to detect (see Fig. 3A).…”

“…In the absence of GTP, the SAC concentrations needed for the PAPS synthesis reactions to reach completion within several hours are higher than the quantities of product formed. The 35 SO 4 assays measure the total product formed (i.e. enzyme-bound and solution-phase product); thus, it is possible that a significant fraction of the product is bound to SAC, a situation that prevents using the reaction end points to calculate solutionphase equilibrium constants.…”

“…The isomerization, which precedes and partially rate-limits both GTP hydrolysis and APS synthesis, is a central energy-coupling step in the mechanism (34,35). The isomerization commits the chemistries to forward reaction and appears to bring residues at both active sites into their catalytic positions, from which chemistry occurs quickly (35).…”

“…The system has isomerized and is stalled because it cannot cleave the ␤,␥-imido bond of GMPPNP. Cleavage of the ␤,␥-bond of GTP produces the GDP⅐E⅐AMP⅐PP i complex, which is not isomerized (34,35). AMP and PP i are needed to experimentally observe the isomerization, and these activators increase the equilibrium of affinity of mGMPPNP for the E. coli enzyme 4700-fold (27 M to 5.8 nM) (34).…”

5′-Adenosinephosphosulfate Lies at a Metabolic Branch Point in Mycobacteria

“…The enzyme that performs this task, ATP sulfurylase (CysD), forms part of a heterodimer. The other component is a GTPase (CysN) that couples the hydrolysis of GTP to the sulfurylation of ATP, thereby providing energy to shift the equilibrium so as to favor the formation of the product (15,16). APS kinase (CysC) then converts APS to PAPS.…”