GTPase Activation of ATP Sulfurylase: The Mechanism

Chang-xian, Liu; Martin, Eugene L.; Leyh, Thomas S.

doi:10.1021/bi00174a009

Cited by 36 publications

(37 citation statements)

References 26 publications

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“…We have previously shown that GTP and ATP bind randomly to the enzyme and that GTP is hydrolyzed in the absence of activators Liu et al, 1994). Hence, the allosteric model used in the interpretation of the current results is one in which GTP and the activator bind randomly to the enzyme, and the substrate complexes with and without activator can produce product (see Figure 1).…”

Section: Resultsmentioning

confidence: 72%

Allosteric Regulation of the ATP Sulfurylase Associated GTPase

1995

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ATP sulfurylase catalyzes and chemically links the hydrolysis of GTP and the synthesis of activated sulfate (APS). Like many GTPases, its GTPase activity is allosterically regulated, in this case, by APS-forming reactants and their analogues. Using these activators, we have been able to mimic many of the complexes that form in the native reaction, including an DAMP intermediate. The effects of each of these complexes on GTP hydrolysis are determined. The results of pre-steady-state and isotope trapping studies demonstrate that the binding of activator and substrate to the enzyme are near equilibrium and that the rate-determining step appears to be scission of the /?,y-bond of GTP. These properties of the system allow the energetic consequences of activator binding on the ground-and transition-state complexes to be evaluated. Activation occurs predominantly by transition-state stabilization, resulting in kcat increases.The values for kcat span a 180-fold range and vary with each activator. Km, or ground-state, effects are relatively small, -3-fold, and are uniform throughout the activator series. These studies provide an indepth view of the energetic interactions between the two active sites at each step of the APS-forming reaction.

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Section: Resultsmentioning

confidence: 72%

Allosteric Regulation of the ATP Sulfurylase Associated GTPase

1995

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“…[ 35 S]-PAPS was purified chromatographically by anion exchange HPLC (AX300, SynChropak) using an isocratic eluant (NaPO 4 , 0.30 M, pH 6.4). The purified [ 35 S]-PAPS was desalted and concentrated to dryness as described previously (32). The compounds was dissolved in Hepes (50 mM, pH 8.0) and stored at −70°C.…”

Section: Methodsmentioning

confidence: 99%

The Human Estrogen Sulfotransferase: A Half-Site Reactive Enzyme

Sun

Leyh

2010

Biochemistry

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The affinity of 17β-estradiol (E 2 ) for the estrogen receptor is weakened beyond the point of physiological relevance by transfer of the sulfuryl-moiety (-SO 3 ) from PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 3'-hydroxyl of E 2 . The mechanism of this transfer reaction, catalyzed by estrogen sulfotransferase (EST), is investigated here in detail. The enzyme (a dimer of identical protomers) presents a clear example of half-sites reactivity -only one of the subunits of the dimer produces product during the catalytic cycle. This is the first example of halfsites reactivity in the sulfotransferase family. A burst of product, with an amplitude that corresponds to one-half of the available active sites, reveals that the mechanism is rate-limited by product release. The equilibrium constant governing interconversion of the substrate (E·PAPS·E 2 ) and product (E·PAP·E 2 S) central complexes was determined and is strongly biased toward product (K eq int ~49). Slow product release allows the interconversion of the central complexes to approach equilibrium, with the result that K eq int becomes nearly linearly coupled to K m , and contributes a factor of ~ 30 to the steady-state affinity of the enzyme for substrate. Typical of its family, estrogen sulfotransferase is partially k cat -inhibited by its acceptor substrate, E 2 . This inhibition does not influence the burst kinetics, and thus occurs after formation of the product central complex -a finding consistent with slow escape of PAP from the non-reactive E·PAP·E 2 complex. Keywords estrogen sulfotransferase; SULT1E1; substrate selectivity; catalytic mechanism Estrogen sulfotransferase (EC 2.8.2.4) catalyzes transfer of the sulfuryl moiety (-SO 3 ) from PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 3'-hydroxyl of 17β-estradiol (E 2 ), Reaction 1. The remarkably large chemical potential of the phosphoric-sulfuric acid anhydride (1) bond found in PAPS (1,2) facilitates the thermodynamically favorable transfer of the sulfuryl moiety to estradiol (K eq = 4200 (3)). Initial-rate and ligandbinding studies indicate the catalysis occurs via a random Bi-Bi mechanism (3). Chemical † Corresponding

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“…The mechanism of helix-induction by TFE is not fully understood and is debated in current literature [4][5][6]. More recently, the effect of TFE on the structure of intact proteins was investigated using circular dichroism (CD) and proton nuclear magnetic resonance (NMR) spectroscopy [7][8][9][10]. The general observation emerged from these studies is that the TFEinduced state of a protein is characterized by a significant content of helical secondary structure, but lacking the specific tertiary interactions of the native protein.…”

Section: Introductionmentioning

confidence: 99%

Limited proteolysis of cytochrome c in trifluoroethanol

et al. 1995

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Horse heart cytochrome c is cleaved by thermolysin in 50% aqueous TFE (v/v) at neutral pH (25°C, 24 h) at the Gly56-Ile 57 peptide bond of the 104-residue chain of the protein.Additional, but anyway minor, fragmentation at the Gly4S-Phe 46 and MetS°-Ile s~ peptide bonds is also observed. On the other hand, in buffer only and in the absence of TFE, cytochrome c is digested by thermolysin to numerous small peptides. Considering the broad substrate specificity of the TFE-resistant thermolysin, clearly the conformational state of the protein substrate dictates the observed selective proteolysis. It is proposed that the highly helical secondary structure acquired by cytochrome c when dissolved in aqueous TFE hampers binding and adaptation of the protein substrate at the active site of the protease and that peptide bond fission occurs at flexible chain segments characterized by a low a-helix propensity. globular proteins are characterized by enhanced chain flexibility, since a correlation exists between sites of proteolysis and sites of high segmental mobility, these last determined crystallographically [13].Here we show that horse heart cytochrome c in the presence of aqueous TFE is cleaved by the TFE-resistant proteolytic enzyme thermolysin [18] to few fragments only, whereas in aqueous solution and in the absence of alcohol many small peptides are formed. The preferential proteolysis was interpreted on the basis of the conformational properties of cytochrome c dissolved in aqueous TFE, as deduced from circular dichroism (CD) and fluorescence emission measurements. Materials and methods

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GTPase Activation of ATP Sulfurylase: The Mechanism

Cited by 36 publications

References 26 publications

Allosteric Regulation of the ATP Sulfurylase Associated GTPase

Allosteric Regulation of the ATP Sulfurylase Associated GTPase

The Human Estrogen Sulfotransferase: A Half-Site Reactive Enzyme

Limited proteolysis of cytochrome c in trifluoroethanol

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