Estrogen sulfotransferase (EST) catalyzes the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to 17beta-estradiol (E2). The sulfation of E2 prevents it from binding to, and thereby activating, the estrogen receptor. The regulation of EST appears to be causally linked to tumorigenesis in the breast and endometrium. In this study, recombinant human EST is characterized, and the catalytic mechanism of the transfer reaction is investigated in ligand binding and initial rate experiments. The native enzyme is a dimer of 35-kDa subunits. The apparent equilibrium constant for transfer to E2 is (4.5 +/- 0.2) x 10(3) at pH 6.3 and T = 25 +/- 2 degrees C. Initial rate studies provide the kinetic constants for the reaction and suggest a sequential mechanism. E2 is a partial substrate inhibitor (Ki = 80 +/- 5 nM). The binding of two E2 per EST subunit suggests that the partial inhibition occurs through binding at an allosteric site. In addition to providing the dissociation constants for the ligand-enzyme complexes, binding studies demonstrate that each substrate binds independently to the enzyme and that both the E.PAP.E2S and E.PAP.E2 dead-end complexes form. These results strongly suggest a Random Bi Bi mechanism with two dead-end complexes.

Human cytosolic sulfotransferases (SULTs) transfer the sulfuryl-moiety (-SO3) from activated sulfate (3′-phosphoadenosine 5′-phosphosulfate, PAPS) to the hydroxyls and primary amines of numerous metabolites, drugs and xenobiotics. Receipt of the sulfuryl-group often radically alters acceptor-target interactions. How these enzymes select particular substrates from the hundreds of candidates in a complex cytosol remains an important question. Recent work reveals PAPS binding causes SULT2A1to undergo an isomerization that controls selectivity by constricting the opening through which acceptors must pass to enter the active site. The enzyme maintains an affinity for large substrates by isomerizing between the open and closed states with nucleotide bound. Here, the molecular basis of the nucleotide-induced closure is explored in equilibrium and non-equilibrium molecular dynamics simulations. The simulations predict that the active-site “cap,” which covers both the nucleotide and acceptor binding sites, opens and closes in response to nucleotide. The cap subdivides into nucleotide and acceptor halves whose motions, while coupled, exhibit an independence that can explain the isomerization. In-silico weakening of electrostatic interactions between the cap and base of the active site causes the acceptor-half of the cap to open and close while the nucleotide lid remains shut. Simulations predict that SULT1A1, the most abundant SULT in human liver, will utilize a similar selection mechanism. This prediction is tested using fulvestrant, an antiestrogen too large to pass through the closed pore, and estradiol, which is not restricted by closure. Equilibrium and presteady state binding studies confirm that SULT1A1 undergoes a nucleotide induced isomerzation that controls substrate selection.

Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosis universal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-Å-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

The cellular translational machinery (TM) synthesizes proteins using exclusively L-or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aatRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells.A lthough the ribosome catalyzes protein synthesis using more than 20 chemically diverse natural amino acids, these natural amino acids are all of L-or achiral configurations. As a consequence, the ability of the ribosome to incorporate L-aminoacyltRNAs (L-aa-tRNAs) with both a high degree of speed and accuracy has been the focus of decades of intense mechanistic and structural investigations (1-3). In contrast, the response of the ribosome to D-aa-tRNAs has not been as well characterized. Nonetheless, a comprehensive mechanistic understanding of how the translational machinery (TM) responds to D-aa-tRNAs is of interest for several reasons. First, improved incorporation of D-amino acids by the TM would be useful for protein engineering applications that seek to use the synthetic power of the ribosome to create novel polymers (4) as well as for mechanistic applications that seek to probe protein structure and folding with unnatural amino acids (5). Second, there is growing evidence that ribosomes may have to contend with D-aa-tRNAs in vivo: D-amino acids are synthesized by racemase enzymes (6) and can be found at high concentrations in cells (7), and a growing number of aa-tRNA synthetase (aaRS) enzymes exhibit the ability to misacylate tRNAs with D-amino acids (8...

Nature often colocalizes successive steps in a metabolic pathway. Such organization is predicted to increase the effective concentration of pathway intermediates near their recipient active sites and to enhance catalytic efficiency. Here, the pathway of a two-step reaction is modeled using a simple spherical approximation for the enzymes and substrate particles. Brownian dynamics are used to simulate the trajectory of a substrate particle as it diffuses between the active site zones of two different enzyme spheres. The results approximate distances for the most effective reaction pathways, indicating that the most effective reaction pathway is one in which the active sites are closely aligned. However, when the active sites are too close, the ability of the substrate to react with the first enzyme was hindered, suggesting that even the most efficient orientations can be improved for a system that is allowed to rotate or change orientation to optimize the likelihood of reaction at both sites.

The initial steps in assimilation of sulfate during cysteine biosynthesis entail sulfate uptake and sulfate activation by formation of adenosine 5'-phosphosulfate, conversion to 3'-phosphoadenosine 5'-phosphosulfate, and reduction to sulfite. Mutations in a previously uncharacterized Escherichia coli gene, cysQ, which resulted in a requirement for sulfite or cysteine, were obtained by in vivo insertion of transposons TnStacl and TnSsupF and by in vitro insertion of resistance gene cassettes. cysQ is at chromosomal position 95.7 min (kb 4517 to 4518) and is transcribed divergently from the adjacent cpdB gene. A TnStacl insertion just inside the 3' end of cysQ, with its isopropyl-4-D-thiogalactopyranoside-inducible tac promoter pointed toward the cysQ promoter, resulted in auxotrophy only when isopropyl-4-D-thiogalactopyranoside was present; this conditional phenotype was ascribed to collision between converging RNA polymerases or interaction between complementary antisense and cysQ mRNAs. The auxotrophy caused by cysQ null mutations was leaky in some but not all E. coli strains and could be compensated by mutations in unlinked genes. cysQ mutants were prototrophic during anaerobic growth. Mutations in cysQ did not affect the rate of sulfate uptake or the activities of ATP sulfurylase and its protein activator, which together catalyze adenosine 5'-phosphosulfate synthesis. Some mutations that compensated for cysQ null alleles resulted in sulfate transport defects. cysQ is identical to a gene called amtA, which had been thought to be needed for ammonium transport. Computer analyses, detailed elsewhere, revealed significant amino acid sequence homology between cysQ and suhB of E. coli and the gene for mammalian inositol monophosphatase. Previous work had suggested that 3'-phosphoadenoside 5'-phosphosulfate is toxic if allowed to accumulate, and we propose that CysQ helps control the pool of 3'-phosphoadenoside 5'-phosphosulfate, or its use in sulfite synthesis.The cysteine biosynthetic pathway (Fig. 1), a principal route of sulfur assimilation, involves more than 15 genes in at least five chromosomal regions in Escherichia coli and Salmonella typhimurium. It has been studied since the early days of physiological genetics in order to elucidate the roles of the individual genes, the control of their expression, and how the flow of metabolic intermediates is regulated (for a review, see reference 25). The transcription of most cys genes is positively controlled by the protein product of cysB and its coinducer, O-acetyl serine (also a cysteine precursor), during aerobic growth; transcription is repressed by sulfide, which is generated by reversal of the final biosynthetic step (Fig. 1). CysB seems not to be needed during anaerobic growth (3). The cysQ gene described here is also needed only during aerobic growth. It is inferred to act before sulfite formation, and hence this early part of the cysteine pathway is reviewed briefly below.The initial step, sulfate uptake, is mediated by a permease encoded by the cysT, cysW,...

Human SULT2A1 is one of two predominant sulfotransferases in liver and catalyzes transfer of the sulfuryl moiety (−SO 3 ) from activated sulfate (PAPS, 3′-phosphoadenosine 5-phosphosulfate) to hundreds of acceptors (metabolites and xenobiotics). Sulfation recodes the biologic activity of acceptors by altering their receptor interactions. The molecular basis on which these enzymes select and sulfonate specific acceptors from complex mixtures of competitors in vivo is a long-standing issue in the SULT field. Raloxifene, a synthetic steroid used in the prevention of osteoporosis, and dehydroepiandrosterone (DHEA), a ubiquitous steroid precusor, are reported to be sulfated efficiently by SULT2A1 in vitro, yet unlike DHEA, raloxifene is not sulfated in vivo. This selectivity was explored in initial rate and equilibrium binding studies that demonstrate pronounced binding antisynergy (21-fold) between PAPS and raloxifene, but not DHEA. Analysis of crystal structures suggests that PAP binding restricts access to the acceptor-binding pocket by restructuring a nine-residue segment of the pocket edge that constricts the active site opening, or "pore", that sieves substrates on the basis of their geometries. In silico docking predicts that raloxifene, which is considerably larger than DHEA, can bind only to the unliganded (open) enzyme, whereas DHEA binds both the open and closed forms. The predictions of these structures with regard to substrate binding are tested using equilibrium and pre-steady-state ligand binding studies, and the results confirm that a nucleotide-driven isomerization controls access to the acceptor-binding pocket and plays an important role in substrate selection by SULT2A1 and possibly other sulfotransferases.

This article is an overview of current research in the area of sulfate activation. Emphasis is placed on presenting unresolved issues in an appropriate context for critical evaluation by the reader. The energetics of sulfate activation is reevaluated in light of recent findings that demonstrate that the synthesis of activated sulfate is thermodynamically driven by GTP hydrolysis. The structural and functional bases of this GTPase activation are discussed in detail. The bonding and hydrolysis of the high-energy, phosphoric-sulfuric acid anhydride bond of activated sulfate are presented along with an analysis of the importance of the divalent cation and pyrophosphate protonation in the equilibria governing activated sulfate formation. The molecular genetics of sulfate assimilation in prokaryotes is reviewed with an emphasis on the regulation of the pathway. Recent discoveries connecting sulfate activation to plant/microbe symbiogenesis are presented, as are several examples of the importance of activated sulfate in human metabolism and disease.