Thomas Leyh - Academia.edu (original) (raw)

Papers by Thomas Leyh

Research paper thumbnail of Allosteric Regulation of the ATP Sulfurylase Associated GTPase

Biochemistry Usa, 1995

ATP sulfurylase catalyzes and chemically links the hydrolysis of GTP and the synthesis of activat... more 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.

Research paper thumbnail of Superhyperfine coupling between metal ions at the active site of S-adenosylmethionine synthetase

Journal of the American Chemical Society, 1987

599 ordered oxygen overlayer. These perturbations are also manifest in the observed a-bonding of ... more 599 ordered oxygen overlayer. These perturbations are also manifest in the observed a-bonding of molecular ethylene at 200 K, as opposed to the di-0-bonded molecular ethylene that is observed on the Ru(001) surface.

Research paper thumbnail of The sulfate activation locus of Escherichia coli K12: Cloning, genetic, and enzymatic characterization

Journal of Biological Chemistry

The sulfate activation locus of Escherichia coli K12 has been cloned by complementation. The gene... more The sulfate activation locus of Escherichia coli K12 has been cloned by complementation. The genes and gene products of this locus have been characterized by correlating the enzyme activity, complementation patterns, and polypeptides associated with subclones of the cloned DNA. The enzymes of the sulfate activation pathway, ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (ATP:adenosine-5'-phosphosulfate 3'-phosphotransferase, EC 2.7.1.25) have been overproduced approximately 100-fold. Overproduction of ATP sulfurylase requires the expression of both the cysD gene, encoding a 27-kDa polypeptide, and a previously unidentified gene, denoted cysN, which encodes a 62-kDa polypeptide. Purification of ATP sulfurylase to homogeneity reveals that the enzyme is composed of two types of subunits which are encoded by cysD and cysN. Insertion of a kanamycin resistance gene into plasmid or chromosomal cysN prevents sulfate activation and decreases expression of the downstream cysC gene. cysC appears to be the APS kinase structural gene and encodes a 21-kDa polypeptide. The genes are adjacent and are transcribed counterclockwise on the E. coli chromosome in the order cysDNC. cysN and cysC are within the same operon and cysDNC are not in an operon containing cysHIJ.

Research paper thumbnail of γ-Phosphate Protonation and pH-dependent Unfolding of the Ras·GTP·Mg2+ Complex

Journal of Biological Chemistry

ABSTRACT

Research paper thumbnail of H-dependent unfolding of the Ras.GTP.Mg2 + complex: a vibrational spectroscopy study. J Biol Chem

Journal of Biological Chemistry

ABSTRACT

Research paper thumbnail of Synthesis of mevalonate- and fluorinated mevalonate prodrugs and their in vitro human plasma stability

European journal of medicinal chemistry, Jan 27, 2015

The mevalonate pathway is essential for the production of many important molecules in lipid biosy... more The mevalonate pathway is essential for the production of many important molecules in lipid biosynthesis. Inhibition of this pathway is the mechanism of statin cholesterol-lowering drugs, as well as the target of drugs to treat osteoporosis, to combat parasites, and to inhibit tumor cell growth. Unlike the human mevalonate pathway, the bacterial pathway appears to be regulated by diphosphomevalonate (DPM). Enzymes in the mevalonate pathway act to produce isopentenyl diphosphate, the product of the DPM decarboxylase reaction, utilize phosphorylated (charged) intermediates, which are poorly bioavailable. It has been shown that fluorinated DPMs (6-fluoro- and 6,6,6-trifluoro-5-diphosphomevalonate) are excellent inhibitors of the bacterial pathway; however, highly charged DPM and analogs are not bioavailable. To increase cellular permeability of mevalonate analogs, we have synthesized various prodrugs of mevalonate and 6-fluoro- and 6,6,6-trifluoromevalonate that can be enzymatically tr...

Research paper thumbnail of Standards for Reporting Enzyme Data: The STRENDA Consortium: What it aims to do and why it should be helpful

Perspectives in Science, 2014

Data on enzyme activities and kinetics have often been reported with insufficient experimental de... more Data on enzyme activities and kinetics have often been reported with insufficient experimental detail to allow their repetition. This paper discusses the objectives and recommendations of the Standards for Reporting Enzyme Data (STRENDA) project to define minimal experimental standards for the reporting enzyme functional data.

Research paper thumbnail of A large-scale protein-function database

Nature Chemical Biology, 2010

Research paper thumbnail of The Mycobacterium tuberculosis cysD and cysNC genes form a stress-induced operon that encodes a tri-functional sulfate-activating complex

Microbiology, 2004

Sulfur metabolism has been implicated in the virulence, antibiotic resistance and anti-oxidant de... more Sulfur metabolism has been implicated in the virulence, antibiotic resistance and anti-oxidant defence of Mycobacterium tuberculosis. Despite its human disease relevance, sulfur metabolism in mycobacteria has not yet been fully characterized. ATP sulfurylase catalyses the synthesis of activated sulfate (adenosine 59-phosphosulfate, APS), the first step in the reductive assimilation of sulfate. Expression of the M. tuberculosis cysD gene, predicted to encode the adenylyl-transferase subunit of ATP sulfurylase, is upregulated by the bacilli inside its preferred host, the macrophage. This study demonstrates that cysD and cysNC orthologues exist in M. tuberculosis and constitute an operon whose expression is induced by sulfur limitation and repressed by the presence of cysteine, a major end-product of sulfur assimilation. The cysDNC genes are also induced upon exposure to oxidative stress, suggesting regulation of sulfur assimilation by M. tuberculosis in response to toxic oxidants. To ensure that the cysDNC operon encoded the activities predicted by its primary sequence, and to begin to characterize the products of the operon, they were expressed in Escherichia coli, purified to homogeneity, and tested for their catalytic activities. The CysD and CysNC proteins were shown to form a multifunctional enzyme complex that exhibits the three linked catalytic activities that constitute the sulfate activation pathway.

Research paper thumbnail of Superhyperfine coupling between metal ions at the active site of S-adenosylmethionine synthetase

Journal of the American Chemical Society, 1987

599 ordered oxygen overlayer. These perturbations are also manifest in the observed a-bonding of ... more 599 ordered oxygen overlayer. These perturbations are also manifest in the observed a-bonding of molecular ethylene at 200 K, as opposed to the di-0-bonded molecular ethylene that is observed on the Ru(001) surface.

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

Journal of Biological Chemistry, 2005

The sulfate activation pathway is essential for the assimilation of sulfate and, in many bacteria... more The sulfate activation pathway is essential for the assimilation of sulfate and, in many bacteria, is comprised of three reactions: the synthesis of adenosine 5'-phosphosulfate (APS), the hydrolysis of GTP, and the 3'-phosphorylation of APS to produce 3'-phosphoadenosine 5'-phosphosulfate (PAPS), whose sulfuryl group is reduced or transferred to other metabolites. The entire sulfate activation pathway is organized into a single complex in Mycobacterium tuberculosis. Although present in many bacteria, these tripartite complexes have not been studied in detail. Initial rate characterization of the mycobacterial system reveals that it is poised for extremely efficient throughput: at saturating ATP, PAPS synthesis is 5800 times more efficient than APS synthesis. The APS kinase domain of the complex does not appear to form the covalent E.P intermediate observed in the closely related APS kinase from Escherichia coli. The stoichiometry of GTP hydrolysis and APS synthesis is 1:1, and the APS synthesis reaction is driven 1.1 x 10(6)-fold further during GTP hydrolysis; the system harnesses the full chemical potential of the hydrolysis reaction to the synthesis of APS. A key energy-coupling step in the mechanism is a ligand-induced isomerization that enhances the affinity of GTP and commits APS synthesis and GTP hydrolysis to the completion of the catalytic cycle. Ligand-induced increases in guanine nucleotide affinity observed in the mycobacterial system suggest that it too undergoes the energy-coupling isomerization.

Research paper thumbnail of Regulating Energy Transfer in the ATP Sulfurylase−GTPase System †

Biochemistry, 1998

ATP sulfurylase, isolated from Escherichia coli K-12, is a GTPase-target complex that catalyzes a... more 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 × 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‚PP i complex converts it into the central P i ‚GDP‚E‚APS‚PP i complex which catalyzes the energy-transfer reaction. Thus, P i 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 energycoupling 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.

Research paper thumbnail of GTPase Activation of ATP Sulfurylase: The Mechanism

Biochemistry, 1994

ATP sulfurylase from Escherichia coli K12 catalyzes two, coupled reactions: the hydrolysis of GTP... more ATP sulfurylase from Escherichia coli K12 catalyzes two, coupled reactions: the hydrolysis of GTP and the formation of activated sulfate (APS). At saturating levels of GTP, the initial rate of APS formation is stimulated 116-fold. The mechanism of this activation has been investigated using isotope trapping, mass spectrometry, and initial velocity kinetic techniques. In the presence of GTP, APS formation proceeds via nucleophilic attack of sulfate at the alpha-phosphoryl group of ATP. Isotope-trapping experiments demonstrate productive, random binding of ATP and GTP. ATP is hydrolyzed to yield AMP and PPi. AMP production requires GTP and is suppressible by sulfate, suggesting GTP-dependent formation of an E*AMP intermediate in the synthesis of APS. Studies using the hydrolysis-resistant nucleotide analogues AMPCPP and GMPPNP demonstrate that GTP hydrolysis precedes scision of the alpha-beta bond of ATP. Product inhibition studies indicate that PPi release occurs prior to the addition of sulfate and APS formation. These results are used to construct a proposed mechanism for the GTP-activated synthesis of APS.

Research paper thumbnail of Product Release during the First Turnover of the ATP Sulfurylase−GTPase †

Biochemistry, 2001

ATP sulfurylase, from Escherichia coli Kappa-12, is a GTPase target complex that catalyzes and co... more ATP sulfurylase, from Escherichia coli Kappa-12, is a GTPase target complex that catalyzes and couples the chemical potentials of two reactions: GTP hydrolysis and activated sulfate (APS) synthesis. Previous work suggested that the product release branch of the GTPase mechanism might include rate-determining release and/or isomerization step(s). Such steps are known to couple chemical potentials in other energy transducing systems. Rate-determining, product release step(s) were confirmed in the ATP sulfurylase-GTPase reaction by a burst of product in pre-steady-state, rapid-quench experiments. Classical rapid-quench experiments, which measure total product formation, do not allow the slow steps to be assigned to the release of a specific product, or to slow isomerization, because they do not distinguish solution-phase from enzyme-bound product. Assay systems that exclusively monitor solution-phase P(i) and GDP were used to obtain free product progress curves during the first turnover of ATP sulfurylase. Together, the free and total product data describe how the products partition between the enzyme surface and solution during the first turnover. In combination, the data provide the time dependence of the concentrations of specific product intermediates, AMP.PP(i).E.GDP.P(i) and AMP.PP(i).E.GDP, the rate constants for the release of P(i) (4.2 s(-1)) and GDP (4.8 s(-1)) from these complexes, respectively, and the equilibrium constant for the enzyme-bound, beta,gamma-bond cleavage reaction: [AMP.PP(i).E.GTP']/[AMP.PP(i).E.GDP.P(i)] = 0.7. The data are fit, using global analysis, to obtain a complete kinetic and energetic description of this GTPase reaction.

Research paper thumbnail of Allosteric Regulation of the ATP Sulfurylase Associated GTPase

Biochemistry, 1995

ATP sulfurylase catalyzes and chemically links the hydrolysis of GTP and the synthesis of activat... more 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.

Research paper thumbnail of Vibrational Structure of GDP and GTP Bound to RAS:  An Isotope-Edited FTIR Study †

Biochemistry, 2001

A complete vibrational description of the bonding of a ligand to a protein requires the assignmen... more A complete vibrational description of the bonding of a ligand to a protein requires the assignment of both symmetric and antisymmetric vibrational modes. The symmetric modes of isotopically enriched enzyme-bound ligands can be obtained by Raman difference spectroscopy, but until now, the antisymmetric modes, which require IR difference spectroscopy, have not been generally accessible. We have developed the methodology needed to perform IR difference spectroscopy, assign the antisymmetric modes, and accurately describe bonding. The method is used to assess the bonding changes that occur as Mg‚GDP and Mg‚GTP move from solution into the active site of RAS. Binding to RAS opens the nonbridging, O-P-O angle of the γ-phosphate of GTP by 2.7°, yet the angular freedom (dispersion of the O-P-O angle) of the γ-phosphate is comparable to that in solution. In contrast, the motion of the -phosphate of GDP is highly restricted, suggesting that it positions the γ-phosphate for nucleophilic attack. The ,γbridging O-P bond of bound GTP is slightly weakened, being lengthened by 0.005 Å in the active site, corresponding to a bond order decrease of 0.012 valence unit (vu). The observed binding changes are consistent with a RAS-mediated hydrolysis mechanism that parallels that for solution hydrolysis.

Research paper thumbnail of Isozyme Specific Allosteric Regulation of Human Sulfotransferase 1A1

Biochemistry, Jan 29, 2016

The human cytosolic sulfotransferases (SULTs) comprise a 13-member enzyme family that regulates t... more The human cytosolic sulfotransferases (SULTs) comprise a 13-member enzyme family that regulates the activities of hundreds, perhaps thousands of signaling small molecules via regiospecific transfer of the sulfuryl-moiety (-SO3) from PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the hydroxyls and amines of acceptors. Signaling molecules regulated by sulfonation include numerous steroid and thyroid hormones, epinephrine, serotonin, and dopamine. SULT1A1, a major phase II metabolism SULT isoform, is found at high concentration in liver and has recently been show to harbor two allosteric-binding sites, each of which binds a separate and complex class of compounds - the catechins (naturally occurring polyphenols) and NSAIDS. Among catechins, epigallocatechin gallate (EGCG) displays high affinity and specificity toward SULT1A1. The allosteric network associated with either site has yet to be defined. Here, using equilibrium binding and presteady state studies, the network is sho...

Research paper thumbnail of The Gate That Governs Sulfotransferase Selectivity

Biochemistry, Dec 20, 2012

Human cytosolic sulfotransferases (SULTs) transfer the sulfuryl moiety (-SO(3)) from activated su... more Human cytosolic sulfotransferases (SULTs) transfer the sulfuryl moiety (-SO(3)) 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 SULT2A1 to 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 nonequilibrium 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 anti-estrogen too large to pass through the closed pore, and estradiol, which is not restricted by closure. Equilibrium and pre-steady-state binding studies confirm that SULT1A1 undergoes a nucleotide-induced isomerzation that controls substrate selection.

Research paper thumbnail of Defining the Mandate of Proteomics in the Post-Genomics Era: Workshop Report ©2002 National Academy of Sciences, Washington, D.C., USA. Reprinted with permission from the National Academies Press for the National Academy of Sciences. All rights reserved. The original report may be viewed online a...

Molecular Amp Cellular Proteomics, Oct 1, 2002

Research in proteomics is the next logical step after genomics in understanding life processes at... more Research in proteomics is the next logical step after genomics in understanding life processes at the molecular level. In the largest sense proteomics encompasses knowledge of the structure, function and expression of all proteins in the biochemical or biological contexts of all organisms. Since that is an impossible goal to achieve, at least in our lifetimes, it is appropriate to set more realistic, achievable goals for the field. Up to now, primarily for reasons of feasibility, scientists have tended to concentrate on accumulating information about the nature of proteins and their absolute and relative levels of expression in cells (the primary tools for this have been 2D gel electrophoresis and mass spectrometry). Although these data have been useful and will continue to be so, the information inherent in the broader definition of proteomics must also be obtained if the true promise of the growing field is to be realized. Acquiring this knowledge is the challenge for researchers in proteomics and the means to support these endeavors need to be provided. An attempt has been made to present the major issues confronting the field of proteomics and two clear messages come through in this report. The first is that the mandate of proteomics is and should be much broader than is frequently recognized. The second is that proteomics is much more complicated than sequencing From the

Research paper thumbnail of Structure of the Ternary Complex of Phosphomevalonate Kinase: The Enzyme and Its Family t

Biochemistry Usa, Jul 1, 2009

The Galacto-, Homoserine-, Mevalonate-, Phosphomevalonate-kinase (GHMP) superfamily encompases a ... more The Galacto-, Homoserine-, Mevalonate-, Phosphomevalonate-kinase (GHMP) superfamily encompases a wide-range of protein function. Three members of the family (mevalonate kinase, phosphomevalonate kinase and diphosphomevalonate decarboxylase) comprise the mevalonate pathway found in S. pneumoniae and other organisms. We have determined the 1.9 Å crystal structure of phosphomevalonate kinase (PMK) from S. pneumoniae in complex with phosphomevalonate and AMPPNP·Mg 2+ . Comparison of the apo and ternary PMK structures suggests that ligand binding reverses the side-chain orientations of two anti-parallel lysines residues (100 and 101) with the result that lys101 is "switched" into a position in which its ammonium ion is in direct contact with the β,γ-bridging atom of the nucleotide, where it is expected to stabilize both the ground and transition states of the reaction. Analysis of all available GHMP kinase ternary-complex structures reveals that while their C α -scaffolds are highly conserved, their substrates bind in one of two conformations, which appear to be either reactive or non-reactive. The active site of PMK seems spacious enough to accommodate interconversion of the reactive and nonreactive conformers. A substantial fraction of the PMK active site is occupied by ordered water, which clusters near the charged regions of substrate. Notably, a water pentamer that interacts extensively with the reactive groups of both substrates was discovered at the active site.

Research paper thumbnail of Allosteric Regulation of the ATP Sulfurylase Associated GTPase

Biochemistry Usa, 1995

ATP sulfurylase catalyzes and chemically links the hydrolysis of GTP and the synthesis of activat... more 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.

Research paper thumbnail of Superhyperfine coupling between metal ions at the active site of S-adenosylmethionine synthetase

Journal of the American Chemical Society, 1987

599 ordered oxygen overlayer. These perturbations are also manifest in the observed a-bonding of ... more 599 ordered oxygen overlayer. These perturbations are also manifest in the observed a-bonding of molecular ethylene at 200 K, as opposed to the di-0-bonded molecular ethylene that is observed on the Ru(001) surface.

Research paper thumbnail of The sulfate activation locus of Escherichia coli K12: Cloning, genetic, and enzymatic characterization

Journal of Biological Chemistry

The sulfate activation locus of Escherichia coli K12 has been cloned by complementation. The gene... more The sulfate activation locus of Escherichia coli K12 has been cloned by complementation. The genes and gene products of this locus have been characterized by correlating the enzyme activity, complementation patterns, and polypeptides associated with subclones of the cloned DNA. The enzymes of the sulfate activation pathway, ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (ATP:adenosine-5'-phosphosulfate 3'-phosphotransferase, EC 2.7.1.25) have been overproduced approximately 100-fold. Overproduction of ATP sulfurylase requires the expression of both the cysD gene, encoding a 27-kDa polypeptide, and a previously unidentified gene, denoted cysN, which encodes a 62-kDa polypeptide. Purification of ATP sulfurylase to homogeneity reveals that the enzyme is composed of two types of subunits which are encoded by cysD and cysN. Insertion of a kanamycin resistance gene into plasmid or chromosomal cysN prevents sulfate activation and decreases expression of the downstream cysC gene. cysC appears to be the APS kinase structural gene and encodes a 21-kDa polypeptide. The genes are adjacent and are transcribed counterclockwise on the E. coli chromosome in the order cysDNC. cysN and cysC are within the same operon and cysDNC are not in an operon containing cysHIJ.

Research paper thumbnail of γ-Phosphate Protonation and pH-dependent Unfolding of the Ras·GTP·Mg2+ Complex

Journal of Biological Chemistry

ABSTRACT

Research paper thumbnail of H-dependent unfolding of the Ras.GTP.Mg2 + complex: a vibrational spectroscopy study. J Biol Chem

Journal of Biological Chemistry

ABSTRACT

Research paper thumbnail of Synthesis of mevalonate- and fluorinated mevalonate prodrugs and their in vitro human plasma stability

European journal of medicinal chemistry, Jan 27, 2015

The mevalonate pathway is essential for the production of many important molecules in lipid biosy... more The mevalonate pathway is essential for the production of many important molecules in lipid biosynthesis. Inhibition of this pathway is the mechanism of statin cholesterol-lowering drugs, as well as the target of drugs to treat osteoporosis, to combat parasites, and to inhibit tumor cell growth. Unlike the human mevalonate pathway, the bacterial pathway appears to be regulated by diphosphomevalonate (DPM). Enzymes in the mevalonate pathway act to produce isopentenyl diphosphate, the product of the DPM decarboxylase reaction, utilize phosphorylated (charged) intermediates, which are poorly bioavailable. It has been shown that fluorinated DPMs (6-fluoro- and 6,6,6-trifluoro-5-diphosphomevalonate) are excellent inhibitors of the bacterial pathway; however, highly charged DPM and analogs are not bioavailable. To increase cellular permeability of mevalonate analogs, we have synthesized various prodrugs of mevalonate and 6-fluoro- and 6,6,6-trifluoromevalonate that can be enzymatically tr...

Research paper thumbnail of Standards for Reporting Enzyme Data: The STRENDA Consortium: What it aims to do and why it should be helpful

Perspectives in Science, 2014

Data on enzyme activities and kinetics have often been reported with insufficient experimental de... more Data on enzyme activities and kinetics have often been reported with insufficient experimental detail to allow their repetition. This paper discusses the objectives and recommendations of the Standards for Reporting Enzyme Data (STRENDA) project to define minimal experimental standards for the reporting enzyme functional data.

Research paper thumbnail of A large-scale protein-function database

Nature Chemical Biology, 2010

Research paper thumbnail of The Mycobacterium tuberculosis cysD and cysNC genes form a stress-induced operon that encodes a tri-functional sulfate-activating complex

Microbiology, 2004

Sulfur metabolism has been implicated in the virulence, antibiotic resistance and anti-oxidant de... more Sulfur metabolism has been implicated in the virulence, antibiotic resistance and anti-oxidant defence of Mycobacterium tuberculosis. Despite its human disease relevance, sulfur metabolism in mycobacteria has not yet been fully characterized. ATP sulfurylase catalyses the synthesis of activated sulfate (adenosine 59-phosphosulfate, APS), the first step in the reductive assimilation of sulfate. Expression of the M. tuberculosis cysD gene, predicted to encode the adenylyl-transferase subunit of ATP sulfurylase, is upregulated by the bacilli inside its preferred host, the macrophage. This study demonstrates that cysD and cysNC orthologues exist in M. tuberculosis and constitute an operon whose expression is induced by sulfur limitation and repressed by the presence of cysteine, a major end-product of sulfur assimilation. The cysDNC genes are also induced upon exposure to oxidative stress, suggesting regulation of sulfur assimilation by M. tuberculosis in response to toxic oxidants. To ensure that the cysDNC operon encoded the activities predicted by its primary sequence, and to begin to characterize the products of the operon, they were expressed in Escherichia coli, purified to homogeneity, and tested for their catalytic activities. The CysD and CysNC proteins were shown to form a multifunctional enzyme complex that exhibits the three linked catalytic activities that constitute the sulfate activation pathway.

Research paper thumbnail of Superhyperfine coupling between metal ions at the active site of S-adenosylmethionine synthetase

Journal of the American Chemical Society, 1987

599 ordered oxygen overlayer. These perturbations are also manifest in the observed a-bonding of ... more 599 ordered oxygen overlayer. These perturbations are also manifest in the observed a-bonding of molecular ethylene at 200 K, as opposed to the di-0-bonded molecular ethylene that is observed on the Ru(001) surface.

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

Journal of Biological Chemistry, 2005

The sulfate activation pathway is essential for the assimilation of sulfate and, in many bacteria... more The sulfate activation pathway is essential for the assimilation of sulfate and, in many bacteria, is comprised of three reactions: the synthesis of adenosine 5'-phosphosulfate (APS), the hydrolysis of GTP, and the 3'-phosphorylation of APS to produce 3'-phosphoadenosine 5'-phosphosulfate (PAPS), whose sulfuryl group is reduced or transferred to other metabolites. The entire sulfate activation pathway is organized into a single complex in Mycobacterium tuberculosis. Although present in many bacteria, these tripartite complexes have not been studied in detail. Initial rate characterization of the mycobacterial system reveals that it is poised for extremely efficient throughput: at saturating ATP, PAPS synthesis is 5800 times more efficient than APS synthesis. The APS kinase domain of the complex does not appear to form the covalent E.P intermediate observed in the closely related APS kinase from Escherichia coli. The stoichiometry of GTP hydrolysis and APS synthesis is 1:1, and the APS synthesis reaction is driven 1.1 x 10(6)-fold further during GTP hydrolysis; the system harnesses the full chemical potential of the hydrolysis reaction to the synthesis of APS. A key energy-coupling step in the mechanism is a ligand-induced isomerization that enhances the affinity of GTP and commits APS synthesis and GTP hydrolysis to the completion of the catalytic cycle. Ligand-induced increases in guanine nucleotide affinity observed in the mycobacterial system suggest that it too undergoes the energy-coupling isomerization.

Research paper thumbnail of Regulating Energy Transfer in the ATP Sulfurylase−GTPase System †

Biochemistry, 1998

ATP sulfurylase, isolated from Escherichia coli K-12, is a GTPase-target complex that catalyzes a... more 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 × 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‚PP i complex converts it into the central P i ‚GDP‚E‚APS‚PP i complex which catalyzes the energy-transfer reaction. Thus, P i 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 energycoupling 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.

Research paper thumbnail of GTPase Activation of ATP Sulfurylase: The Mechanism

Biochemistry, 1994

ATP sulfurylase from Escherichia coli K12 catalyzes two, coupled reactions: the hydrolysis of GTP... more ATP sulfurylase from Escherichia coli K12 catalyzes two, coupled reactions: the hydrolysis of GTP and the formation of activated sulfate (APS). At saturating levels of GTP, the initial rate of APS formation is stimulated 116-fold. The mechanism of this activation has been investigated using isotope trapping, mass spectrometry, and initial velocity kinetic techniques. In the presence of GTP, APS formation proceeds via nucleophilic attack of sulfate at the alpha-phosphoryl group of ATP. Isotope-trapping experiments demonstrate productive, random binding of ATP and GTP. ATP is hydrolyzed to yield AMP and PPi. AMP production requires GTP and is suppressible by sulfate, suggesting GTP-dependent formation of an E*AMP intermediate in the synthesis of APS. Studies using the hydrolysis-resistant nucleotide analogues AMPCPP and GMPPNP demonstrate that GTP hydrolysis precedes scision of the alpha-beta bond of ATP. Product inhibition studies indicate that PPi release occurs prior to the addition of sulfate and APS formation. These results are used to construct a proposed mechanism for the GTP-activated synthesis of APS.

Research paper thumbnail of Product Release during the First Turnover of the ATP Sulfurylase−GTPase †

Biochemistry, 2001

ATP sulfurylase, from Escherichia coli Kappa-12, is a GTPase target complex that catalyzes and co... more ATP sulfurylase, from Escherichia coli Kappa-12, is a GTPase target complex that catalyzes and couples the chemical potentials of two reactions: GTP hydrolysis and activated sulfate (APS) synthesis. Previous work suggested that the product release branch of the GTPase mechanism might include rate-determining release and/or isomerization step(s). Such steps are known to couple chemical potentials in other energy transducing systems. Rate-determining, product release step(s) were confirmed in the ATP sulfurylase-GTPase reaction by a burst of product in pre-steady-state, rapid-quench experiments. Classical rapid-quench experiments, which measure total product formation, do not allow the slow steps to be assigned to the release of a specific product, or to slow isomerization, because they do not distinguish solution-phase from enzyme-bound product. Assay systems that exclusively monitor solution-phase P(i) and GDP were used to obtain free product progress curves during the first turnover of ATP sulfurylase. Together, the free and total product data describe how the products partition between the enzyme surface and solution during the first turnover. In combination, the data provide the time dependence of the concentrations of specific product intermediates, AMP.PP(i).E.GDP.P(i) and AMP.PP(i).E.GDP, the rate constants for the release of P(i) (4.2 s(-1)) and GDP (4.8 s(-1)) from these complexes, respectively, and the equilibrium constant for the enzyme-bound, beta,gamma-bond cleavage reaction: [AMP.PP(i).E.GTP']/[AMP.PP(i).E.GDP.P(i)] = 0.7. The data are fit, using global analysis, to obtain a complete kinetic and energetic description of this GTPase reaction.

Research paper thumbnail of Allosteric Regulation of the ATP Sulfurylase Associated GTPase

Biochemistry, 1995

ATP sulfurylase catalyzes and chemically links the hydrolysis of GTP and the synthesis of activat... more 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.

Research paper thumbnail of Vibrational Structure of GDP and GTP Bound to RAS:  An Isotope-Edited FTIR Study †

Biochemistry, 2001

A complete vibrational description of the bonding of a ligand to a protein requires the assignmen... more A complete vibrational description of the bonding of a ligand to a protein requires the assignment of both symmetric and antisymmetric vibrational modes. The symmetric modes of isotopically enriched enzyme-bound ligands can be obtained by Raman difference spectroscopy, but until now, the antisymmetric modes, which require IR difference spectroscopy, have not been generally accessible. We have developed the methodology needed to perform IR difference spectroscopy, assign the antisymmetric modes, and accurately describe bonding. The method is used to assess the bonding changes that occur as Mg‚GDP and Mg‚GTP move from solution into the active site of RAS. Binding to RAS opens the nonbridging, O-P-O angle of the γ-phosphate of GTP by 2.7°, yet the angular freedom (dispersion of the O-P-O angle) of the γ-phosphate is comparable to that in solution. In contrast, the motion of the -phosphate of GDP is highly restricted, suggesting that it positions the γ-phosphate for nucleophilic attack. The ,γbridging O-P bond of bound GTP is slightly weakened, being lengthened by 0.005 Å in the active site, corresponding to a bond order decrease of 0.012 valence unit (vu). The observed binding changes are consistent with a RAS-mediated hydrolysis mechanism that parallels that for solution hydrolysis.

Research paper thumbnail of Isozyme Specific Allosteric Regulation of Human Sulfotransferase 1A1

Biochemistry, Jan 29, 2016

The human cytosolic sulfotransferases (SULTs) comprise a 13-member enzyme family that regulates t... more The human cytosolic sulfotransferases (SULTs) comprise a 13-member enzyme family that regulates the activities of hundreds, perhaps thousands of signaling small molecules via regiospecific transfer of the sulfuryl-moiety (-SO3) from PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the hydroxyls and amines of acceptors. Signaling molecules regulated by sulfonation include numerous steroid and thyroid hormones, epinephrine, serotonin, and dopamine. SULT1A1, a major phase II metabolism SULT isoform, is found at high concentration in liver and has recently been show to harbor two allosteric-binding sites, each of which binds a separate and complex class of compounds - the catechins (naturally occurring polyphenols) and NSAIDS. Among catechins, epigallocatechin gallate (EGCG) displays high affinity and specificity toward SULT1A1. The allosteric network associated with either site has yet to be defined. Here, using equilibrium binding and presteady state studies, the network is sho...

Research paper thumbnail of The Gate That Governs Sulfotransferase Selectivity

Biochemistry, Dec 20, 2012

Human cytosolic sulfotransferases (SULTs) transfer the sulfuryl moiety (-SO(3)) from activated su... more Human cytosolic sulfotransferases (SULTs) transfer the sulfuryl moiety (-SO(3)) 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 SULT2A1 to 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 nonequilibrium 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 anti-estrogen too large to pass through the closed pore, and estradiol, which is not restricted by closure. Equilibrium and pre-steady-state binding studies confirm that SULT1A1 undergoes a nucleotide-induced isomerzation that controls substrate selection.

Research paper thumbnail of Defining the Mandate of Proteomics in the Post-Genomics Era: Workshop Report ©2002 National Academy of Sciences, Washington, D.C., USA. Reprinted with permission from the National Academies Press for the National Academy of Sciences. All rights reserved. The original report may be viewed online a...

Molecular Amp Cellular Proteomics, Oct 1, 2002

Research in proteomics is the next logical step after genomics in understanding life processes at... more Research in proteomics is the next logical step after genomics in understanding life processes at the molecular level. In the largest sense proteomics encompasses knowledge of the structure, function and expression of all proteins in the biochemical or biological contexts of all organisms. Since that is an impossible goal to achieve, at least in our lifetimes, it is appropriate to set more realistic, achievable goals for the field. Up to now, primarily for reasons of feasibility, scientists have tended to concentrate on accumulating information about the nature of proteins and their absolute and relative levels of expression in cells (the primary tools for this have been 2D gel electrophoresis and mass spectrometry). Although these data have been useful and will continue to be so, the information inherent in the broader definition of proteomics must also be obtained if the true promise of the growing field is to be realized. Acquiring this knowledge is the challenge for researchers in proteomics and the means to support these endeavors need to be provided. An attempt has been made to present the major issues confronting the field of proteomics and two clear messages come through in this report. The first is that the mandate of proteomics is and should be much broader than is frequently recognized. The second is that proteomics is much more complicated than sequencing From the

Research paper thumbnail of Structure of the Ternary Complex of Phosphomevalonate Kinase: The Enzyme and Its Family t

Biochemistry Usa, Jul 1, 2009

The Galacto-, Homoserine-, Mevalonate-, Phosphomevalonate-kinase (GHMP) superfamily encompases a ... more The Galacto-, Homoserine-, Mevalonate-, Phosphomevalonate-kinase (GHMP) superfamily encompases a wide-range of protein function. Three members of the family (mevalonate kinase, phosphomevalonate kinase and diphosphomevalonate decarboxylase) comprise the mevalonate pathway found in S. pneumoniae and other organisms. We have determined the 1.9 Å crystal structure of phosphomevalonate kinase (PMK) from S. pneumoniae in complex with phosphomevalonate and AMPPNP·Mg 2+ . Comparison of the apo and ternary PMK structures suggests that ligand binding reverses the side-chain orientations of two anti-parallel lysines residues (100 and 101) with the result that lys101 is "switched" into a position in which its ammonium ion is in direct contact with the β,γ-bridging atom of the nucleotide, where it is expected to stabilize both the ground and transition states of the reaction. Analysis of all available GHMP kinase ternary-complex structures reveals that while their C α -scaffolds are highly conserved, their substrates bind in one of two conformations, which appear to be either reactive or non-reactive. The active site of PMK seems spacious enough to accommodate interconversion of the reactive and nonreactive conformers. A substantial fraction of the PMK active site is occupied by ordered water, which clusters near the charged regions of substrate. Notably, a water pentamer that interacts extensively with the reactive groups of both substrates was discovered at the active site.