Transient-state Kinetic Analysis Research Articles

Mechanism of the Multistep Catalytic Cycle of 6-Hydroxynicotinate 3-Monooxygenase Revealed by Global Kinetic Analysis

The class A flavoenzyme 6-hydroxynicotinate 3-monooxygenase (NicC) catalyzes a rare decarboxylative hydroxylation reaction in the degradation of nicotinate by aerobic bacteria. While the structure and critical residues involved in catalysis have been reported, the mechanism of this multistep enzyme has yet to be determined. A kinetic understanding of the NicC mechanism would enable comparison to other phenolic hydroxylases and illuminate its bioengineering potential for remediation of N-heterocyclic aromatic compounds. Toward these goals, transient state kinetic analyses by stopped-flow spectrophotometry were utilized to follow rapid changes in flavoenzyme absorbance spectra during all three stages of NicC catalysis: (1) 6-HNA binding; (2) NADH binding and FAD reduction; and (3) O2 binding with C4a-adduct formation, substrate hydroxylation, and FAD regeneration. Global kinetic simulations by numeric integration were used to supplement analytical fitting of time-resolved data and establish a kinetic mechanism. Results indicate that 6-HNA binding is a two-step process that substantially increases the affinity of NicC for NADH and enables the formation of a charge-transfer-complex intermediate to enhance the rate of flavin reduction. Singular value decomposition of the time-resolved spectra during the reaction of the substrate-bound, reduced enzyme with dioxygen provides evidence for the involvement of C4a-hydroperoxy-flavin and C4a-hydroxy-flavin intermediates in NicC catalysis. Global analysis of the full kinetic mechanism suggests that steady-state catalytic turnover is partially limited by substrate hydroxylation and C4a-hydroxy-flavin dehydration to regenerate the flavoenzyme. Insights gleaned from the kinetic model and determined microscopic rate constants provide a fundamental basis for understanding NicC's substrate specificity and reactivity.

Transient-State Kinetic Analysis of the RNA Polymerase II Nucleotide Incorporation Mechanism.

Eukaryotic RNA polymerase II (Pol II) is an essential enzyme that lies at the core of eukaryotic biology. Due to its pivotal role in gene expression, Pol II has been subjected to a substantial number of investigations. We aim to further our understanding of Pol II nucleotide incorporation by utilizing transient-state kinetic techniques to examine Pol II single nucleotide addition on the millisecond time scale. We analyzed Saccharomyces cerevisiae Pol II incorporation of ATP or an ATP analog, Sp-ATP-α-S. Here we have measured the rate constants governing individual steps of the Pol II transcription cycle in the presence of ATP or Sp-ATP-α-S. These results suggest that Pol II catalyzes nucleotide incorporation by binding the next cognate nucleotide and immediately catalyzes bond formation and bond formation is either followed by a conformational change or pyrophosphate release. By comparing our previously published RNA polymerase I (Pol I) and Pol I lacking the A12 subunit (Pol I ΔA12) results that we collected under the same conditions with the identical technique, we show that Pol II and Pol I ΔA12 exhibit similar nucleotide addition mechanisms. This observation indicates that removal of the A12 subunit from Pol I results in a Pol II like enzyme. Taken together, these data further our collective understanding of Pol II's nucleotide incorporation mechanism and the evolutionary divergence of RNA polymerases across the three domains of life.

Investigating Substrate Specificity For 6‐Hydroxynicotinate 3‐Monooxygenase (NicC) With Coumalic Acid: Consequences To Catalysis Of Replacing or Removing The Ring Nitrogen

6‐Hydroxynicotinate 3‐monooxygenase (NicC) is a group A flavin dependent monooxygenase (FDM) that catalyzes the regioselective decarboxylative hydroxylation of 6‐HNA into 2,5‐dihydroxypyridine (DHP) with the concomitant oxidation of NADH to NAD+. This enzyme‐catalyzed reaction involves three segments: (1) substrate binding, (2) FAD reduction by NADH, and (3) hydroxylation‐decarboxylation of 6‐HNA through a hydroperoxy‐flavin intermediate. Past studies have shown that NicC can catalyze this reaction with alternate aromatic substrates including 4‐hydroxybenzoic acid (4‐HBA), a substrate analogue with a homocyclic ring (i.e. lacking the ring nitrogen). However, 4‐HBA is bound weakly (355 ± 66 μM) and only 50% of the reaction progresses to substrate hydroxylation, versus hydroperoxy‐flavin degradation to H2O2, suggesting the importance of the ring nitrogen in the coupling efficiency of NicC. To further explore the role of the ring nitrogen, the efficiency of the reaction with coumalic acid (CA) was determined. The NicC‐CA dissociation constant measured by monitoring the change in absorbance at 450nm (flavin) shows that NicC binds CA (Kd = 5 ± 1 μM) 10x more tightly than 6‐HNA (Kd = 58 ± 12 μM). Binding of CA by NicC is an isothermal process (∆H = 0), indicating that it is driven by a favorable change in entropy. Analysis of the reaction products by Q‐TOF MS indicates that NicC catalyzes the decarboxylative hydroxylation of CA into 5‐hydroxy‐2H‐pyrane‐2‐one. However, coupling experiments by HPLC analysis and detection of H2O2 by a fluorescence assay both indicate that the reaction with CA is highly uncoupled with only minor 5‐hydroxy‐2H‐pyrane‐2‐one formation. Steady‐state kinetic analyses show that CA bound to NicC enhances its rate of NADH oxidation 16‐fold (by comparison, 6‐HNA enhances the rate of NicC NADH oxidation 30‐fold). Transient state kinetic analyses by stopped flow under anaerobic conditions to permit single‐turnover kinetics shows that, unlike in the binding of 6‐HNA, the binding of CA does not enable the formation of a charge‐transfer complex prior to FAD reduction. These results indicate that an H‐bond accepting group in the aromatic ring at position 1 is important for establishing the correct conformation of the ES complex to enable effective binding, flavin reduction and, ultimately, substrate hydroxylation.

Transient-state kinetic analysis of multi-nucleotide addition catalyzed by RNA polymerase I

RNA polymerases execute the first step in gene expression: transcription of DNA into RNA. Eukaryotes, unlike prokaryotes, express at least three specialized nuclear multisubunit RNA polymerases (Pol I, Pol II, and Pol III). RNA polymerase I (Pol I) synthesizes the most abundant RNA, ribosomal RNA. Nearly 60% of total transcription is devoted to ribosomal RNA synthesis, making it one of the cell’s most energy consuming tasks. While a kinetic mechanism for nucleotide addition catalyzed by Pol I has been reported, it remains unclear to what degree different nucleotide sequences impact the incorporation rate constants. Furthermore, it is currently unknown if the previous investigation of a single-nucleotide incorporation was sensitive to the translocation step. Here, we show that Pol I exhibits considerable variability in both kmax and K1/2values using an in vitro multi-NTP incorporation assay measuring AMP and GMP incorporations. We found the first two observed nucleotide incorporations exhibited faster kmax-values (∼200 s−1) compared with the remaining seven positions (∼60 s−1). Additionally, the average K1/2 for ATP incorporation was found to be approximately threefold higher compared with GTP, suggesting Pol I has a tighter affinity for GTP compared with ATP. Our results demonstrate that Pol I exhibits significant variability in the observed rate constant describing each nucleotide incorporation. Understanding of the differences between the Pol enzymes will provide insight on the evolutionary pressures that led to their specialized roles. Therefore, the findings resulting from this work are critically important for comparisons with other polymerases across all domains of life.

Staphylopine and pseudopaline dehydrogenase from bacterial pathogens catalyze reversible reactions and produce stereospecific metallophores

Pseudopaline and staphylopine are opine metallophores biosynthesized by Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The final step in opine metallophore biosynthesis is the condensation of the product of a nicotianamine (NA) synthase reaction (i.e. l-HisNA for pseudopaline and d-HisNA for staphylopine) with an α-keto acid (α-ketoglutarate for pseudopaline and pyruvate for staphylopine), which is performed by an opine dehydrogenase. We hypothesized that the opine dehydrogenase reaction would be reversible only for the opine metallophore product with (R)-stereochemistry at carbon C2 of the α-keto acid (prochiral prior to catalysis). A kinetic analysis using stopped-flow spectrometry with (R)- or (S)-staphylopine and kinetic and structural analysis with (R)- and (S)-pseudopaline confirmed catalysis in the reverse direction for only (R)-staphylopine and (R)-pseudopaline, verifying the stereochemistry of these two opine metallophores. Structural analysis at 1.57-1.85 Å resolution captured the hydrolysis of (R)-pseudopaline and allowed identification of a binding pocket for the l-histidine moiety of pseudopaline formed through a repositioning of Phe-340 and Tyr-289 during the catalytic cycle. Transient-state kinetic analysis revealed an ordered release of NADP+ followed by staphylopine, with staphylopine release being the rate-limiting step in catalysis. Knowledge of the stereochemistry for opine metallophores has implications for future studies involving kinetic analysis, as well as opine metallophore transport, metal coordination, and the generation of chiral amines for pharmaceutical development.

Transient-State Kinetic Analysis of the RNA Polymerase I Nucleotide Incorporation Mechanism

Eukaryotes express three or more multisubunit nuclear RNA polymerases (Pols) referred to as Pols I, II, and III, each of which synthesizes a specific subset of RNAs. Consistent with the diversity of their target genes, eukaryotic cells have evolved divergent cohorts of transcription factors and enzymatic properties for each RNA polymerase system. Over the years, many trans-acting factors that orchestrate transcription by the individual Pols have been described; however, little effort has been devoted to characterizing the molecular mechanisms of Pol I activity. To begin to address this gap in our understanding of eukaryotic gene expression, here we establish transient-state kinetic approaches to characterize the nucleotide incorporation mechanism of Pol I. We collected time courses for single turnover nucleotide incorporation reactions over a range of substrate ATP concentrations that provide information on both Pol I’s nucleotide addition and nuclease activities. The data were analyzed by model-independent and model-dependent approaches, resulting in, to our knowledge, the first minimal model for the nucleotide addition pathway for Pol I. Using a grid searching approach we provide rigorous bounds on estimated values of the individual elementary rate constants within the proposed model. This work reports the most detailed analysis of Pol I mechanism to date. Furthermore, in addition to their use in transient state kinetic analyses, the computational approaches applied here are broadly applicable to global optimization problems.

Transient-State Kinetic Analysis of the RNA Polymerase I Nucleotide Addition Cycle

RNA polymerases (Pols) are a diverse class of enzymes that fulfill an array of biological requirements. In eukaryotes the task of nuclear RNA synthesis is apportioned among three distinct Pols. Unlike all other cellular Pols, Pol I recognizes a single promoter and synthesizes a single target transcript. Pol I's RNA product is ultimately processed into the three largest RNAs of the ribosome. It has long been recognized that cellular protein synthetic capacity is linked to cell proliferation and transformation. In summary, Pol I is centrally located in a process requisite to tumorgenesis. Recently, the Pol I system has received attention as a target of chemotherapeutic intervention. Despite Pol I's involvement in cancer, there are no mechanistic data available to further its utility as a drug target. In addition, it is not clear why eukaryotes have evolved three distinct Pols to perform the task of nuclear RNA synthesis. A thorough understanding of the molecular mechanisms governing Pol I transcription will aid the development of Pol I - specific inhibitors as well as further our understanding of the necessity of three nuclear Pols.We have developed techniques that enable the reconstitution of Pol I elongation complexes from purified protein and nucleic acid components. Combining reconstituted elongation complexes with transient-state kinetic approaches we have characterized the Pol I nucleotide addition cycle and developed a minimal kinetic framework describing the reaction pathway. Combining these approaches with structure-based mutagenesis will enable elucidation of the molecular mechanisms governing Pol I - catalyzed RNA synthesis.

A mechanistic and structural investigation of modified derivatives of the diaryltriazine class of NNRTIs targeting HIV-1 reverse transcriptase

BackgroundNon-nucleoside reverse transcriptase inhibitors (NNRTIs) are vital in treating HIV-1 infection by inhibiting reverse transcriptase (RT). Drug toxicity and resistance drive the need for effective new inhibitors with improved physiochemical properties and potent antiviral activity. Computer-aided and structure-based drug design have guided the addition of solubilizing substituents to the diaryltriazine scaffold. These derivatives have markedly improved solubility and maintain low nanomolar antiviral activity against RT. The molecular and structural basis of inhibition for this series was determined to facilitate future inhibitor development with improved pharmacological profiles. MethodsThe molecular mechanism of inhibition was investigated using transient-state kinetic analysis. Crystal structures of RT in complex with each inhibitor were obtained to investigate the structural basis of inhibition. ResultsThe diaryltriazine and its morpholine derivative have RT inhibition constants of 9±2nM and 14±4nM, respectively. They adopt differential binding modes within the non-nucleoside inhibitor binding pocket to distort the catalytic site geometry and primer grip regions. The novel morpholinopropoxy substituent extends into the RT/solvent interface of the NNIBP. ConclusionsKinetic and structural analyses show that these inhibitors behave as conventional NNRTIs and inhibit the polymerization step. This study confirms that appending solubilizing substituents on the azine ring of diaryltriazine class of NNRTIs that extend into the RT/solvent interface effectively maintains low nanomolar potency and improves physiochemical properties. General significanceThe modification of NNRTI scaffolds with solubilizing substituents, which extend into the RT/solvent interface, yields potent antivirals and is an effective strategy for developing novel inhibitors with improved pharmacological properties.

Kinetic Study of the Inhibition Mechanism of Dehaloperoxidase-Hemoglobin A by 4-Bromophenol

The mechanism of dehaloperoxidase-hemoglobin (DHP) inhibition by 4-bromophenol (4-BP) was investigated using Michealis-Menten and transient-state kinetic analyses. Transient-state kinetics using the stopped-flow technique to mix DHP and H2O2 in the presence of inhibitor concentrations less than 10-fold greater than the enzyme concentration show that 4-BP does not fully impede H2O2 entering the distal pocket to activate DHP. It is not clear whether an oxoferryl intermediate is formed under these conditions and there may be alternative pathways for H2O2 reaction in the 4-BP bound form of DHP. Two new species have been identified during the reaction of 4-BP bound form of DHP in the transient-state kinetic experiment by using Singular Value Decomposition (SVD) and global-fitting analysis. Rather than forming Compound ES in the unbound form, an inhibitor bound intermediate that possesses blue-shifted Soret band and a double peaked Q-band is observed. This intermediate is subsequently converted to the end-point species that is distinguished from Compound RH formed in the uninhibited enzyme. Bench-top mixing kinetics of DHP were conducted in order to determine the inhibitor binding constant and to understand the enzyme inhibition mechanism from a thermodynamic perspective. It was found that the inhibition constant, Ki, decreased from 2.56 mM to 0.15 mM over the temperature range from 283 to 298 K, which permits determination of the enthalpy and entropy for inhibitor binding as -135.5 ± 20.9 kJ/mol and 526.1 ± 71.9 J/(mol·K), respectively, leading to the conclusion that inhibitor binding is entropically driven.

The Catalytic Serine of meta-Cleavage Product Hydrolases Is Activated Differently for C–O Bond Cleavage Than for C–C Bond Cleavage

meta-Cleavage product (MCP) hydrolases catalyze C-C bond fission in the aerobic catabolism of aromatic compounds by bacteria. These enzymes utilize a Ser-His-Asp triad to catalyze hydrolysis via an acyl-enzyme intermediate. BphD, which catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) in biphenyl degradation, catalyzed the hydrolysis of an ester analogue, p-nitrophenyl benzoate (pNPB), with a k(cat) value (6.3 ± 0.5 s(-1)) similar to that of HOPDA (6.5 ± 0.5 s(-1)). Consistent with the breakdown of a shared intermediate, product analyses revealed that BphD catalyzed the methanolysis of both HOPDA and pNPB, partitioning the products to benzoic acid and methyl benzoate in similar ratios. Turnover of HOPDA was accelerated up to 4-fold in the presence of short, primary alcohols (methanol > ethanol > n-propanol), suggesting that deacylation is rate-limiting during catalysis. In the steady-state hydrolysis of HOPDA, k(cat)/K(m) values were independent of methanol concentration, while both k(cat) and K(m) values increased with methanol concentration. This result was consistent with a simple model of nucleophilic catalysis. Although the enzyme could not be saturated with pNPB at methanol concentrations of >250 mM, k(obs) values from the steady-state turnover of pNPB at low methanol concentrations were also consistent with a nucleophilic mechanism of catalysis. Finally, transient-state kinetic analysis of pNPB hydrolysis by BphD variants established that substitution of the catalytic His reduced the rate of acylation by more than 3 orders of magnitude. This suggests that for pNPB hydrolysis, the serine nucleophile is activated by the His-Asp dyad. In contrast, rapid acylation of the H265Q variant during C-C bond cleavage suggests that the serinate forms via a substrate-assisted mechanism. Overall, the data indicate that ester hydrolysis proceeds via the same acyl-enzyme intermediate as that of the physiological substrate but that the serine nucleophile is activated via a different mechanism.

Assembly, Purification, and Pre-steady-state Kinetic Analysis of Active RNA-dependent RNA Polymerase Elongation Complex

NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase·RNA complex suitable for mechanistic and structural studies has remained elusive. Here, we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B·primer·template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so that the purified complex was suitable for pre-steady-state kinetic analyses of polymerase activity. Single turnover kinetic studies showed that CTP is incorporated with apparent K(d) and k(pol) values of 39 ± 3 μM and 16 ± 1 s(-1), respectively, giving a specificity constant of k(pol)/K(d) of 0.41 μM(-1) s(-1). The kinetics of multiple nucleotide incorporation during processive elongation also were determined. This work establishes a novel way to generate a highly active elongation complex of the medically important NS5B polymerase for structural and functional studies.

Kinetic Mechanism of Active Site Assembly and Chemical Catalysis of DNA Polymerase β

It has been inferred from structural and computational studies that the mechanism of DNA polymerases involves subtle but important discrete steps that occur between binding and recognition of the correct dNTP and chemical catalysis. These steps potentially include local conformational changes involving active site residues, reorganization of Mg(2+)-coordinating ligands, and proton transfer. Here we address this broad issue by conducting extensive transient state kinetic analyses of DNA polymerase β (Pol β). We also performed kinetic simulations to evaluate alternative kinetic models. These studies provide some support for two-step subdomain closing and define constraints under which a kinetically significant prechemistry step can occur. To experimentally identify additional microscopic steps, we developed a stopped flow absorbance assay to measure proton formation that occurs during catalysis. These studies provide direct evidence that formation of the enzyme-bound 3'-O(-) nucleophile is rate determining for chemistry. We additionally show that at low pH the chemical step is rate limiting for catalysis, but at high pH, a postchemistry conformational step is rate limiting due to a pH-dependent increase in the rate of nucleotidyl transfer. Finally, we performed exhaustive analyses of [Mg(2+)] and pH effects. In contrast to published studies, the results suggest an irregular pH dependence of k(pol), which is consistent with general base catalysis involving cooperativity between two or more protonic residues. Overall, the results represent significant advancement in the kinetic mechanism of Pol β and also reconcile some computational and experimental findings.

A Transient Kinetic Analysis of PRMT1 Catalysis

Post-translational modifications (PTMs) are important strategies used by eukaryotic organisms to modulate their phenotypes. One of the well-studied PTMs, arginine methylation, is catalyzed by protein arginine methyltransferases (PRMTs) with SAM as the methyl donor. The functions of PRMTs have been broadly studied in different biological processes and diseased states, but the molecular basis for arginine methylation is not well-defined. In this study, we report the transient-state kinetic analysis of PRMT1 catalysis. The fast association and dissociation rates suggest that PRMT1 catalysis of histone H4 methylation follows a rapid equilibrium sequential kinetic mechanism. The data give direct evidence that the chemistry of methyl transfer is the major rate-limiting step and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, from the stopped-flow fluorescence measurements, we have identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. These results provide new insights into the mechanism of arginine methylation and the rational design of PRMT inhibitors.

Transient-state Kinetic Analysis of Transcriptional Activator·DNA Complexes Interacting with a Key Coactivator

Several lines of evidence suggest that the prototypical amphipathic transcriptional activators Gal4, Gcn4, and VP16 interact with the key coactivator Med15 (Gal11) during transcription initiation despite little sequence homology. Recent cross-linking data further reveal that at least two of the activators utilize the same binding surface within Med15 for transcriptional activation. To determine whether these three activators use a shared binding mechanism for Med15 recruitment, we characterized the thermodynamics and kinetics of Med15·activator·DNA complex formation by fluorescence titration and stopped-flow techniques. Combination of each activator·DNA complex with Med15 produced biphasic time courses. This is consistent with a minimum two-step binding mechanism composed of a bimolecular association step limited by diffusion, followed by a conformational change in the Med15·activator·DNA complex. Furthermore, the equilibrium constant for the conformational change (K(2)) correlates with the ability of an activator to stimulate transcription. VP16, the most potent of the activators, has the largest K(2) value, whereas Gcn4, the least potent, has the smallest value. This correlation is consistent with a model in which transcriptional activation is regulated at least in part by the rearrangement of the Med15·activator·DNA ternary complex. These results are the first detailed kinetic characterization of the transcriptional activation machinery and provide a framework for the future design of potent transcriptional activators.

Heat Maps for Intramolecular Communication in an RNP Enzyme Encoding Glutamine

Allosteric signaling within large ribonucleoproteins modulates both catalytic function and biological specificity, but the spatial extent and quantitative magnitudes of long-distance free-energy couplings have yet to be well characterized. Here, we employ pre-steady-state kinetics to generate a comprehensive mapping of intramolecular communication in the glutaminyl-tRNA synthetase:tRNA(Gln) complex. Alanine substitution at 29 positions across the protein-RNA interface reveals distinct coupling amplitudes for glutamine binding and aminoacyl-tRNA formation on the enzyme, respectively, implying the existence of multiple signaling pathways. Structural models suggest that long-range signal propagation from the tRNA anticodon is dynamically driven, whereas shorter pathways are mediated by induced-fit rearrangements. Seven protein contacts with the distal tRNA vertical arm each weaken glutamine binding affinity across distances up to 40 Å, demonstrating that negative allosteric coupling plays a key role in enforcing the selective RNA-amino acid pairing at the heart of the genetic code.

Histidine 197 in Release Factor 1 Is Essential for A Site Binding and Peptide Release

Class I peptide release factors 1 and 2 (RF1 and RF2, respectively) recognize the stop codons in the ribosomal decoding center and catalyze peptidyl-tRNA hydrolysis. High-fidelity stop codon recognition by these release factors is essential for accurate peptide synthesis and ribosome recycling. X-ray crystal structures of RF1 and RF2 bound to the ribosome have identified residues in the mRNA-protein interface that appear to be critical for stop codon recognition. Especially interesting is a conserved histidine in all bacterial class I release factors that forms a stacking interaction with the second base of the stop codon. Here we analyzed the functional significance of this conserved histidine (position 197 in Escherichia coli) of RF1 by point mutagenesis to alanine. Equilibrium binding studies and transient-state kinetic analysis have shown that the histidine is essential for binding with high affinity to the ribosome. Furthermore, analysis of the binding data indicates a conformational change within the RF1·ribosome complex that results in a more tightly bound state. The rate of peptidyl-tRNA hydrolysis was also reduced significantly, more than the binding data would suggest, implying a defect in the orientation of the GGQ domain without the histidine residue.

Global Fit Analysis of Glucose Binding Curves Reveals a Minimal Model for Kinetic Cooperativity in Human Glucokinase

Human pancreatic glucokinase is a monomeric enzyme that displays kinetic cooperativity, a feature that facilitates enzyme-mediated regulation of blood glucose levels in the body. Two theoretical models have been proposed to describe the non-Michaelis-Menten behavior of human glucokinase. The mnemonic mechanism postulates the existence of one thermodynamically favored enzyme conformation in the absence of glucose, whereas the ligand-induced slow transition model (LIST) requires a preexisting equilibrium between two enzyme species that interconvert with a rate constant slower than turnover. To investigate whether either of these mechanisms is sufficient to describe glucokinase cooperativity, a transient-state kinetic analysis of glucose binding to the enzyme was undertaken. A complex, time-dependent change in enzyme intrinsic fluorescence was observed upon exposure to glucose, which is best described by an analytical solution comprised of the sum of four exponential terms. Transient-state glucose binding experiments conducted in the presence of increasing glycerol concentrations demonstrate that three of the observed rate constants decrease with increasing viscosity. Global fit analyses of experimental glucose binding curves are consistent with a kinetic model that is an extension of the LIST mechanism with a total of four glucose-bound binary complexes. The kinetic model presented herein suggests that glucokinase samples multiple conformations in the absence of ligand and that this conformational heterogeneity persists even after the enzyme associates with glucose.

Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia

Giardia lamblia arginine deiminase (GlAD), the topic of this paper, belongs to the hydrolase branch of the guanidine-modifying enzyme superfamily, whose members employ Cys-mediated nucleophilic catalysis to promote deimination of l-arginine and its naturally occurring derivatives. G. lamblia is the causative agent in the human disease giardiasis. The results of RNAi/antisense RNA gene-silencing studies reported herein indicate that GlAD is essential for G. lamblia trophozoite survival and thus, a potential target for the development of therapeutic agents for the treatment of giardiasis. The homodimeric recombinant protein was prepared in Escherichia coli for in-depth biochemical characterization. The 2-domain GlAD monomer consists of a N-terminal domain that shares an active site structure (depicted by an in silico model) and kinetic properties (determined by steady-state and transient state kinetic analysis) with its bacterial AD counterparts, and a C-terminal domain of unknown fold and function. GlAD was found to be active over a wide pH range and to accept l-arginine, l-arginine ethyl ester, N α-benzoyl- l-arginine, and N ω-amino- l-arginine as substrates but not agmatine, l-homoarginine, N α-benzoyl- l-arginine ethyl ester or a variety of arginine-containing peptides. The intermediacy of a Cys424–alkylthiouronium ion covalent enzyme adduct was demonstrated and the rate constants for formation ( k 1 = 80 s −1) and hydrolysis ( k 2 = 35 s −1) of the intermediate were determined. The comparatively lower value of the steady-state rate constant ( k cat = 2.6 s −1), suggests that a step following citrulline formation is rate-limiting. Inhibition of GlAD using Cys directed agents was briefly explored. S-Nitroso- l-homocysteine was shown to be an active site directed, irreversible inhibitor whereas N ω-cyano- l-arginine did not inhibit GlAD but instead proved to be an active site directed, irreversible inhibitor of the Bacillus cereus AD.

Mechanism of 4-Chlorobenzoate:Coenzyme A Ligase Catalysis

Within the accompanying paper in this issue (Reger et al. (2008) Biochemistry, 47, 8016-8025) we reported the X-ray structure of 4-chlorobenzoate:CoA ligase (CBL) bound with 4-chlorobenzoyl-adenylate (4-CB-AMP) and the X-ray structure of CBL bound with 4-chlorophenacyl-CoA (4-CP-CoA) (an inert analogue of the product 4-chlorobenzoyl-coenzyme A (4-CB-CoA)) and AMP. These structures defined two CBL conformational states. In conformation 1, CBL is poised to catalyze the adenylation of 4-chlorobenzoate (4-CB) with ATP (partial reaction 1), and in conformation 2, CBL is poised to catalyze the formation of 4-CB-CoA from 4-CB-AMP and CoA (partial reaction 2). These two structures showed that, by switching from conformation 1 to conformation 2, the cap domain rotates about the domain linker and thereby changes its interface with the N-terminal domain. The present work was carried out to determine the contributions made by each of the active site residues in substrate/cofactor binding and catalysis, and also to test the role of domain alternation in catalysis. In this paper, we report the results of steady-state kinetic and transient state kinetic analysis of wild-type CBL and of a series of site-directed CBL active site mutants. The major findings are as follows. First, wild-type CBL is activated by Mg (2+) (a 12-75-fold increase in activity is observed depending on assay conditions) and its kinetic mechanism (ping-pong) supports the structure-derived prediction that PP i dissociation must precede the switch from conformation 1 to conformation 2 and therefore CoA binding. Also, transient kinetic analysis of wild-type CBL identified the rate-limiting step of the catalyzed reaction as one that follows the formation of 4-CB-CoA (viz. CBL conformational change and/or product dissociation). The single turnover rate of 4-CB and ATP to form 4-CB-AMP and PP i ( k = 300 s (-1)) is not affected by the presence of CoA, and it is approximately 3-fold faster than the turnover rate of 4-CB-AMP and CoA to form 4-CB-CoA and AMP ( k = 120 s (-1)). Second, the active site mutants screened via steady-state kinetic analysis were ranked based on the degree of reduction observed in any one of the substrate k cat/ K m values, and those scoring higher than a 50-fold reduction in k cat/ K m value were selected for further evaluation via transient state kinetic analysis. The single-turnover time courses, measured for the first partial reaction, and then for the full reaction, were analyzed to define the microscopic rate constants for the adenylation reaction and the thioesterification reaction. On the basis of our findings we propose a catalytic mechanism that centers on a small group of key residues (some of which serve in more than one role) and that includes several residues that function in domain alternation.

Nucleic Acid Unwinding by Hepatitis C Virus and Bacteriophage T7 Helicases Is Sensitive to Base Pair Stability

Helicases are motor enzymes that convert the chemical energy of NTP hydrolysis into mechanical force for motion and nucleic acid strand separation. Within the cell, helicases process a range of nucleic acid sequences. It is not known whether this composite rate of moving and opening the strands of nucleic acids depends on the base sequence. Our presteady state kinetic studies of helicases from two classes, the ring-shaped T7 helicase and two forms of non-ring-shaped hepatitis C virus (HCV) helicase, show that both the unwinding rate and processivity depend on the sequence and decrease as the nucleic acid stability increases. The DNA unwinding activity of T7 helicase and the RNA unwinding activity of HCV helicases decrease steeply with increasing base pair stability. On the other hand, the DNA unwinding activity of HCV helicases is less sensitive to base pair stability. These results predict that helicases will fall into a spectrum of modest to high sensitivity to base pair stability depending on their biological role in the cell. Modeling of the dependence provided the degree of the active involvement of helicase in base pair destabilization during the unwinding process and distinguished between passive and active mechanisms of unwinding.