Analysis of the substrate specificity of human sulfotransferases SULT1A1 and SULT1A3: Site-directed mutagenesis and kinetic studies

Sulfonation is an important metabolic process involved in the excretion and in some cases activation of various endogenous compounds and xenobiotics. This reaction is catalyzed by a family of enzymes named sulfotransferases. The cytosolic human sulfotransferases SULT1A1 and SULT1A3 have overlapping yet distinct substrate specificities. SULT1A1 favors simple phenolic substrates such as p-nitrophenol, whereas SULT1A3 prefers monoamine substrates such as dopamine. In this study we have used a variety of phenolic substrates to functionally characterize the role of the amino acid at position 146 in SULT1A1 and SULT1A3. First, the mutation A146E in SULT1A1 yielded a SULT1A3-like protein with respect to the Michaelis constant for simple phenols. The mutation E146A in SULT1A3 resulted in a SULT1A1-like protein with respect to the Michaelis constant for both simple phenols and monoamine compounds. When comparing the specificity of SULT1A3 toward tyramine with that for p-ethylphenol (which differs from tyramine in having no amine group on the carbon side chain), we saw a 200-fold preference for tyramine. The kinetic data obtained with the E146A mutant of SULT1A3 for these two substrates clearly showed that this protein preferred substrates without an amine group attached. Second, changing the glutamic acid at position 146 of SULT1A3 to a glutamine, thereby neutralizing the negative charge at this position, resulted in a 360-fold decrease in the specificity constant for dopamine. The results provide strong evidence that residue 146 is crucial in determining the substrate specificity of both SULT1A1 and SULT1A3 and suggest that there is a direct interaction between glutamic acid 146 in SULT1A3 and monoamine substrates.

A kinetic study of the thermally initiated free radical reactions of styrene with octanedithiol as a model for the reactions of multifunctional thiols with divinylbenzene

The kinetics of chain reactions of octanedithiol with styrene, thermally initiated with TX29B50 (a 50:50 wt% solution of TX29 diperoxy initiator in a phthalate plasticizer), have been studied over a range of initiator concentrations, a range of mixture formulations and a range of temperatures. This system has been investigated as a model system for the reactions of polyfunctional thiols with divinyl benzene. The reactions have been shown to follow first-order kinetics for both the thiol and the ene species and to be characterized by a dependence on the initiator concentration to the power of one half. The kinetic rate parameters have been shown to adhere to Arrhenius behaviour. A kinetic model for the chain reactions for this system has been proposed. (C) 2003 Society of Chemical Industry.

The peroxisomal Acyl-CoA thioesterase Pte1p from Saccharomyces cerevisiae is required for efficient degradation of short straight chain and branched chain fatty acids.

The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.

Kinetic modeling of mechanisms of industrially important organic reactions in gas and liquid phase

This dissertation is based on 5 articles which deal with reaction mechanisms of the following selected industrially important organic reactions: 1. dehydrocyclization of n-butylbenzene to produce naphthalene 2. dehydrocyclization of 1-(p-tolyl)-2-methylbutane (MB) to produce 2,6-dimethylnaphthalene 3. esterification of neopentyl glycol (NPG) with different carboxylic acids to produce monoesters 4. skeletal isomerization of 1-pentene to produce 2-methyl-1-butene and 2-methyl-2-butene The results of initial- and integral-rate experiments of n-butylbenzene dehydrocyclization over selfmade chromia/alumina catalyst were applied when investigating reaction 2. Reaction 2 was performed using commercial chromia/alumina of different acidity, platina on silica and vanadium/calcium/alumina as catalysts. On all catalysts used for the dehydrocyclization, major reactions were fragmentation of MB and 1-(p-tolyl)-2-methylbutenes (MBes), dehydrogenation of MB, double bond transfer, hydrogenation and 1,6-cyclization of MBes. Minor reactions were 1,5-cyclization of MBes and methyl group fragmentation of 1,6- cyclization products. Esterification reactions of NPG were performed using three different carboxylic acids: propionic, isobutyric and 2-ethylhexanoic acid. Commercial heterogeneous gellular (Dowex 50WX2), macroreticular (Amberlyst 15) type resins and homogeneous para-toluene sulfonic acid were used as catalysts. At first NPG reacted with carboxylic acids to form corresponding monoester and water. Then monoester esterified with carboxylic acid to form corresponding diester. In disproportionation reaction two monoester molecules formed NPG and corresponding diester. All these three reactions can attain equilibrium. Concerning esterification, water was removed from the reactor in order to prevent backward reaction. Skeletal isomerization experiments of 1-pentene were performed over HZSM-22 catalyst. Isomerization reactions of three different kind were detected: double bond, cis-trans and skeletal isomerization. Minor side reaction were dimerization and fragmentation. Monomolecular and bimolecular reaction mechanisms for skeletal isomerization explained experimental results almost equally well. Pseudohomogeneous kinetic parameters of reactions 1 and 2 were estimated by usual least squares fitting. Concerning reactions 3 and 4 kinetic parameters were estimated by the leastsquares method, but also the possible cross-correlation and identifiability of parameters were determined using Markov chain Monte Carlo (MCMC) method. Finally using MCMC method, the estimation of model parameters and predictions were performed according to the Bayesian paradigm. According to the fitting results suggested reaction mechanisms explained experimental results rather well. When the possible cross-correlation and identifiability of parameters (Reactions 3 and 4) were determined using MCMC method, the parameters identified well, and no pathological cross-correlation could be seen between any parameter pair.

Adsorption kinetic investigations of phthalocyanine derivatives self assembled monolayers (SAMs) on gold: Temperature influence on the SAM formation process and quality

The ordered nano-structured surfaces, like self-assembled monolayers (SAMs) are of a great scientific interest, due to the low cost, simplicity, and versatility of this method. SAMs found numerous of applications in molecular electronics, biochemistry and optical devices. Phthalocyanine (Pc) complexes are of particular interest for the SAM preparation. These molecules exhibit fascinating physical properties and are chemically and thermally stable. Moreover their complex structure is advantageous for the fabrication of switchable surfaces. In this work the adsorption process of Pcs derivatives, namely, subphthalocyanines (SubPcB) and terbium (2TbPc) sandwich complexes on gold has been investigated. The influence of the molecular concentration, chain length of peripheral groups, and temperature on the film formation process has been examined using a number of techniques. The SAMs formation process has been followed in situ and in real time by means of second harmonic generation (SHG) and surface plasmon resonance (SPR) spectroscopy. To investigate the quality of the SAMs prepared at different temperatures atomic force microscopy (AFM) and X-Ray photoelectron spectroscopy (XPS)measurements were performed. Valuable information about SubPcB and 2TbPc adsorbtion process has been obtained in the frame of this work. The kinetic data, obtained with SHG and SPR, shows the best conformance with the first order Langmuir kinetic model. Comparing SHG and SPR results, it has been found, that the film formation occurs faster than the formation of chemical bonds. Such, the maximum amount of molecules on the surface is reached after 6 min for SubPcB and 30 min for 2TbPc. However, at this time the amount of formed chemicals bonds is only 10% and 40% for SubPcB and 2TbPc, respectively. The most intriguing result, among others, was obtained at T = 2 °C, where the formation of the less dense SAMs have been detected with SHG.However, analyzing XPS and AFM data, it has been revealed, that there is the same amount of molecules on the surface at both temperature T = 2 °C, and T = 21 °C, but the amount of formed chemicals bond is different. At T = 2 °C molecules form aggregates, therefore many of available anchor groups stay unattached.

Kinetic study of crystallization of PHB in presence of hydroxy acids

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Subcellular localization and kinetic characterization of a gill (Na +, K+)-ATPase from the giant freshwater prawn macrobrachium rosenbergii

The stimulation by Mg2+, Na+, K+, NH 4 +, and ATP of (Na+, K+)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na +, K+)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg2+, Na+, and K+ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V M = 115.0 ± 2.3 U mg-1, K 0.5 = 0.10 ± 0.01 mmol L-1. Stimulation by Na+ (V M = 110.0 ± 3.3 U mg-1, K 0.5 = 1.30 ± 0.03 mmol L -1), Mg2+ (V M = 115.0 ± 4.6 U mg -1, K 0.5 = 0.96 ± 0.03 mmol L-1), NH4 + (V M = 141.0 ± 5.6 U mg -1, K 0.5 = 1.90 ± 0.04 mmol L-1), and K+ (V M = 120.0 ± 2.4 U mg-1, K M = 2.74 ± 0.08 mmol L-1) followed single saturation curves and, except for K+, exhibited site-site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K I = 12.4 ± 1.3 mol L-1. Complementary inhibition studies suggest the presence of F0F1-, Na+-, or K +-ATPases, but not V(H+)- or Ca2+-ATPases, in the gill microsomal preparation. K+ and NH4 + synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4 +, and K+ of the gill enzyme. © 2013 Springer Science+Business Media New York.

A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide election

The enzymatically catalyzed template-directed extension of ssDNA/primer complex is an impor-tant reaction of extraordinary complexity. The DNA polymerase does not merely facilitate the insertion of dNMP, but it also performs rapid screening of substrates to ensure a high degree of fidelity. Several kinetic studies have determined rate constants and equilibrium constants for the elementary steps that make up the overall pathway. The information is used to develop a macro-scopic kinetic model, using an approach described by Ninio [Ninio J., 1987. Alternative to the steady-state method: derivation of reaction rates from first-passage times and pathway probabili-ties. Proc. Natl. Acad. Sci. U.S.A. 84, 663–667]. The principle idea of the Ninio approach is to track a single template/primer complex over time and to identify the expected behavior. The average time to insert a single nucleotide is a weighted sum of several terms, in-cluding the actual time to insert a nucleotide plus delays due to polymerase detachment from ei-ther the ternary (template-primer-polymerase) or quaternary (+nucleotide) complexes and time delays associated with the identification and ultimate rejection of an incorrect nucleotide from the binding site. The passage times of all events and their probability of occurrence are ex-pressed in terms of the rate constants of the elementary steps of the reaction pathway. The model accounts for variations in the average insertion time with different nucleotides as well as the in-fluence of G+C content of the sequence in the vicinity of the insertion site. Furthermore the model provides estimates of error frequencies. If nucleotide extension is recognized as a compe-tition between successful insertions and time delaying events, it can be described as a binomial process with a probability distribution. The distribution gives the probability to extend a primer/template complex with a certain number of base pairs and in general it maps annealed complexes into extension products.

Efficiency of DNA replication in the polymerase chain reaction

A detailed quantitative kinetic model for the polymerase chain reaction (PCR) is developed, which allows us to predict the probability of replication of a DNA molecule in terms of the physical parameters involved in the system. The important issue of the determination of the number of PCR cycles during which this probability can be considered to be a constant is solved within the framework of the model. New phenomena of multimodality and scaling behavior in the distribution of the number of molecules after a given number of PCR cycles are presented. The relevance of the model for quantitative PCR is discussed, and a novel quantitative PCR technique is proposed.

The kinetic mechanism of myosin V

Myosin V is an unconventional myosin proposed to be processive on actin filaments, analogous to kinesin on a microtubule [Mehta, A. D., et al. (1999) Nature (London) 400, 590–593]. To ascertain the unique properties of myosin V that permit processivity, we undertook a detailed kinetic analysis of the myosin V motor. We expressed a truncated, single-headed myosin V construct that bound a single light chain to study its innate kinetics, free from constraints imposed by other regions of the molecule. The data demonstrate that unlike any previously characterized myosin a single-headed myosin V spends most of its kinetic cycle (>70%) strongly bound to actin in the presence of ATP. This kinetic tuning is accomplished by increasing several of the rates preceding strong binding to actin and concomitantly prolonging the duration of the strongly bound state by slowing the rate of ADP release. The net result is a myosin unlike any previously characterized, in that ADP release is the rate-limiting step for the actin-activated ATPase cycle. Thus, because of a number of kinetic adaptations, myosin V is tuned for processive movement on actin and will be capable of transporting cargo at lower motor densities than any other characterized myosin.

Kinetic and structural analysis of mutant CD4 receptors that are defective in HIV gp120 binding

The T-cell antigen coreceptor CD4 also serves as the receptor for the envelope glycoprotein gp120 of HIV. Extensive mutational analysis of CD4 has implicated residues from a portion of the extracellular amino-terminal domain (D1) in gp120 binding. However, none of these proteins has been fully characterized biophysically, and thus the precise effects on molecular structure and binding interactions are unknown. In the present study, we produced soluble versions of three mutant CD4 molecules (F43V, G47S, and A55F) and characterized their structural properties, thermostability, and ability to bind gp120. Crystallographic and thermodynamic analysis showed minimal structural alterations in the F43V and G47S mutant proteins, which have solvent-exposed mutant side chains. In contrast, some degree of disorder appears to exist in the folded state of A55F, as a result of mutating a buried side chain. Real time kinetic measurements of the interaction of the mutant proteins with gp120 showed affinity decreases of 5-fold for G47S, 50-fold for A55F, and 200-fold for F43V. Although both rate constants for the binding reaction were affected by these mutations, the loss in affinity was mainly due to a decrease in on rates, with less drastic changes occurring in the off rates. These observations suggest the involvement of conformational adaptation in the CD4–gp120 interaction. Together, the structural and kinetic data confirm that F43V is a critical residue in gp120 recognition site, which may also include main chain interactions at residue Gly-47.

Transition-state structure as a unifying basis in protein-folding mechanisms: Contact order, chain topology, stability, and the extended nucleus mechanism

I attempt to reconcile apparently conflicting factors and mechanisms that have been proposed to determine the rate constant for two-state folding of small proteins, on the basis of general features of the structures of transition states. Φ-Value analysis implies a transition state for folding that resembles an expanded and distorted native structure, which is built around an extended nucleus. The nucleus is composed predominantly of elements of partly or well-formed native secondary structure that are stabilized by local and long-range tertiary interactions. These long-range interactions give rise to connecting loops, frequently containing the native loops that are poorly structured. I derive an equation that relates differences in the contact order of a protein to changes in the length of linking loops, which, in turn, is directly related to the unfavorable free energy of the loops in the transition state. Kinetic data on loop extension mutants of CI2 and α-spectrin SH3 domain fit the equation qualitatively. The rate of folding depends primarily on the interactions that directly stabilize the nucleus, especially those in native-like secondary structure and those resulting from the entropy loss from the connecting loops, which vary with contact order. This partitioning of energy accounts for the success of some algorithms that predict folding rates, because they use these principles either explicitly or implicitly. The extended nucleus model thus unifies the observations of rate depending on both stability and topology.

Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage

Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76Δ) ribozymes in D2O and H2O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76Δ mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under kcat/KM conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (β) of 0.51 was determined for the base-rescued reaction of C76Δ. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage.

Kinetic analysis of peptide loading onto HLA-DR molecules mediated by HLA-DM.

The nonclassical major histocompatibility complex class II molecule HLA-DM (DM) has recently been shown to play a central role in the class II-associated antigen presentation pathway: DM releases invariant chain-derived CLIP peptides (class II-associated invariant chain protein peptide) from HLA-DR (DR) molecules and thereby facilitates loading with antigenic peptides. Some observations have led to the suggestion that DM acts in a catalytic manner, but so far direct proof is missing. Here, we investigated in vitro the kinetics of exchange of endogenously bound CLIP for various peptides on DR1 and DR2a molecules: we found that in the presence of DM the peptide loading process follows Michaelis-Menten kinetics with turnover numbers of 3-12 DR molecules per minute per DM molecule, and with KM values of 500-1000 nM. In addition, surface plasmon resonance measurements showed that DM interacts efficiently with DR-CLIP complexes but only weakly with DR-peptide complexes isolated from DM-positive cells. Taken together, our data provide evidence that DM functions as an enzyme-like catalyst of peptide exchange and favors the generation of long-lived DR-peptide complexes that are no longer substrates for DM.

NusG is required to overcome a kinetic limitation to Rho function at an intragenic terminator.

Rho-dependent transcription termination at certain terminators in Escherichia coli also depends on the presence of NusG [Sullivan, S. L. & Gottesman, M. E. (1992) Cell 68, 989-994]. We have found that termination at the first intragenic terminator in lacZ (tiZ1) is strongly dependent on NusG when transcription is done in vitro with the concentrations of NTPs found in vivo. With a lower level of NTPs, and consequently a slower rate of RNA-chain growth, Rho causes some termination by itself that is enhanced with NusG. These results suggest that NusG serves to overcome a kinetic limitation of Rho to function at certain terminators. At a second intragenic terminator within the lacZ reading frame (tiZ2) the efficiency of Rho-mediated termination was unaffected by either NusG or by RNA polymerase elongation kinetics. Thus, using purified components and intracellular levels of NTPs, we have confirmed the in vivo finding that certain Rho-dependent terminators also depend on NusG, whereas others do not.