Carotenoid Chemistry and Biochemistry : Edited by G. Britton and T.W. Goodwin Pergamon Press; Oxford, 1982 ix + 399 pages. £47.50

The dimeric tryptophanyl-tRNA synthetase from beef pancreas has been found to activate 2 tryptophans/mol enzyme [Eur. J. Biochem. (1982) 128, 389–398]. By using quenched-flow and stopped-flow methods under presteady-state conditions, we show that only one enzyme subunit operates at a time in the aminoacylation of tRNA^Trp and that the transfer reaction is not the rate-limiting step in the overall aminoacylation process.     

Studies on chromophore coupling in isolated phycobiliproteins. I. Picosecond fluorescence kinetics of energy transfer in phycocyanin 645 from Chroomonas sp.

By applying the single-photon timing method the fluorescence kinetics of phycocyanin 645 from Chroomonas sp. has been measured as a function of both the excitation and emission wavelength using low-intensity excitation. The fluorescence kinetics were found to be dominated by a fast (15 ps) and a slow (1.44 ns) decay component. The relative yields and amplitudes of these components depended strongly on both the excitation and emission wavelengths. A component with a small relative amplitude and a lifetime (τ) in the range of 360–680 ps has been found as well. The fast decay component is attributed to intramolecular energy transfer from sensitizing to fluorescing chromophores. Our results are discussed in relation to a chromophore coupling model suggested previously (Jung, J., Song, P.-S., Paxton, R.J., Edelstein, M.S., Swanson, R. and Hazen, E.E. (1980) Biochemistry 19, 24–32).     

GAT1 (GABA:Na+:Cl-) cotransport function. Kinetic studies in giant Xenopus oocyte membrane patches.

To explain cotransport function, the "alternating access" model requires that conformational changes of the empty transporter allow substrates to bind alternatively on opposite membrane sides. To test this principle for the GAT1 (GABA:Na+:Cl-) cotransporter, we have analyzed how its charge-moving partial reactions depend on substrates on both membrane sides in giant Xenopus oocyte membrane patches. (a) "Slow" charge movements, which require extracellular Na+ and probably reflect occlusion of Na+ by GAT1, were defined in three ways with similar results: by application of the high-affinity GAT1 blocker (NO-711), by application of a high concentration (120 mM) of cytoplasmic Cl-, and by removal of extracellular Na+ via pipette perfusion. (b) Three results indicate that cytoplasmic Cl- and extracellular Na+ bind to the transporter in a mutually exclusive fashion: first, cytoplasmic Cl- (5-140 mM) shifts the voltage dependence of the slow charge movement to more negative potentials, specifically by slowing its "forward" rate (i.e., extracellular Na+ occlusion); second, rapid application of cytoplasmic Cl- induces an outward current transient that requires extracellular Na+, consistent with extracellular Na+ being forced out of its binding site; third, fast charge-moving reactions, which can be monitored as a capacitance, are "immobilized" both by cytoplasmic Cl- binding and by extracellular Na+ occlusion (i.e., by the slow charge movement). (c) In the absence of extracellular Na+, three fast (submillisecond) charge movements have been identified, but no slow components. The addition of cytoplasmic Cl- suppresses two components (tau < 1 ms and 13 micros) and enables a faster component (tau < 1 micros). (d) We failed to identify charge movements of fully loaded GAT1 transporters (i.e., with all substrates on both sides). (e) Under zero-trans conditions, inward (forward) GAT1 current shows pronounced pre-steady state transients, while outward (reverse) GAT1 current does not. (f) Turnover rates for reverse GAT1 transport (33 degrees C), calculated from the ratio of steady state current magnitude to total charge movement magnitude, can exceed 60 s(-1) at positive potentials.     

A generalized compartmental model to estimate the fibre mass in the ruminoreticulum: 1. Estimating parameters of digestion

Parameters related to the microbial digestion of nutrients in the ruminoreticulum have been estimated by fitting mathematical models to degradation profiles generated from kinetic studies. In the present paper, we propose a generalized compartmental model of digestion (GCMD) based on implicit theoretical concepts and the gamma probability density function to estimate fibre digestion parameters. The proposed model is consistent to a broader compartmental model presented in a companion paper that integrates aspects of fibre digestion and passage. Different versions of the GCMD were generated by increasing the integer order of time dependency of the gamma function. These versions were fitted to 192 published fibre degradation profiles that were obtained using an in vitro fermentation technique. The quality of fit was evaluated based on the frequency of minimum sum of squares of errors (SSE), the number of runs of signs of residuals, and its likelihood probability calculated according to the Akaike's Information Criterion. The likelihood of the proposed model was also compared to a discrete lag time model (DLT), which is commonly used to interpret fibre degradation profiles. The GCMD had superior quality of fit compared to the DLT and was considered more likely in describing 68.75% of the profiles evaluated. Only 9.38% of the degradation profiles that were fitted to the DLT model had a lower SSE. Even though the degradation profiles studied were generated by incubating feed samples up to 96 h, the true asymptotic limit of fibre degradation can only be achieved by long-term fermentations. This fact leads to questioning the uniformity of the potentially digestible fibre fraction and a further approach based on GCMD-type model was used to account for its heterogeneous nature.     

Mechanism and evolution of protein dimerization.

We have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure-kinetics-evolution relationship for a collection of symmetric homodimers classified into three groups: (1) 14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2-state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3-state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain. (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomeric state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers, and can usually predict whether a dimer has domain swapping. The kinetics of dimerization indicates that some dimers which have been assigned in the literature as domain swapping cases, dimerize through the 2-state kinetics, rather than through swapping segments of performed monomers. The compactness profiles indicate a wide spectrum in the kinetics of dimerization: dimers having no intermediate stable monomers; dimers having an intermediate with a stable monomer structure; and dimers having an intermediate with a stable structure in part of the monomer. These correspond to the multiple evolutionary pathways for dimer formation. The evolutionary mechanisms proposed here for dimers are applicable to other oligomers as well.     

NUTRITIONAL STUDIES OF TWO RED ALGAE. II. KINETICS OF AMMONIUM AND NITRATE UPTAKE1, 2

Similar NH₄₊ and NO₃^?.uptake kinetic patterns were observed in Neoagardhiella baileyi (Harvey ex Kiitzing) Wyinne & Taylor and Gracilaria foliifera (Forssk?l) Borgesen. NO₃^? was taken up in a rate-sturating fashion described by the Michaelis-Menten equation. NH₄₊ uptake was multicomponent: a saturable component was accompanied by a diffusive or a high K component showing no evidence of saturation (at ≤50 μM [NH₄₊]). Nitrogen starved plantsi(C/N atom ratios > ca. 10) showed higher transient rates of NH₄₊ uptake at a given concentration than plants not N-Iimited. Only plants with high N content exhibited diel changes inNH₄₊ uptake rates, and showed transient rates of NH₄₊ accumulation which did not greatly exceed the capacity to incorporate N in steady-state growth. NH₄₊ was preferred over NO_3?even in plants preconditioned on NO_3?as the sole N. source, NO_3? uptake was suppressed at 5μM [NH₄₊], but simultaneous uptake occurred at unsurpressed rates at lower concentrations. Potential for N accumulation was greater via NH₄₊uptake than via NO_3?uptake. Changing capacity for NH₄₊ uptake with N content appears to be a mechanism whereby excessive accumulation of N was avoided by N-.satiated plants but a large accumulation was possible for N-depleted plants.     

Stereoselective degradation of metalaxyl and metalaxyl-M in soil and sunflower plants.

A high proportion of agrochemicals are chiral compounds. Since stereoisomers often show different biological and physiological properties, the biological and metabolic responses to these compounds and their fate in the environment are expected to be different. In this work we investigate a possible stereo and/or enantioselective degradation in soil and plants (sunflower) of the fungicide Metalaxyl (rac-Metalaxyl) and the new compound Metalaxyl-M ((-)-(R)-Metalaxyl) and propose procedures for extraction, cleanup, chromatographic separation of enantiomers, and determination of the R : S ratio by using an HPLC chiral column. The degradation of the two stereoisomers of Metalaxyl proved to be enantioselective and dependent on the media: the (+)-(S)-enantiomer showed a faster degradation in plants, while the (-)-(R)-enantiomer showed a faster degradation in soil. In this study there was no evidence that racemization of Metalaxyl-M took place either in soil or in sunflowers.     

The use of reaction timecourses to determine the level of minor contaminants in enzyme preparations

Enzyme mutagenesis is a commonly used tool to investigate the structure and activity of enzymes. However, even minute contamination of a weakly active mutant enzyme by a considerably more active wild-type enzyme can partially or completely obscure the activity of the mutant enzyme. In this work, we propose a theoretical approach using reaction timecourses and initial velocity measurements to determine the actual contamination level of an undesired wild-type enzyme. To test this method, we applied it to a batch of the Q215A/R235A double mutant of orotidine 5′-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae that was inadvertently contaminated by the more active wild-type OMPDC from Escherichia coli. The enzyme preparation showed significant deviations from the expected kinetic behavior at contamination levels as low as 0.093 mol%. We then confirmed the origin of the unexpected kinetic behavior by deliberately contaminating a sample of the mutant OMPDC from yeast that was known to be pure, with 0.015% wild-type OMPDC from E. coli and reproducing the same hybrid kinetic behavior.     

NUTRITIONAL STUDIES OF TWO RED ALGAE. I. GROWT RATE AS A FUNCTION OF NITROGEN SOURCE AND CONCENTRATION1

Gracilaria foliifera (Forsskal) Borgesen and Neoagardhiella bailiyi (Harvey ex Kiitzing) Wynne & Taylor were grown in continuous-flow culture under controlled environmental conditions in 15 liter experimental chambers. Growth rate was related to the source and concentration of nitrogen enrichment supplied to the plants, Growth rate appeared to follow saturation-type nutrient uptake kinetics for plants receiving ammonium, nitrate, urea of sewage effluent enrichments. Ammonium enrichment produced higher growth rates than nitrate of sewage enrichment. The lowest growth rates occurred in the chambers receiving unenriched seawater or urea. The low estimated constants (K) for growth were in the range of 0.2–0.4 (M N for all N enrichments examined. The low estimated values of K compare closely with those found for microalgae and indicate that both species possess the ability to utilize very low concentrations of N.     

The glutaredoxin mono- and di-thiol mechanisms for deglutathionylation are functionally equivalent: implications for redox systems biology

Glutathionylation plays a central role in cellular redox regulation and anti-oxidative defence. Grx (Glutaredoxins) are primarily responsible for reversing glutathionylation and their activity therefore affects a range of cellular processes, making them prime candidates for computational systems biology studies. However, two distinct kinetic mechanisms involving either one (monothiol) or both (dithiol) active-site cysteines have been proposed for their deglutathionylation activity and initial studies predicted that computational models based on either of these mechanisms will have different structural and kinetic properties. Further, a number of other discrepancies including the relative activity of active-site mutants and contrasting reciprocal plot kinetics have also been reported for these redoxins. Using kinetic modelling, we show that the dithiol and monothiol mechanisms are identical and, we were also able to explain much of the discrepant data found within the literature on Grx activity and kinetics. Moreover, our results have revealed how an apparently futile side-reaction in the monothiol mechanism may play a significant role in regulating Grx activity in vivo.     

Fastin situ determination of the biomass of anchorage-dependent cells

The lengthy and cumbersome protocol used to establish the growth kinetics characteristics of anchorage-dependent cells (ADC's)in situ (i.e. while the cells adhere on their microsupport) by Aperture Impedance Pulse Spectroscopy (AIPS) can be replaced by an accelerated procedure. This we have named Turbo AIPS whereby the same results can be obtained without actually performing the manipulations leading to the determination of the biomass.     

Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74‐compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k_cat/K_m values >10,000 M^?1 s^?1: Q92EH0_LISIN of Listeria innocua serovar 6a against ADP‐ribose, Q5LBB1_BACFN of Bacillus fragilis against 5‐Me‐CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8‐oxo‐dATP and 3'‐dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty‐two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k_cat/K_m values exhibited against these canonical substrates are well under 10⁵ M^?1 s^?1. By contrast, several Nudix enzymes show much larger k_cat/K_m values (in the range of 10⁵ to >10⁷ M^?1 s^?1) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810–1822. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance

Family D DNA polymerases (polDs) have been implicated as the major replicative polymerase in archaea, excluding the Crenarchaeota branch, and bear little sequence homology to other DNA polymerase families. Here we report a detailed kinetic analysis of nucleotide incorporation and exonuclease activity for a Family D DNA polymerase from Thermococcus sp. 9°N. Pre-steady-state single-turnover nucleotide incorporation assays were performed to obtain the kinetic parameters, k_pol and K_d, for correct nucleotide incorporation, incorrect nucleotide incorporation, and ribonucleotide incorporation by exonuclease-deficient polD. Correct nucleotide incorporation kinetics revealed a relatively slow maximal rate of polymerization (k_pol ∼2.5 s⁻¹) and especially tight nucleotide binding (K_d(dNTP) ∼1.7 μm), compared with DNA polymerases from Families A, B, C, X, and Y. Furthermore, pre-steady-state nucleotide incorporation assays revealed that polD prevents the incorporation of incorrect nucleotides and ribonucleotides primarily through reduced nucleotide binding affinity. Pre-steady-state single-turnover assays on wild-type 9°N polD were used to examine 3′-5′ exonuclease hydrolysis activity in the presence of Mg²⁺ and Mn²⁺. Interestingly, substituting Mn²⁺ for Mg²⁺ accelerated hydrolysis rates >40-fold (k_exo ≥110 s⁻¹ versus ≥2.5 s⁻¹). Preference for Mn²⁺ over Mg²⁺ in exonuclease hydrolysis activity is a property unique to the polD family. The kinetic assays performed in this work provide critical insight into the mechanisms that polD employs to accurately and efficiently replicate the archaeal genome. Furthermore, despite the unique properties of polD, this work suggests that a conserved polymerase kinetic pathway is present in all known DNA polymerase families.     

Sensitivity of bovine retinal acetylcholinesterase (E.C. 3.1.1.7) toward tacrine: Kinetic characterization

This work addresses the kinetic analysis of the interaction of tacrine with bovine retina acetylcholinesterase (AChE, E.C. 3.1.1.7). It was found that the tacrine effect was reversible in nature. Tacrine inhibited bovine retinal AChE activity in a concentration-dependent manner; IC₅₀ was found to be 8.07 nM. The Michaelis-Menten constant (K_a) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.061 mM in the control system, and this value was increased by 54–67% in the tacrine-treated systems. The V_max was 0.701 μ mole/min per milligram protein for the control system, but it was decreased by 26–69% in the tacrine-treated systems. The Lineweaver–Burk plot, Dixon plot, and their secondary replots indicated that the nature of the inhibition was of the partial mixed type, that is, a mixture of competitive and noncompetitive inhibition. The values of K_i and K_t were estimated to be as 4.475 and 8.517 nM, respectively. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 245–251, 1998     

Neuromuscular glutamatergic and GABAergic channels

Our laboratory has worked extensively on glutamatergic and GABA-ergic channels, predominantly in crayfish, but also in locust,Drosophila and recentlyAscaris. Channel currents were recorded in the different modes of the patch-clamp technique (Hamillet al., 1981). The opening kinetics of the channels were derived from open and closed time histograms obtained from single channel recordings. From these, channel conductances could also be evaluated. The most relevant data were obtained by very rapidly rising and falling pulses (time of change about 0.1 ms) of agonists applied to outside-out patches containing the respective channels (Frankeet al., 1987). From such recordings we constructed dose-response curves for peak and steady-state currents, for the rise times of the currents and for the time constants of desensitization. In double-pulse experiments we measured recovery from desensitization and predesensitization due to low agonist concentrations. For most of the channel types, we succeeded in constructing a reaction scheme which in computer simulations mimicked channel behaviour to a good approximation.     

Mechanism of inhibition of wt-dihydrofolate reductase from E. coli by tea epigallocatechin-gallate

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme involved in major biological process, including DNA synthesis and cancer inhibition, and its modulation is the object of extensive structural, kinetic, and pharmacological studies. In particular, earlier studies showed that green tea catechins are powerful inhibitors of bovine liver and chicken liver DHFR. In this article, we report the results of inhibition kinetics for the enzyme from another source (DHFR from E. coli) exerted by (-)-epigallocatechingallate (EGCG). Using different analytical techniques, we reported that EGCG acts as a bisubstrate inhibitor on the bacterial DHFR. Moreover, the combined approach of biosensor, kinetic, and molecular modelling analysis disclosed the ability of EGCG to bind to the enzyme both on substrate (DHF) and cofactor (NADPH) site. Collectively, our data have confirmed the selectivity of antifolate compounds with respect to the different source of enzyme (bacterial or mammalian DHFR) and the possible role of tea catechins as chemopreventive agents.     

5′‐Single‐stranded/duplex DNA junctions are loading sites for E. coli UvrD translocase

Escherichia coli UvrD is a 3′–5′ superfamily 1A helicase/translocase involved in a variety of DNA metabolic processes. UvrD can function either as a helicase or only as an single‐stranded DNA (ssDNA) translocase. The switch between these activities is controlled in vitro by the UvrD oligomeric state; a monomer has ssDNA translocase activity, whereas at least a dimer is needed for helicase activity. Although a 3′‐ssDNA partial duplex provides a high‐affinity site for a UvrD monomer, here we show that a monomer also binds with specificity to DNA junctions possessing a 5′‐ssDNA flanking region and can initiate translocation from this site. Thus, a 5′‐ss–duplex DNA junction can serve as a high‐affinity loading site for the monomeric UvrD translocase, whereas a 3′‐ss–duplex DNA junction inhibits both translocase and helicase activity of the UvrD monomer. Furthermore, the 2B subdomain of UvrD is important for this junction specificity. This highlights a separation of helicase and translocase function for UvrD and suggests that a monomeric UvrD translocase can be loaded at a 5′‐ssDNA junction when translocation activity alone is needed.     

Inhibition kinetics of acetosyringone on xylanase in hydrolysis of hemicellulose

ABSTRACT

Many phenolic compounds, derived from lignin during the pretreatment of lignocellulosic biomass, could obviously inhibit the activity of cellulolytic and hemicellulolytic enzymes. Acetosyringone (AS) is one of the phenolic compounds produced from lignin degradation. In this study, we investigated the inhibitory effects of AS on xylanase activity through kinetic experiments. The results showed that AS could obviously inhibit the activity of xylanase in a reversible and noncompetitive binding manner (up to 50% activity loss). Inhibitory kinetics and constants of xylanase on AS were conducted by the HCH-1 model (β = 0.0090 ± 0.0009 mM^?1). Furthermore, intrinsic and 8-anilino-1-naphthalenesulfonic (ANS)-binding fluorescence results showed that the tertiary structure of AS-mediated xylanase was altered. These findings provide new insights into the role of AS in xylanase activity. Our results also suggest that AS was an inhibitor of xylanase and targeting AS was a potential strategy to increase xylose production.     

Determination of hepatitis delta virus ribozyme N(–1) nucleobase and functional group specificity using internal competition kinetics

Biological catalysis involves interactions distant from the site of chemistry that can position the substrate for reaction. Catalysis of RNA 2′-O-transphosphorylation by the hepatitis delta virus (HDV) ribozyme is sensitive to the identity of the N(–1) nucleotide flanking the reactive phosphoryl group. However, the interactions that affect the conformation of this position, and in turn the 2′O nucleophile, are unclear. Here, we describe the application of multiple substrate internal competition kinetic analyses to understand how the N(–1) nucleobase contributes to HDV catalysis and test the utility of this approach for RNA structure–function studies. Internal competition reactions containing all four substrate sequence variants at the N(–1) position in reactions using ribozyme active site mutations at A77 and A78 were used to test a proposed base-pairing interaction. Mutants A78U, A78G, and A79G retain significant catalytic activity but do not alter the specificity for the N(–1) nucleobase. Effects of nucleobase analog substitutions at N(–1) indicate that U is preferred due to the ability to donate an H-bond in the Watson–Crick face and avoid minor groove steric clash. The results provide information essential for evaluating models of the HDV active site and illustrate multiple substrate kinetic analyses as a practical approach for characterizing structure–function relationships in RNA reactions.     

Regulation of E. coli Rep helicase activity by PriC

Stalled DNA replication forks can result in incompletely replicated genomes and cell death. DNA replication restart pathways have evolved to deal with repair of stalled forks and E. coli Rep helicase functions in this capacity. Rep and an accessory protein, PriC, assemble at a stalled replication fork to facilitate loading of other replication proteins. A Rep monomer is a rapid and processive single stranded (ss) DNA translocase but needs to be activated to function as a helicase. Activation of Rep in vitro requires self-assembly to form a dimer, removal of its auto-inhibitory 2B sub-domain, or interactions with an accessory protein. Rep helicase activity has been shown to be stimulated by PriC, although the mechanism of activation is not clear. Using stopped flow kinetics, analytical sedimentation and single molecule fluorescence methods, we show that a PriC dimer activates the Rep monomer helicase and can also stimulate the Rep dimer helicase. We show that PriC can self-assemble to form dimers and tetramers and that Rep and PriC interact in the absence of DNA. We further show that PriC serves as a Rep processivity factor, presumably co-translocating with Rep during DNA unwinding. Activation is specific for Rep since PriC does not activate the UvrD helicase. Interaction of PriC with the C-terminal acidic tip of the ssDNA binding protein, SSB, eliminates Rep activation by stabilizing the PriC monomer. This suggests a likely mechanism for Rep activation by PriC at a stalled replication fork.