The reaction of amino-oxyacetate with pyridoxal phosphate-dependent enzymes.

The two transmitter binding sites of the neuromuscular acetylcholine (ACh) receptor channel contain several aromatic residues, including a tryptophan located on the complementary, negative face of each binding pocket. These two residues, Trp-55 in the epsilon subunit and Trp-57 in the delta subunit, were mutated (AEFHILRVY), and for most constructs the rate constants for acetylcholine binding and channel gating were estimated by using single channel kinetic analyses. The rate constants for unliganded channel opening and closing were also estimated for some mutants. From these measurements we calculated all of the equilibrium constants of the "allosteric" cycle as follows: diliganded gating, unliganded gating, dissociation from the C(losed) conformation, and dissociation from the O(pen) conformation. The results indicate the following. (i) These aromatic side chains play a relatively minor role in ACh receptor channel activation. (ii) The main consequence of mutations is to reduce the affinity of the O conformation of the binding site for ACh, with the effect being greater at the epsilon subunit. (iii) In epsilon (but not delta) the aromatic nature of the side chain is important in determining affinity, to a slightly greater degree in the O conformation. Phi value analyses (of both tryptophan residues) show Phi approximately 1 for both the ACh binding and diliganded gating reactions. (iv) This suggests that the structural boundaries of the dynamic elements of the gating conformational change may not be subunit-delimited, and (v) the mutated tryptophan residues experience energy changes that occur relatively early in both the ligand-binding and channel-gating reactions.

The flowing afterglow technique has been employed in the measurement of rate and equilibrium constants at 296 ± 2 K for unsolvated proton transfer reactions of the type [Formula: see text] and several solvated proton transfer reactions of the type [Formula: see text] where X and Y may be H2O, H2S, HCN, or H2CO. Where possible, direct comparisons are made with similar measurements performed with other techniques. The equilibrium constant measurements provide a measure of the relative proton affinities of H2O, H2S, HCN, and H2CO and absolute values for PA(H2O) = 166.4 ± 2.4 kcal mol−1, PA(H2S) = 170.2 ± 1.8 kcal mol−1, and PA(HCN) = 171.0 ± 1.7 kcal mol−1 when reference is made to PA(H2CO) = 170.9 ± 1.2 kcal mol−1 which can be derived from available thermochemical information. The rate constant measurements reinforce the generalization that unsolvated proton transfer involving simple molecules proceeds with high efficiency and provide information about the influence of solvation on this efficiency.

The rapid growth of the organometallic chemistry of the transition metals during the last 15–20 years owes much to the development of homogeneous catalyst systems which are capable of synthesizing organic molecules under mild conditions and occasionally with remarkable selectivities. Several have been commercialized and are now used on a large scale. (1) A few have received considerable detailed study—including spectroscopic identification of the species present in solution under reaction conditions, isolation of reactive intermediates in some cases, determination of the overall rate law and measurement of rate and equilibrium constants of several individual steps, and isotopic labeling studies—so that we have a reasonably clear picture of how they operate. It is these systems that form the focus of this chapter. For more general reviews the reader is referred to some recent books,(1-3) which also discuss the special electronic properties of transition metals which are in part responsible for their catalytic behavior.†

Pyridoxal phosphate (PLP) dependent enzymes are essential proteins that catalyze a diverse number of reactions: transamination, racemization, phosphorylation, decarboxylation, aldol cleavage, elimination, and replacement. Despite completing a myriad of chemical reactions, these proteins have a unifying Schiff base catalytic intermediate but the overall mechanisms remain ambiguous. Based upon previous X‐ray structures and abundant literature of the different families of PLP dependent enzymes, one question must be answered: How do the different families of PLP dependent enzymes utilize different protonation profiles of pyridoxal phosphate that enable them to promote catalysis of a specific reaction? The objective of this research will be development of detailed model mechanisms for the different classes of PLP dependent enzymes, based on the protonation state of the coenzyme. Aspartate aminotransferase (AAT) and tryptophan synthase (TS) are two model PLP dependent enzymes that have shown to grow neutron diffraction quality crystals (1mm3). Neutron crystallography is an unparalleled tool to determine the atomic coordinates of the hydrogen/deuterium in macromolecules undetectable by other means. Initial neutron data collection and refinement of these proteins gives insight to the protonation state of PLP during catalysis.

Fundamental considerations, such as kinetics, thermodynamics and various computational methods, are not well suited to describe the reactivity of organic molecules containing a large number of carbon atoms, such as natural products and similar synthetic compounds [1]. In fact, of the different, complementary experimental methods used in the determination of a mechanism (e.g. labelling, measurements of rate and equilibrium constants, etc.), the analysis of stereochemical effects constitutes a suitable, alternative approach [2] and appears to be more appropriate for organic chemists concerned with the elucidation of gas-phase reaction mechanisms. Indeed, the analysis of stereochemical effects can be of assistance in (i) the determination of the molecularity of gas phase processes (e.g. unimolecular or bimolecular pathways), and (ii) distinguishing the relative importance of thermochemical and kinetic control of ion-molecule reactions [3] without extensive calculations. The instrumentation employed for such investigations may be a low pressure system [4] such as ion cyclotron resonance or an ion trap, or a high pressure source of a more conventional mass spectrometer, including tandem instruments [5].KeywordsProton TransferProton AffinityCyclohexane DiolStereochemical EffectChemical Ionisation ConditionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A central dogma in immunology is that antibody specificity is a function of the variable (V) region. However serological analysis of IgG(1), IgG(2a), and IgG(2b) switch variants of murine monoclonal antibody (mAb) 3E5 IgG(3) with identical V domains revealed apparent specificity differences for Cryptococcus neoformans glucuronoxylomannan (GXM). Kinetic and thermodynamic binding properties of mAbs 3E5 to a 12-mer peptide mimetic of GXM revealed differences in the affinity of these mAbs for a monovalent ligand, a result that implied that the constant (C) region affects the secondary structure of the antigen binding site, thus accounting for variations in specificity. Structural models of mAbs 3E5 suggested that isotype-related differences in binding resulted from amino acid sequence polymorphisms in the C region. This study implies that isotype switching is another mechanism for generating diversity in antigen binding and that isotype restriction of certain antibody responses may reflect structural constraints imposed by C region on V region binding. Furthermore, isotype affected the polyreactivity of V region identical antibodies, implying a role for C region in determining self-reactivity.

The acetylcholine receptor (AChR) from vertebrate skeletal muscle initiates voluntary movement, and its kinetics of activation are crucial for maintaining the safety margin for neuromuscular transmission. Furthermore, the kinetic mechanism of the muscle AChR serves as an archetype for understanding activation mechanisms of related receptors from the Cys-loop superfamily. Here we record currents through single muscle AChR channels with improved temporal resolution approaching half an order of magnitude over our previous best. A range of concentrations of full and partial agonists are used to elicit currents from human wild-type and gain-of-function mutant AChRs. For each agonist-receptor combination, rate constants are estimated from maximum likelihood analysis using a kinetic scheme comprised of agonist binding, priming, and channel gating steps. The kinetic scheme and rate constants are tested by stochastic simulation, followed by incorporation of the experimental step response, sampling rate, background noise, and filter bandwidth. Analyses of the simulated data confirm all rate constants except those for channel gating, which are overestimated because of the established effect of noise on the briefest dwell times. Estimates of the gating rate constants were obtained through iterative simulation followed by kinetic fitting. The results reveal that the agonist association rate constants are independent of agonist occupancy but depend on receptor state, whereas those for agonist dissociation depend on occupancy but not on state. The priming rate and equilibrium constants increase with successive agonist occupancy, and for a full agonist, the forward rate constant increases more than the equilibrium constant; for a partial agonist, the forward rate and equilibrium constants increase equally. The gating rate and equilibrium constants also increase with successive agonist occupancy, but unlike priming, the equilibrium constants increase more than the forward rate constants. As observed for a full and a partial agonist, the gain-of-function mutation affects the relationship between rate and equilibrium constants for priming but not for channel gating. Thus, resolving brief single channel currents distinguishes priming from gating steps and reveals how the corresponding rate and equilibrium constants depend on agonist occupancy.

Dopamine has been proposed as an intrarenal natriuretic hormone. We reported previously that inner medullary collecting duct (IMCD) cells express a novel DA2-like dopamine receptor (namely, DA2K) that is linked to stimulation of prostaglandin E2 (PGE2) production. In this study we examined whether locally formed dopamine could stimulate PGE2 production in cultured IMCD cells. L-Dopa stimulated PGE2 production dose dependently in cultured IMCD cells (concentration for half-maximal stimulation, 54.3 microM; maximal stimulation, 212.7% of basal), with the maximal stimulation similar to that obtained with dopamine. This effect was blocked by aromatic L-amino acid decarboxylase (AADC) inhibitors and DA2-receptor antagonists. IMCD cells also had measurable AADC activity and produced dopamine from exogenously added L-dopa. AADC inhibitors and DA2 antagonists also lowered basal PGE2 levels, suggesting that dopamine was being formed constitutively in culture. These results suggest that cultured IMCD cells have the capacity to take up and convert L-dopa to dopamine, which then stimulates PGE2 production via DA2K receptors. These results further suggest that locally formed dopamine could act as an autocrine/paracrine hormone in the kidney inner medulla to regulate PGE2 synthesis and water and electrolyte excretion.

Positron emission tomography (PET) following intravenous administration of beta-[11C]-L-DOPA provides a method of assessing regional cerebral uptake and utilization of levodopa. Cerebral levodopa kinetics in the rhesus monkey were investigated after the inhibition of catechol-O-methyltransferase (COMT) with RO 40-7592, and after coadministration of the peripheral aromatic L-amino acid decarboxylase (AADC) inhibitors benserazide and carbidopa. Pretreatment with RO 40-7592 (10 mg/kg), benserazide (10 mg/kg) or carbidopa (3.5 mg/kg) did not change striatal k3, which mainly reflects the ability for the brain tissue to convert [11C]-L-DOPA to [11 C]-dopamine, although the brain's uptake of radioactivity increased substantially after pretreatment with the AADC inhibitors. When benserazide was coadministered with RO 40-7592 (10 mg/kg) a dose-dependent decrease in striatal k3 was measured with an apparent ED50 of 3 mg/kg. No such effect was indicated after pretreatment with the combination of RO 40-7592 (10 mg/kg) and carbidopa (3.5 mg/kg). The possible negative interactions of coadministration with COMT inhibitors and predominantly peripherally acting AADC inhibitors must be considered when used in the therapy of Parkinson's disease.

The catalytic site of Escherichia coli F1-ATPase is reviewed in terms of structure and function. Structural prediction, biochemical analyses, and mutagenesis experiments suggest that the catalytic site is formed primarily by residues 137-335 of beta-subunit. Subdomains of the site involved in phosphate-bond cleavage/synthesis and adenine-ring binding are discussed. Ambiguities inherent in steady-state catalytic measurements due to catalytic site cooperativity are discussed, and the advantages of pre-steady-state ("unisite") techniques are emphasized. The emergence of a single high-affinity catalytic site occurs as a result of F1-oligomer assembly. Measurements of unisite catalysis rate and equilibrium constants, and their modulation by varied pH, dimethylsulfoxide, and mutations, are described and conclusions regarding the nature of the high-affinity catalytic site and mechanism of catalysis are presented.

Equilibrium and rate constants of immobilized concanavalin a determined by high-performance affinity chromatography

Similarity between serine hydroxymethyltransferase and other pyridoxal phosphate-dependent enzymes

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

Thapsigargin is a high affinity inhibitor of sarco- and endoplasmic reticulum (SERCA) type ATPases. We have used kinetics to determine the dissociation constant of thapsigargin-sarcoplasmic reticulum Ca(2+)-ATPase interaction in the absence and presence of non-ionic detergent. The observed "off" rate constant was measured as 0.0052 s-1 at 26 degrees C by the kinetics of inhibition of ATPase activity following transfer from an inactivated thapsigargin-ATPase complex to native ATPase. Inactive ATPase was produced by cross-linking the active site with glutaraldehyde. The observed dissociation rate constant was increased 7-fold by 0.1% Triton X-100, indicating that perturbation of the transmembrane and stalk region by detergent altered the binding parameters of the inhibitor. In addition, thapsigargin stabilized the ATPase against inactivation caused by detergent in the absence of Ca2+. The observed "on" rate constant of thapsigargin was measured at 26 degrees C as 25 s-1 irrespective of thapsigargin concentration, by the kinetics of thapsigargin- induced change in intrinsic fluorescence. An Arrhenius plot showed a temperature dependence of this rate constant, indicative of a conformational change in the protein with an activation energy of 9.5 kcal/mol for thapsigargin binding. The affinity of the Ca(2+)-ATPase for thapsigargin was calculated to be greater than 2 pM at pH 7.0 and 26 degrees C.

Backround: Parkinson's disease is a pathology involving the progressive degeneration of dopaminergic neurons in the substantia nigra of the brain. L-DOPA combined with an inhibitor of DOPA decarboxylase, a pyridoxal 5'-phosphate-dependent enzyme, is still the most effective treatment for symptoms of Parkinson's disease. LDOPA increases synaptic dopamine, while the inhibitor of peripheral DOPA decarboxylase reduces the conversion of L-DOPA to dopamine in the systemic circulation, allowing for greater L-DOPA distribution into the central nervous system. CarbiDOPA and benserazide are the inhibitors currently used in Parkinson's disease treatment. However, carbiDOPA and trihydroxybenzylhydrazine, the active metabolite of benserazide, are substrate analogues both endowed with a hydrazine function, which irreversibly bind not only to DDC but also to free pyridoxal 5'-phosphate and pyridoxal 5'-phosphate-dependent enzymes. Therefore, the lack of DOPA decarboxylase specificity, responsible for various side effects and adverse reactions, is a negative factor in such treatment of the disease. Aim of this review is to report on the most recent investigations regarding new DOPA decarboxylase inhibitors that could represent the starting point for possible Parkinson's disease drugs development. We focused on the common chemical features among all the identified inhibitors in order to seek shared structural motifs that could be involved in inhibition. Then, we highlighted the extent of inhibition, measured by means of in vitro and/or cell-based assays. Finally, we pointed out the state of the art in the metabolism of such classes of compounds, and discussed the possible advances in Parkinson's disease pharmacological treatment.