Kinetic Consequences Research Articles

Effect of soluble polymers on the kinetic properties of aldolase and D-glyceraldehyde-3-phosphate dehydrogenase

Abstract The kinetic and thermodynamic properties of aldolase and D-glyceraldehyde-3-phosphate dehydrogenase were studied in the presence of soluble poly(vinyl alcohol), poly(N-vinyl pyrrolidone) and poly(acrylic acid). Two kinds of aldolase preparations were found which did not differ in specific activity, but showed differences in respect of their kinetic response to polymer action. Preparation type a was more active at low concentrations of the polymers, and less active at high concentrations, than in buffer solution. In the case of type b preparation the polymer concentration dependence was detectable only in solutions containing poly(acrylic acid); poly (vinyl alcohol) had no effect in the concentration range studied, whereas poly(N-vinyl pyrrolidone) caused inhibition rather than activation of the enzyme. However, the thermodynamic data indicate that the polymer interacts with the enzyme even in those cases where kinetic effects are absent. Alteration of the steric structure of poly(vinyl alcohol) by intramolecular crosslinks caused qualitative changes in its effect on aldolase activity. In the case of D-glyceraldehyde-3-phosphate dehydrogenase, a dissociable tetramer, the kinetic consequences of polymer action depend on the oligomeric state of the enzyme. It appears that the above non-specific enzyme—macromolecule interactions are considerably influenced by the colloidal state of both interacting partners.

Electron spin resonance study of the decay of methyl radical-bromide ion pairs in acetonitrile at low temperature

Methyl radicals adjacent to cyanide ions are generated upon photolysis of ..gamma..-irradiated, crystalline acetonitrile, and these radicals decay by hydrogen atom abstraction from neighboring matrix molecules. In this paper the kinetic consequences of a slight alteration in the immediate environment of the methyl radical are examined. It is found that replacement of the cyanide ion with a bromide ion causes the observed rate constant for abstraction to be lower by about a factor of 2 at low temperatures. Once again, the low activation energy and large deuterium kinetic isotope effect are indicative of very important tunnel effects in the abstraction reaction.

Kinetic and Structural Consequences Derived from Ageing Effects on Electrochemically Formed Layers

AbstractThe influence of different ageing processes on electrochemical reactions is analysed. Three main types of ageing processes are described: open circuit ageing, potentiostatic ageing and potentiodynamic ageing. The data derived from different electrode processes show that the films are composite systems themselves. They involve various nonequilibrated species which accordingly react to attain either a single equilibrium configuration or a configuration involving equilibria among the various surface species. Surface restructuring and cluster‐type reactions are important contributions toward understanding the dynamic behaviour of electrochemical interfaces.

Circular Dichroic Properties and Conformation of Thionicotinamide Dinucleotides Bound to Horse-Liver Alcohol Dehydrogenase

The interaction between horse liver alcohol dehydrogenase and the oxidized and reduced forms of the 3-thionicotinamide--adenine dinucleotide coenzyme analogues (sNAD and sNADH) has been investigated by ultraviolet absorption, fluorescence and circular dichroism. The fluorescence of sNADH is enhanced when bound to the enzyme, and the protein fluorescence is quenched by both sNADH (60--65%) and sNAD (65%). The possible origin of the larger quenching produced by sNAD with respect to that of NAD is discussed. Coenzyme dissociation constants have been determined by monitoring the quenching of the protein fluorescence, and some kinetic consequences of these dissociation constants are discussed. The dichroic properties of various enzyme complexes have been investigated, and are discussed in terms of conformational changes and environmental changes during coenzyme binding. The conformation of sNAD bound to the enzyme in the presence of trifluorethanol and the conformation of sNADH bound to the enzyme in the presence of isobutyramide have been analyzed in particular detail. Also the circular dichroic spectrum of the apoenzyme is discussed in terms of contributions of the aromatic amino acid residues in the highly resolved 240--310-nm region and in terms of helix content in the 220-nm region.

Fluorescence Properties of Reduced Thionicotinamide - Adenine Dinucleotide and of Its Complex with Octopine Dehydrogenase

Reduced 3-thionicotinamide--adenine dinucleotide (sNADH) is shown to be fluorescent, with an emission maximum at 510 nm when excited in the region of the absorption maximum (398 nm), and with a very low quantum yield, (3.4 +/- 0.5) x 10(-4). The interaction between sNADH and octopine dehydrogenase was investigated by ultraviolet-difference spectroscopy and fluorescence. Some surprising fluorescence features were found when sNADH was bound to the enzyme in the presence of D-octopine, as follows. (a) There is an unusually high enhancement of the dinucleotide fluorescence (by at least a factor of 100) attended by a 40-nm blue shift of the emission maximum. (b) The protein fluorescence is quenched almost completely. (c) The bound coenzyme analog undergoes a photoreaction, which proceeds differently from that occurring the free form. These features appear to be unique to the octopine.sNADH complex, as for example they are not present when sNADH is bound to horse liver alcohol dehydrogenase, or when NADH is bound to octopine dehydrogenase. The possible origin of these fluorescence features is discussed. Binding and kinetic studies were carried out with sNAD and sNADH. It was found that sNAD neither binds nor acts kinetically as a coenzyme. sNADH exhibits relatively good binding, with Km and Ki values close to those of the natural coenzyme, but the turnover number is 460 times smaller than that with NADH. The kinetic consequences of these findings are discussed. The sNADH dissociation constants were determined as a function of temperature, and appear to be practically temperature-independent in the range 10--40 degrees C. It seems thus, in agreement with previous studies, that the interaction between octopine dehydrogenase and coenzymes proceeds athermically, regardless of the structure, affinity, and chemical reactivity of the coenzyme. The possible biological and chemical meaning of this finding is discussed.

The chemical and kinetic consequences of the modification of papain by N-bromosuccinimide.

Nonactivated papain was treated with N-bromosuccinimide at pH 4.75. The N-bromosuccinimide-modified enzyme was characterized by (1) the change in absorbance at 280 nm, (2) amino acid analysis, (3) separate chemical determinations of tryptophan and tyrosine (4) difference spectroscopy, and (5) an N-terminal residue determination. It is concluded that N-bromosuccinimide in sevenfold molar excess oxidizes one tryptophan and two to three tyrosine residues per molecule of nonactivated papain, without causing peptide chain cleavage. Kinetic studies with several substrates and competitive peptide inhibitors were performed at pH6 using the N-bromosuccinimide-modified papain. In addition, the kinetics of the modified enzyme with the substrate alpha-N-benzoyl-L-arginine ethl ester were studied in the region of pH 3.5-9.0. All substrates (and inhibitors) test, with the exception of alpha-N-benzyoyl-L-arginine p-nitroanilide, displayed approximately a two fold decrease in both kcat and Km (or Ki), relative to the native enzyme. It is concluded that the key tryptophan residue which is probably Trp-177.

Properties of a CH3-blocked creatine kinase with altered catalytic activity. Kinetic consequences of the presence of the blocking group.

Steady state kinetic parameters for rabbit muscle creatine kinase (EC 2.7.3.2) and this enzyme stoichiometrically blocked at the iodoacetamide-sensitive cysteinyl residue with a CH3S-group have been measured at 30+/-0.1 degrees, pH 9.00, using Mg(II) as the required metal ion cofactor. The double reciprocal plots for the CH3S-blocked enzyme with MgATP as the variable substrate are biphasic, each curve showing a break at approximately 1.9 mM MgATP, and suggest the possibility of negative cooperativity in metal-nucleotide binding. Furthermore, extrapolated lines at high MgATP concentrations intersect on the abscissa, indicating loss of synergism in binding of substrates. In contrast, observed Michaelis constants for creatine are, within experimental error, the same for both native and blocked enzymes. The maximal velocity of the CH3S-blocked enzyme is found to be 28.1% of the value of the native enzyme. Product inhibition patterns for both native and blocked enzyme are also compared. Again, these patterns indicate that the CH3S-blocking group alters the nucleotide binding site more than the guanidino substrate binding site. Calculations using the methods of Chou and Fasman (1970) Biochemistry 13, 211-222) lead to the prediction that the active cysteinyl residue occurs at the beginning of a beta turn which separates two portions of beta sheet structure of the enzyme, and so may be in a position to mediate conformational changes in the protein.

Macromolecular interactions in enzyme regulation

The differences observed by several authors in the NaDH-binding ability of the subunits of glyceraldehyde-3-phosphate dehydrogenase are caused by the dissociation of the enzyme. A specific model has been elaborated for the interpretation of the phenomenon; the earlier interpretation in terms of ligand-induced negative cooperatively seems to be erroneous. In the regulation of the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase in the glycolytic pathway the association-dissociation dynamics of the enzyme play the decisive role. In the higher aggregational forms of the enzyme the relative concentration of “functioning active centres” progressively decrease and this may furnish the basis for a novel type of regulatory mechanism. Aldolase forms a complex with glyceraldehyde-3-phosphate dehydrogenase (presumably with the dimeric form) thereby enhancing the activity of the latter. This finding calls the attention to the importance of enzyme-enzyme interactions in the intracellular metabolic regulation. The modelling of enzyme-other macromolecule interactions was performed by studying the behavior of aldolase and glyceraldehyde-3-phosphate dehydrogenase in the solution of synthetic polymers. The change of kinetic parameters suggests that the interaction induces conformational alterations in the enzymes. The kinetic consequences of structural changes depend not only on the enzyme, but also on the structure of the polymer.

Drug Kinetics in Pregnancy

Any drug given to a pregnant woman must be considered as possibly harmful in the fetus, since all drugs administered to the mother cross the placental membrane, although at different rates. Important physiological changes occur in pregnancy, which may influence the kinetics of drugs. Differences in gastrointestinal function are likely to alter drug absorption rates in the stomach or gut. Ventilatory alterations modify pulmonary drug absorption or elimination. Important changes in haemodynamics and alterations in body water compartments influence drug distribution and elimination. Renal drug elimination is generally enhanced, whereas hepatic drug elimination may be modified in different ways. Computerised pharmacokinetic models representing the compartmental aspects of the fetal-maternal unit and fetal-maternal drug interrelationships may be used to predict kinetic consequences of fetal drug exposure. Such information may be a useful guide for the clinical use of drugs during pregnancy, particularly for treatment of fetal disease.

Kinetic consequences of molecular aggregation in the hydrolysis of p-nitrophenyl carboxylates

Alkaline hydrolysis of p-nitrophenyl carboxylates was investigated in mixed aqueous–organic solvents. Esters with a long alkyl chain tend to aggregate by intermolecular hydrophobic interaction above the critical concentration. Among acetate, hexanoate, decanoate, dodecanoate, and hexadecanoate, the last showed the lowest critical concentration, while acetate and hexanoate did not demonstrate any detectable aggregation even in 1.0%(v/v) aqueous dioxan. The reactivity of monomeric esters decreased with increasing alkyl-chain length, due to the selfcoiling behaviour of the alkyl chain which acts to mask the ester bond from hydroxide attack. The critical concentration for dodecanoate decreased in the reaction medium order: 10.9%(v/v) dioxan > 10.9%(v/v) acetonitrile [graphic omitted] 1.0%(v/v) dioxan > 10.9%(v/v) ethanol. The rate increase observed for hydrolysis of monomeric dodecanoate by increasing the organic content of a solvent was attributed to elongation or decoiling of the monomeric ester. As for the effect of hydroxide ion concentration on aggregation behaviour, the monomer fractions of dodecanoate and of hexadecanoate above their critical concentrations were found to decrease with increasing [OH–] due to the structure-making ability of hydroxide ion. Urea acted to raise somewhat the critical concentrations of the esters and to decrease their aggregation numbers as exemplified by the alkaline hydrolysis of dodecanoate. The saltingout effects of inorganic salts gave a reduction in the fraction of monomeric ester and hence in the overall hydrolysis rate.

Kinetic consequences of a slow substrate binding step in group transferases Interpretation of product inhibition experiments

The steady stake kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by “transition state analog” inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random “kinetic mechanisms”. I therefore suggest that the use of these concepts should be abandoned.

Alkane sulphonate preparation by the sulphitation of long chain olefins

AbstractThe free radical addition of sodium bisulphite to linear olefins (sulphitation) has been investigated. It was observed that the kinetic consequences of the probable mechanism of sulphitation were consistent with the experimental results. Thus sulphitation is first order with respect to olefin concentration and above a critical level is independent of bisulphite concentration. In the sulphitation of 1‐dodecene an undesirable alkane–sulphonate–sulphinate forms, the proportion of which, as expected, varies with sulphitation pH; the higher the pH the less the proportion of sulphonate–sulphinate. To prepare alkane sulphonates optimally thereby restricting the sulphonate–sulphinate proportion to an acceptable minimum (15% w/w) sulphitation pH should be the maximum at which the critical bisulphite concentration can exist. Further minimisation of sulphonate–sulphinate formation requires a new approach. It was found that whereas linear olefins give rise to this product, as does 1‐hexyne, vinylidene olefins, styrene and 1,3–hexadiene do not. These observations have been rationalised in terms of the relative thermal stabilities of the intermediate sulphone–sulphonate radicals.

Stepwise tRNA recognition mechanism and its kinetic consequences

The Michaelis equation is widely used for the treatment of enzyme kinetic data, maximum velocity V, and Michaelis constant KM being the final values obtained. The first is usually assumed to be the measure of the velocity of the catalytic step of the process, the latter is regarded as a measure of the affinity of substrate to enzyme. With tRNA’s as substrates of ARSases* (E.C. 6.1.1) or aa-tRNA’s as ribosomal substrates, several points of the enzyme-substrate interaction should be considered. It is reasonable to suggest the enzyme-substrate complex formation to be a multi-step process. This possibility has been discussed [ 1 ] . In the present paper the two-step recognition mechanism is analyzed as the most simple model of the multi-step recognition. It is demonstrated that in this case, the above mentioned simple interpretation of the KM and V, values may be wrong, namely significant changes in V, may occur without any changes in the catalytic rate constant of the process, due to changes in the recognition step. Some consequences for the problem of the specificity of tRNA amino acylation and of translation are discussed.

A Ligand-Induced Conformational Change of Yeast Dipeptidase at Physiological pH: Kinetic Consequences and Possible Regulatory Significance

The hydrolysis of dipeptides by purified yeast dipeptidase (EC 3.4.13.?) shows marked deviations from Michaelis-Menten kinetics over a wide range of pH. Quite anomalous kinetics is observed between pH 6 and 7, indicating a drastic change in the enzyme's properties. A reasonable explanation is provided by the assumption of a conformational transition brought about either by pH shifts or, at a constant pH, by changes in the substrate concentration. The transition, which may have a half-life on the order of minutes under appropriate conditions, is a distinctly cooperative process, with a dependence on ligand concentration higher than first order. The two forms of the enzyme differ clearly from each other with respect to various properties. The magnitudes and pH dependence of the kinetic parameters as well as the type of inhibition (or activation) exerted by amino acids and other ligands are different, as are their heat stabilities and the rates of inactivation by photooxidation of proteolytic degradation. Neither the molecular weight nor the gross conformation of the enzyme changes during the transition, so it seems to be due to a local isomerization affecting mainly the geometry of the active site. The sensitivity of dipeptidase to changes in the concentrations of substrates and other ligands is most pronounced exactly at the values of pH known to prevail in the living yeast cell. Thus the observed effects, which modulate dipeptidase activities within wide and limits, according to the amounts of dipeptides and amino acids present, are likely to play a role in the regulation of the enzyme in vivo.

Consequences of resonance tunnelling in chemical kinetics

The kinetic consequences of resonance tunnelling processes that may occur in chemical reactions are investigated in terms of a multi-centered unsymmetrical Eckart potential barrier. This potential function does not only simulate the possible existence of intermediate wells in the effective potential energy cut along the reaction path, but also is amenable to analytic solutions. The reaction rate as well as its dependence on temperature, reduced mass,Q-value, activation energy and barrier diffuseness are evaluated for successively increasing the number of barrier stages. Comparisons between results due to single and multi-humped potential energy barriers are made and discussed.

Ligand binding kinetics of des arginine haemoglobin

Des arginine 141 a haemoglobin (the haemoglobin in which the C-terminal arginine of the a chain has been removed) has a high affinity for oxygen and a reduced co-operativity in its oxygen equilibrium binding. The kinetic consequences of this modification are investigated in this paper. Deoxy des Arg haemoglobin binds carbon monoxide faster than does haemoglobin A, whilst oxy des Arg haemoglobin loses oxygen more slowly. These results are correlated with the oxygen equilibrium binding properties of des Arg haemoglobin. The carbon monoxide binding kinetics have been interpreted as implying a change in the parameter c (of the allosteric model), as well as L, when this arginine is removed from haemoglobin.

Orbital Theory of Heterolytic Fragmentation and Remote Effects on Nitrogen Inversion Equilibria

AbstractFrom a molecular orbital study of model systems we derive the electronic requirements for the Grob fragmentation. The necessary condition for an allowed fragmentation in an X‐C1‐C2‐C3‐N system, or the amino cation +C1‐C2‐C3‐N is the level ordering A below S. This in turn is set by maximal through‐bond coupling of the empty cation orbital and the nitrogen lone pair. The conformational dependence of through‐bond coupling is exactly that derived by Grob, namely parallel orientation of the cation orbital (or the C‐X bond), the C2‐C3‐σ‐bond, and the N‐lone‐pair. When the C1‐C2‐C3 and C2‐C3‐N angles are small, the through‐space interaction dominates, reversing the level ordering to S below A, and consequently makes the fragmentation forbidden even though the conformational requirements for it are met. Ring closure becomes allowed. Some examples exploiting this result are presented, as well as procedures for enhancing through‐bond coupling and thus fragmentation. The through‐bond‐effect has also kinetic consequences, allowing the definition of a new type of remote neighbouring group participation operative through bonds and not by direct overlap. The position of equilibria in nitrogen inversion processes should also be influenced by remote substituents which are π‐acceptors or donors.

Kinetic consequences of intermolecular attraction. I. Aminolysis of p-nitrophenyl decanoate and acetate by n-decylamine and ethylamine

Kinetic consequences of intermolecular attraction. I. Aminolysis of p-nitrophenyl decanoate and acetate by n-decylamine and ethylamine

Kinetic consequences of intermolecular attraction. II. Hydrolysis of a series of fatty acid p-nitrophenyl esters catalyzed by a series of N-n-alkylimidazoles. A very simple esterase model

Kinetic consequences of intermolecular attraction. II. Hydrolysis of a series of fatty acid p-nitrophenyl esters catalyzed by a series of N-n-alkylimidazoles. A very simple esterase model

Imidazole group-catalyzed reactions in formaldehyde.

The catalysis of p‐nitrophenyl acetate hydrolysis by imidazole compounds in formaldehyde solution occurs at a rate lower than in water. Kinetically determined pK' values of imidazole and its derivatives agree with titrimetric values and the catalytically active species is the free base form of the N‐hydroxymethyl adduct of the imidazole ring. With imidazole as catalyst in formaldehyde, N‐acetyl‐N′‐hydroxymethylimidazolium cation is apparently a short‐lived intermediate in the reaction. The hydroxymethyl adduct, although removable by dilution, is not displaced by substrate during catalysis. These results provide additional support for the occurrence of a reaction between for‐aldehyde and the imidazole ring and its kinetic consequences parallel those seen in the effect of formaldehyde on chymotrypsin‐catalyzed reactions.