Characterization of the exchange reactions of rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase

The Fru-6-P/Fru-2,6-P2 exchange reaction of rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase was almost entirely dependent on the presence of Pi. This exchange was not due to a reversal of the bisphosphatase nor to trace amounts of adenine nucleotide in the enzyme. Exchange activity was maximal at pH 7, activated by ADP, and equal to 10-15 percent of the kinase Vmax. The ADP/ATP exchange reaction was more resistant to various protein modifying agents than the kinase. These studies confirm the existence of both exchange reactions but do not prove they are related to the kinase reaction.     

Modification and inactivation of rhodanese by 2,4,6-trinitrobenzenesulphonic acid

Bovine liver rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) is modified by 2,4,6-trinitrobenzenesulphonic acid, by the use of modifying agent concentrations in large excess over enzyme protein concentration. The end-point of the reaction, viz., the number, n, per enzyme protein molecule, of modifiable amino groups was determined graphically by the Kézdy-Swinbourne procedure. It was found that the value for n depends on the pH of the reaction medium, and ranges from 2, at pH 7.00, to 10.66, at pH 9.00. Again, the value for n increases with an increase in the concentration of 2,4,6-trinitrobenzenesulphonic acid used, with values ranging from 3.52, at 0.10 mM modifying agent, to 8.96, at 2 mM modifying agent. Rhodanese primary amino groups modification by 2,4,6-trinitrobenzenesulphonic acid is described by a summation of exponential functions of reaction time at pH values of 8.00 or higher, while at lower pH values it is described by a single exponential function of reaction time. However, the log of the first derivative, at initial reaction conditions, of the equation describing protein modification, is found to be linearly dependent on the pH of the reaction. An identical linear dependence is also found when the log of the first derivative, at the start of the reaction, of the equation describing modification-induced enzyme inactivation is plotted against the pH values of the medium used. In consequence, the fractional concentration of rhodanese modifiable amino groups essential for enzyme catalytic function is equal to unity at all reaction pH values tested. It is accordingly concluded that, when concentrations of 2,4,6-trinitrobenzenesulphonic acid in excess of protein concentration are used, all rhodanese modifiable amino groups are essential for enzyme activity. A number of approaches were used in order to establish a mechanism for the modification-induced enzyme inactivation observed. These approaches, all of which proved to be negative, include the possible modification of enzyme sulfhydryl groups, disulphide bond formation, enzyme inactivation due to sulphite released during modification, modification-induced enzyme protein polymerization, syncatalytic enzyme modification and hydrogen peroxide-mediated enzyme inactivation.     

Kinetics of protein-modification reactions. Stoichiometry of modification-produced enzyme inactivation: modification of rhodanese by 2,4,6-trinitrobenzenesulphonic acid.

A mathematical treatment is presented for the dependence of enzyme activity loss on the numbers and reactivities of the groups essential for catalytic function, when enzyme protein modification is carried out by the use of concentrations of protein reactive groups well in excess of that of modifying agent. Experimentally obtained data on the modification of rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) by 2,4,6-trinitrobenzenesulphonic acid are presented, and it is shown that, at pH9.00, the fractional concentration of rhodanese groups, or of rhodanese group reactivities, essential for enzyme catalytic function is 0.88; this value is found to decrease with decreasing pH of the reaction medium. The possibility that rhodanese inactivation by 2,4,6-trinitrobenzenesulphonic acid is brought about by modification of groups other than amino groups is ruled out by a comparison of the enzyme-inactivation and protein-modification stoichiometries, for putative reaction models for enzyme and modifying agent.     

On the role of tryptophan residues in the mechanism of action of glyceraldehyde-3phosphate dehydrogenase as tested by specific modification.

A method is described to selectively modify one of the three tryptophan residues of the subunit of glyceraldehyde-3-phosphate dehydrogenase from yeast. As modifying agent dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide was used. The residue which is modified by the procedure described has been identified as Trp-193. There are either one or two molecules of the modifying agent being added to this tryptophan side chain. The modification apparently does not cause a detectable conformational change of the protein as judged from the methods employed. However, the enzymatic activities in the dehydrogenase as well as in the esterase reactions are lost after the modification. It could be established that the modification rendered the enzyme unable to bind the oxidized coenzyme. Also the charge-transfer interaction between enzyme and coenzyme could no longer be observed.     

Interpretation of the temperature dependence of equilibrium and rate constants

The objective of this review is to draw attention to potential pitfalls in attempts to glean mechanistic information from the magnitudes of standard enthalpies and entropies derived from the temperature dependence of equilibrium and rate constants for protein interactions. Problems arise because the minimalist model that suffices to describe the energy differences between initial and final states usually comprises a set of linked equilibria, each of which is characterized by its own energetics. For example, because the overall standard enthalpy is a composite of those individual values, a positive magnitude for DeltaH(o) can still arise despite all reactions within the subset being characterized by negative enthalpy changes: designation of the reaction as being entropy driven is thus equivocal. An experimenter must always bear in mind the fact that any mechanistic interpretation of the magnitudes of thermodynamic parameters refers to the reaction model rather than the experimental system. For the same reason there is little point in subjecting the temperature dependence of rate constants for protein interactions to transition-state analysis. If comparisons with reported values of standard enthalpy and entropy of activation are needed, they are readily calculated from the empirical Arrhenius parameters.     

Inactivation of bacteriophage λ and λ DNA by nitrogen mustard

Bacteriophage λ and λ DNA were treated with alkylating agents. The survival of phage was assayed by infectivity and that of DNA by infectivity of phage particles assembled from the DNA in vitro. Phage λ were more sensitive to nitrogen mustard (Cl(CH₂)₂NMe(CH₂)₂Cl; HN2) than was λ DNA. The inactivation of λ DNA was biphasic; the second component of the inactivation was sensitive to mutations allelic for recA, polA and uvrB. This behaviour was not shown by pBR322 plasmid DNA treated with HN2 nor by λ DNA treated with monofunctional alkylating agents (or HN2 if the second alkylation reaction was stopped by addition of a mercaptan). From Arrhenius plots, the activation energy for the reactions with DNA and intact phage were found to be different. The activation energy for the inactivation of intact phage was the same as that (measured independently) for the predominant reaction (or class of reactions) in which HN2 cross-links DNA to protein in λ particles. From these data we conclude that the inactivation of λ by HN2 is due, primarily, to DNA-protein cross-linking. The implications for the mode of action of DNA-reactive bifunctional anti-viral and cytotoxic compounds are discussed.     

The kinetics and thermodynamics of reversible denaturation of crystalline soybean trypsin inhibitor

Crystalline soybean trypsin inhibitor protein undergoes denaturation on heating which is reversed on cooling. In the range of temperature of 35 to 50 degrees C. a solution of the protein consists of a mixture of native and denatured forms in equilibrium with each other. The equilibrium is only slowly established and its final value at any temperature is the same whether a heated, denatured solution of the protein is cooled to the given temperature or whether a fresh solution is raised to that temperature. The kinetics of reversible denaturation of the soybean protein as well as the reversal of denaturation is that of a reversible unimolecular reaction, each process consisting at a given temperature of the same two simultaneous reactions acting in opposite directions. The experimental data on the effect of temperature on the velocity and the equilibrium constants of the opposing reaction were utilized in evaluating the reaction energies and activation energies. The reaction energies for denaturation were found to be as follows:- Change in total heat of reaction DeltaH = 57,000 calories per mole Change in entropy of reaction DeltaS = 180 calories per degree per mole The heat of activation DeltaH(1) (double dagger) for denaturation = 55,000 The heat of activation DeltaH(2) (double dagger) for the reversal of denaturation = -1900 The entropy DeltaS(1) (double dagger) for denaturation = 95 The entropy DeltaS(2) (double dagger) for reversal of denaturation = -84     

Mechanism of cytochrome c peroxidase. O-benzoylhydroxylamine as an analog of hydrogen peroxide.

A number of reagents, some of which are electronic analogs of hydrogen peroxide, will replace it in the reactions of cytochrome c peroxidase. These compounds include N-bromosuccinimide, sodium hypochlorite, and the novel oxidizing agent O-benzoylhydroxylamine. If fragments of the oxidant played a functional role in the structure of the oxidized form of the enzyme, it would be expected that the product formed from O-benzoylhydroxylamine would differ from that formed from hydrogen peroxide. The products formed on reaction of the two oxidizing agents with cytochrome c peroxidase are indistinguishable. This results carries implications for the structure of the so-called ES compound. The extension in the range of specific substrates for cytochrome c peroxidase allows identification of the structure which compounds must possess to be oxidizing substrates for the enzyme. A mechanism for the first step of the reaction is suggested. O-Benzoylhydroxylamine is also a reducing agent, and its reaction with the enzyme is analogous to that of hydrogen peroxide with catalase. The final product of the reaction is the inert nitric oxide complex of ferrous cytochrome c peroxidase.     

The role of dicarbonyl compounds in non-enzymatic crosslinking: a structure-activity study

The Maillard reaction is a complex network of reactions that has been shown to result in the non-enzymatic crosslinking of proteins. Recent attention has focussed on the role of alpha-dicarbonyl compounds as important in vivo contributors to protein crosslinking but, despite extensive research, the molecular mechanisms of the crosslinking reaction remain open to conjecture. In particular, no relationship between the structure of the carbonyl-containing compounds and their activity as crosslinking agents has been established. In an effort to elucidate a structure-reactivity relationship, a wide range of dicarbonyl compounds, including linear, cyclic, di-aldehyde and di-ketone compounds, were reacted with the model protein ribonuclease A and their crosslinking activity assessed. Methylglyoxal and glutaraldehyde were found to be the most efficient crosslinkers, whilst closely related molecules effected crosslinking at a much lower rate. Cyclopentan-1,2-dione was also shown to be a reactive crosslinking agent. The efficiency of methylglyoxal and glutaraldehyde at crosslinking is thought to be related to their ability to form stable heterocyclic compounds that are the basis of protein crosslinks. The reasons for the striking reactivity of these two compounds, compared to closely related structures is explained by subtle balances between competing pathways in a complex reaction network.     

Influence of modifying agents on enzyme inactivation studies. An analysis using a series mechanism and a form of the Hill-type equations

A series deactivation model is utilized to theoretically examine the influence of different modifying agents on enzyme deactivation kinetics. A form of the Hill-type equation is used to describe the effect of the modifying agents on the model parameters. Modification-induced inactivation equations are presented for the acetylation and succinylation of E. Coli asparaginase, for the site-specific reagent and substrate modification of flavocytochrome b(2) from Baker's yeast, and for the guanidinium chloride inactivation of cathepsin D. The analysis of more data for these and other enzymes would help further substantiate the technique presented and enhance the applicability of the model.     

The active site loop of S-adenosylmethionine synthetase modulates catalytic efficiency

Crystallographic studies of Escherichia coli S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase, MAT) have defined a flexible polypeptide loop that can gate access to the active site without contacting the substrates. The influence of the length and sequence of this active site loop on catalytic efficiency has been characterized in a mutant in which the E. coli MAT sequence (DRADPLEQ) has been replaced with the distinct sequence of the corresponding region of the otherwise highly homologous rat liver enzyme (HDLRNEEDV). Four additional mutants in which the entire DRADPLEQ sequence was replaced by five, six, seven, or eight glycines have been studied to unveil the effects of loop length and the influence of side chains. In all of the mutants, the maximal rate of S-adenosylmethionine formation (k(cat)) is diminished by more than 200-fold whereas the rate of hydrolysis of the tripolyphosphate intermediate is decreased by less than 3-fold. Thus, the function of the loop is localized to the first step in the overall reaction. The K(m) for methionine increases in all of the oligoglycine mutants, whereas the K(m) values for ATP are not substantially different. The k(cat) for the wild-type enzyme is decreased by increases in solution microviscosity with 55% of the maximal dependence. Thus, a diffusional event is coupled to the chemical step of AdoMet formation, which is known to be rate-limiting. The results indicate that a conformational change, possibly loop closure, is associated with AdoMet synthesis. The data integrate a previously discovered conformational change associated with PPP(i) binding to the E x AdoMet complex into the reaction sequence, reflecting a difference in protein conformation in the E x AdoMet x PPP(i) complex whether it is formed from the E x ATP x methionine complex or from binding of exogenous PPP(i). The temperature dependence of the k(cat) for S-adenosylmethionine formation shows that the removal of the side chains in the glycine mutants causes the activation enthalpy of the reaction to approximately double in each case, while the activation entropy changes from negative in the wild-type enzyme to positive in the mutants. The favorable activation entropy in the mutant-catalyzed reactions may reflect release of water during catalysis, while the negative activation entropy in the reaction catalyzed by the wild-type enzyme apparently reflects reorganization of the loop. The observations point to how nature can fine-tune the activity of an enzyme by modifying substrate and product access to the active site rather than by altering the enzyme x substrate contacts or the catalytic machinery itself.     

Effect of the immediate environment on the reactivity of the essential -SH group of papain.

The effect of the microenvironment on the reactivity of the essential -- SH group of papain was studied by alkylation with methyl iodide and with the more polar iodoacetamide. Rate and activation parameters for these reactions were determined with two forms of the -- SH group: the free mercaptide ion at pH 10.0, and the mercaptide-imidazolium ion-pair at pH 5.5. The ion-pair of papain reacts with methyl iodide at a rate 1470 times less than that of thiolsubtilisin. This surprising difference between the reactivities of the two enzymes suggests that in contrast to thiolsubtilisin, where a non-polar environment enhances the rate, in the case of papain a more polar environment somewhat inhibits the reaction with the non-polar methyl iodide. The positive activation entropy for the papain reaction may indicate an 'ordered' structure of bound water around the sulfur atom. The high rate and the low activation entropy (organized transition state) of the reaction of papain with iodoacetamide can be explained in terms of hydrogen-bond formation between the enzyme and the amide group of the alkylating agent.     

非典型角蒽环聚酮氧化开环酶的功能与进化

非典型角蒽环聚酮化合物是一类经过氧化重排反应形成的具有独特骨架结构的芳香聚酮类化合物。近年来的研究表明,尽管此类化合物具有多种多样的骨架结构,它们都是由共同的生物合成中间体Dehydrorabelomycin生成的。一个独特的加氧酶家族(称为非典型角蒽环氧化开环酶)催化了Dehydrorabelomycin的氧化碳-碳键断裂与重排反应。尽管这些酶属于同一个蛋白质家族,催化相同的底物发生氧化开环反应,但是通过不同的重排方式形成了对应于各自生物合成终产物的骨架结构,对这类化合物最终结构的形成起到了关键作用。对这一家族的加氧酶进行深入的催化功能与反应机理研究,不仅有助于对已知芳香聚酮的结构改造与新颖骨架结构芳香聚酮的发现,也有助于加深对于蛋白质序列进化与功能演化的认识。     

Conformational fluctuations and protein reactivity. Determination of the rate-constant spectrum and consequences in elementary biochemical processes

When a protein's active site happens to be strongly coupled with the protein structure, the rate constant of the reaction may eventually be modulated by the conformational fluctuations. Evidence for this effect has long been provided by extensive flash photolysis investigations of liganded hemoproteins and more recently of the non-heme respiratory protein hemerythrin in hydro-organic solvents. Within a given protein conformational substate, an elementary reaction step is characterized by one single free energy barrier and by a first-order rate constant, k, which changes with temperature according to an Arrhenius law. At physiological temperature and low viscosity, ultrafast conformational relaxation causes efficient averaging of the reaction rates and the protein displays exponential kinetics with an average rate constant (k). Under sufficiently general conditions, it can be shown that (k) also follows a simple Arrhenius law with 'effective' values of the pre-exponential factor Aeff and activation enthalpy Heff. It is found that Aeff strongly depends on the overall shape of the rate constant distribution and that Heff actually corresponds to the lower limit of the enthalpy of activation, i.e. the value associated with the highest possible reaction rate. The underlying distribution of rate constants can be reconstructed from a set of experiments in which the kinetics depart from an exponential, i.e. at low temperature and high viscosity. The most probable distribution of exponentials consistent with the observed kinetics of the geminate recombinations of oxygen with photodissociated hemerythrin has been determined by using a new approach, known as the maximum entropy method. The results are consistent with a single pre-exponential value and a distributed enthalpy spectrum. As expected, Heff does not coincide either with the most probable nor with the average value of the enthalpy. The most salient findings are that the probability for any protein molecule to have an enthalpy of activation equal to the effective value Heff vanishes and that Aeff differs by nearly three orders of magnitude from the true value A0. Biochemical reaction rates are actually average values, since protein reactions are measured under physiological conditions, where conformational relaxation is always fast. Our understanding of the significance of Aeff and Heff is therefore entirely dependent on the knowledge of the distribution function of the rate constants. In particular, enthalpy and entropy terms of similar reactions performed by different proteins cannot be compared as long as the distribution of the rate constants remains unknown.     

Characterization of the DNA methylase activity of the restriction enzyme from Escherichia coli K

The restriction endonuclease from Escherichia coli K is a multifunctional protein which efficiently methylates heteroduplex DNA (one strand modified and one strand unmodified) in the presence of S-adenosylmethionine (AdoMet), ATP, and Mg2+. The methylase activity is catalytic, and seems to modify different heteroduplex host specificity sites for E. coli K with equal efficiency. In the methylase reaction, both AdoMet and ATP (or its imido analog) act as allosteric effectors, but AdoMet also serves as a methyl donor. Preincubation of the enzyme with AdoMet eliminates the lag period observed in DNA methylation. The rate of enzyme activation was determined using the AdoMet analog Sinefungin. The result are consistent with the hypothesis that the early steps of AdoMet binding and enzyme activation are common to both restriction and modification reactions.     

Modification of the conformational equilibria in the sodium and potassium dependent adenosinetriphosphatase with glutaraldehyde

Glutaraldehyde treatment of electroplax membrane preparations of Na,K-ATPase leads to irreversible changes in the enzymic behavior of the protein, which are not due to modification of the active site. When the glutaraldehyde treatment is carried out in a medium containing K+ and without Na+, the "K+-modified enzyme" so produced shows the following changes in enzymic properties: The steady-state phosphorylation by ATP and the rate of ATP-ADP exchange are decreased to approximately 40% of control, while Na,K-ATPase activity decreases to approximately 15% of control. Phosphatase activity is decreased very little, but the potassium activation parameters of the reaction are changed, from K0.5 approximately equal to 5 mM and nH = 1.9 in control to K0.5 approximately equal to 0.5 mM and nH = 1 in K+-modified enzyme. KI(app) for nucleotide inhibition of phosphatase activity is increased significantly. Changes in the cation dependence of the ATPase reaction are also observed. All of these effects can be explained by assuming that the cross-linking of surface groups in protein subunits when they are in conformation E2 shifts the intrinsic conformational equilibrium of the enzyme toward E2. We considered the simplest mathematical model for the coupling between K+ binding and the conformational equilibrium, with equivalent potassium sites that must be simultaneously in the same state. If one assumes that the potassium activation of phosphatase activity in the K+-modified enzyme reflects the affinity for K+ of E2, the behavior of the phosphatase activity in the native enzyme can be fit if there are only two potassium sites, whose affinity is 80-fold higher in E2 than in E1, and the equilibrium constant for E2 in equilibrium E1 is about 250. The same sites can explain the activation of dephosphorylation during ATP hydrolysis. Independent of the model chosen, potassium ions must be required for the catalytic action of form E2 and cannot be merely "allosteric activators". The enzyme modified with glutaraldehyde in a medium containing Na+ also has interesting properties, but their rationalization is less straightforward. The Na,K-ATPase activity is inhibited more than the "partial reactions", as in the K+-modified enzyme. We suggest that this is a generally expected result of modifications of the enzyme.     

Radiation modulating properties of derivates of 1,4-dihydropyridine and 1,2,3,4,5,6,7,8,9,10-decahydroacridine-1,8 dione

First attempt to analyse radiation modifying action of 52 compounds--derivatives of 1,4-dihydropyridine is presented. Many of these compounds have electrodonating and antioxidative activities. Local radioprotective effects of the substances has been studied using experimental model with partial beta- and X-rays exposure of sole skin in rats to doses of 40 and 30 Gy. Comparison of drug effectiveness and chemical structure revealed the changes of activity dependence due to modification of peripheral radicals of 1,4-dihydropyridine ring. Radioprotective and radiosensitizing compounds with general low toxicity have been found. It is suggested to use the most active compounds for the development of medicinal forms for prevention of local skin and mucosal radiation injuries. Positive results have been obtained with dieton a radioprotective compound of the same type, in radiotherapy.     

Kinetic analysis of the heparin-enhanced plasmin–antithrombin III reaction. Apparent catalytic role of heparin

Inactivation of plasmin by a 3-4-fold molar excess of antithrombin III follows pseudo-first-order kinetics and the apparent rate constants are proportional to the concentration of the inhibitor. Heparin accelerates the inactivation reaction without changing its pseudo-first-order character, and the apparent rate constants are also proportional to the concentration of the polysaccharide. Heparin results in a minimum 20-fold rate enhancement of the reaction between plasmin and antithrombin III when the concentrations of heparin and plasmin are approx. 0.5mum and 1mum respectively. Heparin at a molar concentration well below that of plasmin still accelerates the reaction: one molecule of the polysaccharide is able to facilitate the inactivation of about 100 molecules of plasmin. Heparin must bind to plasmin to accelerate the plasmin-antithrombin III reaction, since the modification of four to five lysine residues of the enzyme inhibits the rate-enhancement effect of heparin and the dissociation of heparin-plasmin complex decreases the inactivation rate of plasmin. Increasing the concentration of antithrombin III, at a constant amount of heparin, results in increase of the inactivation rate. By contrast, the effect of increasing the amount of plasmin in the presence of constant amount of heparin and antithrombin III is such that higher plasmin-to-heparin ratios are associated with lower rates of inactivation. It seems, therefore, that to obtain ;optimal' conditions for fast enzyme inactivation, the amount of heparin should be matched to plasmin rather than to antithrombin III. Arrhenius plots of the plasmin-antithrombin III reaction are linear both in the absence and presence of heparin, at concentrations of 1 or 2mug/ml, over a range of 26K. Under these experimental conditions, heparin increases activation entropy. The findings show that heparin seems to fulfil some criteria that are characteristic for biological catalysis: binding, reaction-rate enhancement (increasing activation entropy), recycling of heparin (effectiveness of non-stoichiometric amounts of the polysaccharide) and specificity.