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: analysis of modification-induced protein unfolding

A mathematical treatment of a two-sited, modification-induced protein unfolding model is presented, and it is shown that the dependence of the concentration of modified protein groups on reaction time is described by a linear, second-order, differential equation with nonzero right hand side. The analytic solution of this equation consists of a summation of exponential functions of reaction time. By assigning arbitrary values to the modification and isomerization rate constants of these equations, simulated cases of protein modification are presented, and the apparent end-point of the reaction is determined graphically. It is found that the apparent end-point of the reaction is, in most cases studied, different from the true value of two groups modified per protein molecule, and is a function of both the modification, and isomerization rate constants of the model. The first derivative of the protein modification reaction, at the start of the reaction, [E]'mod (0), is determined, for the same simulated cases of protein modification, by two different analytical methods. It is found that the [E]'mod(0) value, obtained from graphical and numerical analysis data, is in most cases in good agreement with the value expected from first principles. Finally, the different irreversible enzyme inhibition forms, contingent upon the different kinds of the enzyme inactivation-protein modification relationships of the protein modification model under consideration, are presented and discussed.     

Kinetics of modification of the mitochondrial succinate-ubiquinone reductase by 5,5′-dithiobis-(2-nitro-benzoic acid)

The kinetic theory of the substrate reaction during modification of enzyme activity previously described by Tsou [Tsou (1988),Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381–436] has been applied to a study of the kinetics of the course of inactivation of the mitochondrial succinate-ubiquinone reductase by 5,5′-dithiobis-(2-nitro-benzoic acid) (DTNB). The results show that the inactivation of this enzyme by DTNB is a conformation-change-type inhibition which involves a conformational change of the enzyme before inactivation. The microscopic rate constants were determined for the reaction of the inactivator with the enzyme. The presence of the substrate provides marked protection of this enzyme against inactivation by DTNB. The modification reaction of the enzyme using DTNB was shown to follow a triphasic course by following the absorption at 412 nm. Among these reactive thiol groups, the fast-reaction thiol group is essential for the enzyme activity. The results suggest that the essential thiol group is situated at the succinate-binding site of the mitochondrial succinate-ubiquinone reductase.     

Affinity labeling of the ATP-binding site of type II calmodulin-dependent protein kinase by 5'-p-fluorosulfonylbenzoyl adenosine

Modification of the type II calmodulin-dependent protein kinase by 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) resulted in a time-dependent inactivation of the enzyme. The reaction followed pseudo-first-order kinetics and showed a nonlinear dependence on reagent concentration. The rate of inactivation was sensitive to Mg2+- and calmodulin-induced conformational changes on the enzyme. However, the enhancing effects of these ligands were not additive; indeed, the kinetic parameters of the Mg2+-stimulated inactivation reaction with FSBA (Kinact = 2.4 mM; kappa max = 0.12 min-1) were almost unaffected by the simultaneous addition of calmodulin (Kinact = 1.5 mM; kappa max = 0.086 min-1). Protection from inactivation by FSBA was provided by Mg2+-ADP which is consistent with modification of the catalytic site. An analysis of the protective effect of Mg2+-ADP in the absence (Kd = 590 microM) and presence (Kd = 68 microM) of calmodulin demonstrated that binding of the modulator protein to the enzyme increases the affinity of the protein kinase for nucleotides. Modification by FSBA resulted in labeling of both Tyr and Lys residues but only labeling of Lys was decreased by Mg2+-ADP which is consistent with the hypothesis that a conserved Lys residue is important in nucleotide binding to the protein kinase. However, the kinetic results of the inactivation reaction suggest that this Lys is not involved in mediating the calmodulin-promoted increase in the affinity of the enzyme for Mg2+-nucleotide complexes.     

A kinetic approach to the evaluation of alkaline phosphatase inactivation by urea

A kinetic approach is described which enables the measurement of the enzyme inactivation rate constant during the reaction course. A mathematical analysis is presented and it is shown that a time-dependent step may be postulated to exist. Reaction kinetics follow an exponential rule with time as the independent variable and enzymatic activity as the dependent variable. A simple procedure of graphical analysis is reported and the influence on the inactivation rate constant of various conditions (temperature and inhibitor concentration) is evaluated. The method is illustrated by an experimental model: the inactivation of bovine kidney alkaline phosphatase by urea.     

Kinetics of inhibition of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide

The inactivation of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide has been studied using the kinetic method of the substrate reaction during modification of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. The results show that inactivation of the enzyme is a slow, reversible reaction. The microscopic rate constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and is situated at the active site of the enzyme.     

Chemical modification of phenol hydroxylase by ethoxyformic anhydride

Phenol hydroxylase was inactivated by ethoxyformic anhydride. Part of the inactivation was related to modification of histidyl residues. The remaining part of the inactivation is proposed to be due to the modification of a lysyl residue which, we suggest, is identical with the one previously described, being essential for the binding of NADPH [Neujahr, H. Y. and Kjellén, K. G. (1980) Biochemistry 19, 4967-4972]. The overall inactivation reaction is biphasic and follows pseudo-first-order kinetics. Numerical analysis of kinetic data was applied to discriminate between simultaneous reactions at different sites. It is proposed that phenol hydroxylase contains two essential histidyl residues, located in or near the NADPH-binding sites. Ethoxyformylation of the lysyl residue(s) caused tightening of the binding of phenol and perturbation of the FAD spectrum of phenol hydroxylase, similar to that caused by phenolic effectors.     

Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the activation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modification but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.     

Kinetics of inhibition of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide

The inactivation of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide has been studied using the kinetic method of the substrate reaction during modification of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. The results show that inactivation of the enzyme is a slow, reversible reaction. The microscopic rate constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and is situated at the active site of the enzyme.Abbreviations ALP alkaline phosphatase - PNPP p-nitrophenyl phosphate - NBS N-bromosuccinimide     

Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the acti-vation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modifica-tion but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.     

Principles of selective inactivation of a viral genome. Comparative kinetic study of modification of the viral RNA and model protein with oligoaziridines

Comparative kinetic analysis of inactivation of bacteriophage MS2 infectivity and aminoalkylation of a model protein (trypsin inhibitor) with oligoaziridines was performed in order to evaluate the selectivity of viral RNA modification with oligocationic reagents. The transition from ethyleneimine monomer to di-, tri-, and tetramer leads to a sharp increase in the rate constant of infectivity inactivation, whereas the rate constant of protein modification changes insignificantly. The selectivity coefficient of the phage RNA aminoalkylation relative to trypsin inhibitor modification increases in this series by more than an order of magnitude. This effect is probably associated with the strengthening of the reagent binding to the nucleic acid, which implies a reaction mechanism that involves the formation of a reactive intermediate. The latter might be an electrostatic complex of the oligocationic reagent and RNA, the only polyanion in the virion. A pronounced decrease in the rate constant of infectivity inactivation in the presence of multiply charged anions (in phosphate buffer) and a biogenic polyamine (spermine) favors this hypothesis. Increasing the reaction temperature increases the rate constant of infectivity inactivation and decreases selectivity of the viral RNA modification.     

Kinetics of Chemical Modification of Arginine Residues in Mitochondrial Creatine Kinase from Bovine Heart: Evidence for Negative Cooperativity

The kinetics of chemical modification of arginine residues in mitochondrial creatine kinase (mit-CK) from beef heart by 4-hydroxy-3-nitrophenylglyoxal (HNPG) have been studied with simultaneous registration of enzyme inactivation. Experiments showed that complete inactivation of mit-CK corresponded to modification of two arginine residues per mit-CK monomer. The data on the modification kinetics can be described by the sum of two exponential terms and suggest strong negative cooperativity in the binding of HNPG to arginine residues. The rate constants for the fast and slow phases of modification differ by a factor of about 50. The corresponding rate constants for inactivation differ by a factor of about 30. The rate constant for the slow stage of inactivation is twice as large as that for the rate constant for the slow stage of modification, i.e., the inactivation process is ahead of the modification process.     

Inactivation of Escherichia coli glycerol kinase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine: protection by the hydrolyzed reagent

Incubation of Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSO2BzAdo) at pH 8.0 and 25 degrees C results in the loss of enzyme activity, which is not restored by the addition of beta-mercaptoethanol or dithiothreitol. The FSO2BzAdo concentration dependence of the inactivation kinetics is described by a mechanism that includes the equilibrium binding of the reagent to the enzyme prior to a first-order inactivation reaction in addition to effects of reagent hydrolysis. The hydrolysis of the reagent has two effects on the observed kinetics. The first effect is deviation from pseudo-first-order kinetic behavior due to depletion of the reagent. The second effect is the novel protection of the enzyme from inactivation due to binding of the sulfonate hydrolysis product. The rate constant for the hydrolysis reaction, determined independently from the kinetics of F- release, is 0.021 min-1 under these conditions. Determinations of the reaction stoichiometry with 3H-labeled FSO2BzAdo show that the inactivation is associated with the covalent incorporation of 1.08 mol of reagent/mol of enzyme subunit. Ligand protection experiments show that ATP, AMP, dAMP, NADH, 5'-adenylyl imidodiphosphate, and the sulfonate hydrolysis product of FSO2BzAdo provide protection from inactivation. The protection obtained with ATP is not dependent on Mg2+. Less protection is obtained with glycerol, GMP, etheno-AMP, and cAMP. No protection is obtained with CMP, UMP, TMP, etheno-CMP, GTP, or fructose 1,6-bisphosphate. The results are consistent with modification by FSO2BzAdo of a single adenine nucleotide binding site per enzyme subunit.     

PAR对氨基酰化酶不可逆抑制动力学研究

本文将邹氏的在酶的活性修饰剂存在下的底物反应动力学理论应用于氨基酰化酶被金属螯合剂PAR脱锌而失活的动力学研究。通过对不同浓度的PAR存在下底物反应过程和含有PAR的不同浓度的底物中酶促反应的分析,讨论了PAR对氨基酰化酶的脱锌机制。这一过程很可能按如下机制进行:首先,PAR与酶分子活性部位的锌结合,形成一复合物,这一步是较快的反应,然后发生一个可逆的构象变化,最后是不可逆的去锌步骤。锌的存在显然稳定了酶活性部位的构象,而这正是酶活性所必需的。     

Inhibition by aminoacyl-chloromethane protease inhibitors of superoxide anion production by phorbol-ester-stimulated human neutrophils. The labeled target is a membrane protein

In a previous paper, we described the kinetic characteristics of the inhibition exerted by the protease inhibitors tosylphenylalanyl and tosyllysyl chloromethanes on superoxide production by human polymorphonuclear leukocytes when stimulated by phorbol esters [E. C. Conseiller & F. Lederer (1989) Eur. J. Biochem. 183, 107-114]. The results suggested the existence of a specific target which was affinity labeled by the inhibitors. The target appeared to be neither a protease, nor intracellular enzymes which can be inhibited in vitro by the chloromethanes (protein kinase C, hexokinase and enzymes of the hexose monophosphate shunt). In the present work, using the cell-free reconstitution assay for superoxide production, we substantiate the hypothesis that the chloromethanes, target is on the plasma membrane. We have radiolabeled the membranes of cells inactivated before or after phorbol ester stimulation, using either [3H]KBH4 reduction after reaction with unlabeled inactivator, or tritiated tosylphenylalanyl chloromethane. In all cases, besides a certain background of non-specific labeling, a radioactive band of Mr 15,000 can be observed upon SDS/PAGE of radiolabeled membranes. We suggest that it is the chemical modification of this protein which is responsible for inactivation of superoxide production. Its identity and its role in the oxidative burst remain to be determined.     

A kinetic analysis of enzyme inactivation as applied to the covalent modification of Na+ + K+ -ATPase and Ca2+ -ATPase

A kinetic analysis of enzyme inactivation due to the covalent binding of chemically modified ligands is presented. Reaction schemes similar to the Michaelis-Menten scheme have been studied as well as schemes with two states of the enzyme or two binding sites. The resulting kinetic equations lead to time courses of inactivation which can be represented by two exponential functions at least in a quasi-steady state approximation. These curves are frequently encountered in inactivation experiments. Since rapid methods for model selection and parameter estimation are desirable, but not available, a technique for a preliminary analysis of the experimental data is presented. A mere glance at the time courses shows what reaction schemes are inapplicable. For each family of inactivation curves, the construction of a line of intersections is proposed. This line contains essential kinetic information and can further be utilized for a rough parameter estimation. The technique is illustrated for three sets of experimental data where Na+ + K+ -ATPase and Ca2+ -ATPase have been inactivated by ATP-analogs.     

Chemical modification of rabbit skeletal muscle phosphorylase kinase with phenylglyoxal

Nonactivated phosphorylase kinase from rabbit skeletal muscle is inactivated by treatment with phenylglyoxal. Under mild reaction conditions, a derivative that retains 10-15% of the pH 8.2 catalytic activity is obtained. The kinetics of inactivation profile, differential effects of modification on pH 6.8 and 8.2 catalytic activities, and the insensitiveness of the modified enzyme to activation by ADP reveal that the 10-15% of catalytic activity remaining is very likely due to intrinsic catalytic activity of the derivative rather than to the presence of unmodified enzyme molecules. The kinetic results also suggest that the inactivation is correlatable with the reaction of one molecule of the reagent with the enzyme without any prior binding of phenylglyoxal. The phenylglyoxal modification reduces the autophosphorylation rate of the kinase. Autophosphorylated phosphorylase kinase is inactivated by phenylglyoxal at a much slower rate than the inactivation of nonactivated kinase. Thus, phenylglyoxal modification influences the phosphorylation and vice versa. The modified enzyme can be reactivated by treatment with trypsin or by dissociation using chatropic salts. The activity of the phenylglyoxal-modified enzyme after trypsin digestion or dissociation with LiBr reaches the same level as that of the native enzyme digested with trypsin or treated with LiBr under identical conditions. The results suggest that the effect of modification is overcome by dissociation of the subunits of phosphorylase kinase and that the catalytic site is not modified under conditions when 85% of the pH 8.2 catalytic activity is lost. Among various nucleotides and metal ions tested, only ADP, with or without Mg2+, afforded effective protection against inactivation with phenylglyoxal. At pH 6.8, 1 mM ADP afforded complete protection against inactivation. Experiments with 14C-labeled phenylglyoxal revealed that ADP seemingly protects one residue from modification. This result is in agreement with the kinetic result that the inactivation seemingly is due to reaction of one molecule of the reagent with the enzyme. The results confirm the existence of a high-affinity ADP binding site on nonactivated phosphorylase kinase and suggest the involvement of a functional arginyl residue at or near the ADP binding site in the regulation of of pH 8.2 catalytic activity of the enzyme.     

Gating kinetics of the alpha1I T-type calcium channel.

The alpha1I T-type calcium channel inactivates almost 10-fold more slowly than the other family members (alpha1G and alpha1H) or most native T-channels. We have examined the underlying mechanisms using whole-cell recordings from rat alpha1I stably expressed in HEK293 cells. We found several kinetic differences between alpha1G and alpha1I, including some properties that at first appear qualitatively different. Notably, alpha1I tail currents require two or even three exponentials, whereas alpha1G tails were well described by a single exponential over a wide voltage range. Also, closed-state inactivation is more significant for alpha1I, even for relatively strong depolarizations. Despite these differences, gating of alpha1I can be described by the same kinetic scheme used for alpha1G, where voltage sensor movement is allosterically coupled to inactivation. Nearly all of the rate constants in the model are 5-12-fold slower for alpha1I, but the microscopic rate for channel closing is fourfold faster. This suggests that T-channels share a common gating mechanism, but with considerable quantitative variability.     

Kinetic Analysis of Maturation and Denaturation of DsRed, a Coral-Derived Red Fluorescent Protein

The red fluorescent protein DsRed recently cloned from Discosoma coral, with its significantly red-shifted excitation and emission maxima (558 and 583 nm, respectively), has attracted great interest because of its spectral complementation to other fluorescent proteins, including the green fluorescent protein and its enhanced mutant EGFP. We demonstrated that the much slower DsRed fluorescence development could be described by a three-step kinetic model, in contrast to the fast EGFP maturation, which was fitted by a one-step model. At pH below 5.0 DsRed fluorescence gradually decreased, and the rate and degree of this fluorescence inactivation depended on the pH value. The kinetics of fluorescence inactivation under acidic conditions was fitted by a two-exponential function where the initial inactivation rate was proportional to the fourth power of proton concentration. Subsequent DsRed alkalization resulted in partial fluorescence recovery, and the rate and degree of such recovery depended on the incubation time in the acid. Recovery kinetics had a lag-time and was fitted minimally by three exponential functions. The DsRed absorbance and circular dichroism spectra revealed that the fluorescence loss was accompanied by protein denaturation. We developed a kinetic mechanism for DsRed denaturation that includes consecutive conversion of the initial state of the protein, protonated by four hydrogen ions, to the denatured one through three intermediates. The first intermediate still emits fluorescence, and the last one is subjected to irreversible inactivation. Because of tight DsRed tetramerization we have suggested that obligatory protonation of each monomer results in the fluorescence inactivation of the whole tetramer.