Kinetics of Inhibition of Green Crab (Scylla serrata) Alkaline Phosphatase by L-Cysteine

The inhibition of alkaline phosphatase from green crab (Scylla serrata) by L-cysteine has been studied. The results show that L-cysteine gives a mixed-type inhibition. The progress-of-substrate-reaction method previously described by Tsou [(1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 391–436] was used to study the inactivation kinetics of the enzyme by L-cysteine. The microscopic rate constants were determined for reaction of the inhibitor with the free enzyme and the enzyme–substrate complex (ES) The results show that inactivation of the enzyme by L-cysteine is a slow, reversible reaction. Comparison of the inactivation rate constants of free enzyme and ES suggests that the presence of the substrate offers marked protection of this enzyme against inactivation by L-cysteine.     

Inactivation kinetics of mushroom tyrosinase in the dimethyl sulfoxide solution

Mushroom tyrosinase (EC 1.14.18.1) is a kind of copper-containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones and then forms brown or black pigments. In the present paper, the effects of dimethyl sulfoxide on the enzyme activity for the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) have been studied. The results show that low concentrations of dimethyl sulfoxide (DMSO) can lead to reversible inactivation of the enzyme, and the IC ₅₀ is estimated to be 2.45 M. Inactivation of the enzyme by DMSO is classified as mixed type. The kinetics of inactivation of mushroom tyrosinase at low concentrations of DMSO solution has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The results show the free enzyme molecule is more fragile than the enzyme–substrate complex in the DMSO solution. It is suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by DMSO.     

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     

Inactivation and conformational changes of aminoacyclase in trifluoroethanol solutions

The inactivation and unfolding of aminoacyclase (EC 3.5.1.14) during denaturation by different concentrations of trifluoroethanol (TFE) have been studied. A marked decrease in enzyme activity was observed at low TFE concentrations. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou [Tsou (1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] was applied to study the kinetics of the inactivation course of aminoacyclase during denaturation by TFE. The inactivation rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method. The inactivation reaction was a monophasic first-order reaction. The kinetics of the unfolding course were a biphasic process consisting of two first-order reactions. At 2% TFE concentration, the inactivation rate of the enzyme was much faster than the unfolding rate. At a higher concentration of TFE (10%), the inactivation rate was too fast to be determined by conventional methods, whereas the unfolding course remained as a biphasic process with fast and slow reactions occurring at measurable rates. The results suggest that the aminoacyclase active site containing Zn²⁺ ions is situated in a limited and flexible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole.     

Kinetics of thermal inactivation of penaeus penicillatus acid phosphatase

The kinetics of thermal inactivation of Penaeus penicillatus acid phosphatase have been studied using a kinetic method related to the substrate reaction during irreversible inhibition of the enzyme activity as previously described by Tsou (Adv. Enzymol. Relat. Areas Mol. Biol. (1988) 61, 381-436). The kinetics of thermal inactivation of the enzyme show that the reaction is irreversible. The microscopic rate constants were determined for thermal inactivation of free enzyme and the enzyme--substrate complex. The results show that the presence of substrate has a significant protective effect against thermal inactivation of the enzyme.     

Comparison of inactivation and unfolding of green crab (Scylla serrata) alkaline phosphatase during denaturation by guanidinium chloride

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, each active site in which contains a tight cluster of two zinc ions and one magnesium ion. Unfolding and inactivation of the enzyme during denaturation in guanidinium chloride (GuHCl) solutions of different concentrations have been compared. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] has been applied to a study on the kinetics of the course of inactivation of the enzyme during denaturation by GuHCl. The rate constants of unfolding and inactivation have been determined. The results show that inactivation occurs before noticeable conformational change can be detected. It is suggested that the active site of green crab alkaline phosphatase containing multiple metal ions is also situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

Inactivation and unfolding of aminoacylase during denaturation in sodium dodecyl sulfate solutions

During denaturation by sodium dodecyl sulfate (SDS), aminoacylase shows a rapid decrease in activity with increasing concentration of the detergent to reach complete inactivation at 1.0 mM SDS. The denatured minus native-enzyme difference spectrum showed two negative peaks at 287 and 295 nm. With the increase of concentration of SDS, both negative peaks increased in magnitude to reach maximal values at 5.0 mM SDS. The fluorescence emission intensity of the enzyme decreased, whereas there was no red shift of emission maximum in SDS solutions of increasing concentration. In the SDS concentration regions employed in the present study, no marked changes of secondary structure of the enzyme have been observed by following the changes in far-ultraviolet CD spectra. The inactivation of this enzyme has been followed and compared with the unfolding observed during denaturation in SDS solutions. A marked inactivation is already evident at low SDS concentration before significant conformational changes can be detected by ultraviolet absorbance and fluorescence changes. The inactivation rate constants of free enzyme and substrate-enzyme complex were determined by the kinetics method of the substrate reaction in the presence of inactivator previously described by Tsou [Tsou (1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. It was found that substrate protects against inactivation and at the same SDS concentrations, the inactivation rate of the free enzyme is much higher than the unfolding rate. The above results show that the active sites of metal enzyme containing Zn²⁺ are also situated in a limited and flexible region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

Irreversible kinetics of β-N-acetyl-d-glucosaminidase from prawn (Penaeus vannamei) inactivated by mercuric ion

Cysteine residues in prawn (Penaeus vannamei) β-N-acetyl-d-glucosaminidase (NAGase, EC 3.2.1.52) have been modified by p-chloromercuribenzoate (PCMB). The results show that sulfhydryl group is essential for the activity of the enzyme. Inactivation kinetics of the enzyme by mercuric chloride (HgCl₂) has been studied using the kinetic method of the substrate reaction during inactivation of enzyme previously described by Tsou. The kinetic results show that the inactivation of the enzyme is an irreversible reaction. The microscopic rate constants for the reaction of Hg²⁺ with free enzyme and with the enzyme-substrate complex are determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by Hg²⁺. The above results suggest that the cysteine residue is essential for activity.     

Investigation of the active site of the extracellular β-<Emphasis Type="SmallCaps">D</Emphasis>-glucosidase from <Emphasis Type="Italic">Aspergillus carbonarius</Emphasis>

Summary The catalytic amino acid residues of the extracellular β-D-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus carbonarius were investigated. The pH dependence curves gave apparent pK values of 2.8 and 5.93 for the free enzyme, and 2.24 and 6.14 for the enzyme–substrate complex using p-nitrophenyl-β-D-glucoside as substrate. Carbodiimide- and Woodward reagent K-mediated chemical modifications suggested that a carboxylate residue, located in the active centre, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.61 in the modification reaction, proving that a carboxylate residue was modified. The A. carbonarius β-glucosidase was irreversibly inactivated by N-bromoacetyl-β-D-glucopyranosylamine. The active site specificity of the inactivation was proved by using the competitive inhibitor p-nitrophenyl-1-thio-β-D-glucopyranoside. pH Dependence studies of inactivation revealed that modification by N-bromoacetyl-β-D-glucopyranosylamine could be directed toward the carboxylate group acting as the catalytic nucleophile, as in the case of the carbodiimide and Woodward reagent K modifications.     

Kinetics of Inhibition of Green Crab (Scylla serrata) Alkaline Phosphatase by L-Cysteine

The inhibition of alkaline phosphatase from green crab (Scylla serrata) by L-cysteine has been studied. The results show that L-cysteine gives a mixed-type inhibition. The progress-of-substrate-reaction method previously described by Tsou [(1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 391–436] was used to study the inactivation kinetics of the enzyme by L-cysteine. The microscopic rate constants were determined for reaction of the inhibitor with the free enzyme and the enzyme–substrate complex (ES) The results show that inactivation of the enzyme by L-cysteine is a slow, reversible reaction. Comparison of the inactivation rate constants of free enzyme and ES suggests that the presence of the substrate offers marked protection of this enzyme against inactivation by L-cysteine.     

Reaction of internal forms of the choline carrier of erythrocytes with N-ethylmaleimide: Evidence for a carrier conformational change on complex formation

Summary The choline carrier of human erythrocyte membranes exists in distinguishable outward-facing and inward-facing conformations, and previous studies demonstrated that only the latter reacts with N-ethylmaleimide, producing an irreversible inhibition of transport. We now report experiments to determine the individual reaction rates for the two inward-facing forms: the free carrier and the complex. The pseudo-first-order rate constant for the complex with a substrate analog, di-n-butylaminoethanol, is found to be nearlydouble that for the free carrier, showing that the carrier conformation is altered following addition of a ligand (with 1mm N-ethylmaleimide at pH 6.8, 37°C, the constants are 0.57±0.05 min^–1 and 0.33±0.02 min^–1, respectively). Hence three different conformational states have been distinguished by experiment: (1) the inward-facing free carrier; (2) the inward-facing complex; and (3) the outward-facing carrier.     

Evidence for a two-state mobile carrier mechanism in erythrocyte choline transport: Effects of substrate analogs on inactivation of the carrier by N-ethylmaleimide

Summary Choline transport in erythrocytes is irreversibly inhibited by N-ethylmaleimide. The hypothesis that the carrier alternates between outwardfacing and inward-facing forms and that only the latter reacts with the inhibitor (Martin, K. (1971)J. Physiol. (London) 213:647–667; Edwards, P.A. (1973)Biochim. Biophys. Acta 311:123–140) is here subjected to a quantitative test. In this test the effects of a series of substrate analogs upon rates of inactivation and rates of choline exit are compared. By hypothesis the effect of an analog in the external solution on the inactivation rate depends only on how it affects the proportion of the inward-facing carrier. Since¹⁴C-choline efflux is necessarily proportional to the concentration of free carrier in the inward-facing form, the analogs should have related effects on the two rates. In every case the observed effects were identical, whether the analogs accelerated transport or inhibited it. Analysis of the results demonstrates that (1) the transport mechanism depends on the operation of a mobile element; (2) distinguishable inward-facing and outward-facing conformations of the free carrier, carrier-substrate complex, and carrier-inhibitor complex exist, and only the inwardfacing forms react at a significant rate with N-ethylmaleimide; (3) carrier mechanisms involving a single form of free carrier or a single form of carriersubstrate complex are ruled out; and (4) dissociation of the carrier-substrate complex is a rapid step with all substrate analogs.     

DNA-(N4-Cytosine)-Methyltransferase from Bacillus amyloliquefaciens: Kinetic and Substrate-binding Properties

Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amyloliquefaciens (BamHI MTase, 49 kDa) with a 20-mer duplex containing a palindromic recognition site GGATCC was studied by methods of steady-state and pre-steady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaraldehyde. In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward the 20-mer substrate. A linear dependence was observed for the reaction rate on the enzyme concentration and a Michaelis dependence of the reaction rate on the concentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl group donor, and DNA, the methyl group acceptor. In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme concentration being substantially lower than the concentrations of substrates. The k _cat values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s^–1. The K _M values for the duplex and SAM are 0.35 and 1.6 M, respectively. An analysis of single turnover kinetics (at limiting concentration of the 20-mer duplex) revealed the following characteristics of the BamHI MTase-dependent methylation of DNA. The value of rate constant of the DNA methylation step at the enzyme saturating concentration is on average 0.085 s^–1, which is only 1.6 times higher than the value determined in steady-state conditions. Only one of two target cytidine residues was methylated in a single turnover of the enzyme, which coincides with the earlier data on EcoRI MTase. Regardless of the order of enzyme preincubation with SAM and DNA, both curves for the single turnover methylation are comparable. These results are consistent with the model of the random order of the productive ternary enzyme–substrate complex formation. In contrast to the relatively simple kinetic behavior of BamHI MTase in the steady-state reaction are the data on the enzyme binding with DNA. In gel retardation experiments, there was no stoichiometrically simple complex with the oligonucleotide duplex even at low enzyme concentrations. The molecular mass of the complexes was so high that they did not enter 12% PAG. In experiments on crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer. Introduction of substoichiometric amounts of DNA into the reaction mixture results in pronounced multimerization of the enzyme. However, addition of SAM in saturating concentration at an excess of the oligonucleotide duplex over BamHI MTase converts most of the enzyme into a monomeric state.     

Binding and incorporation of 4-trans-(N,N-dimethylamino) cinnamaldehyde by aldehyde dehydrogenase

4-trans-(N,N-Dimethylamino)cinnamaldehyde (DACA) is a chromophoric substrate of aldehyde dehydrogenase (EC 1.2.1.3) whose fate can be followed during catalysis. During this investigation we found that DACA also fluoresces and that this fluorescence is enhanced and blue-shifted upon binding to aldehyde dehydrogenase. Binding of DACA to aldehyde dehydrogenase also occurs in the absence of coenzyme. Benzaldehyde (a substrate), acetophenone (a substrate-competitive inhibitor), and the substrate-competitive affinity reagent bromoaceto-phenone interfere with DACA binding. Thus, DACA binds to the active site and can be employed for titration of active aldehyde dehydrogenase. Both E1 and E2 isozymes, which are homotetramers, bind DACA with dissociation constants of 1–4 M with a stoichiometry of 2 mol DACA/mol enzyme. The stoichiometry of enzyme–acyl intermediate was also found to be 2 mol DACA/mol enzyme for both E1 and E2 isozymes. Thus, both enzymes appear to have only two substrate-binding sites which participate in catalysis. The level of enzyme–acyl intermediate remained constant at different pH values, showing that enhancement of velocity with pH was not due to altered DACA–enzyme levels. When the reaction velocity was increased even further by using 150 M Mg²⁺ the intermediate level was decreased, suggesting that both increased pH and Mg²⁺ promote decomposition of the DACA–enzyme intermediate. Titration with DACA permits study of aldehyde substrate catalysis before central complex interconversion.     

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.     

The inhibition of β-lactamases from Gram-negative bacteria by clavulanic acid

The β-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme–clavulanate complex, a transiently stable species (t_½=4min at pH7.3 and 37°C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 β-lactamase and 1:1 for Klebsiella β-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 β-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella β-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t_½=40min at pH7.3 and 37°C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m⁻¹·s⁻¹) to produce a relatively stable complex (t_½=180min at pH7.3 and 37°C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate–enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.     

Inactivation kinetics of mushroom tyrosinase by cetylpyridinium chloride

Cetylpyridinium chloride (CPC) was found to inactivate tyrosinase from mushroom (Agaricus bisporus). CPC can bind to the enzyme molecule and induce the enzyme conformation changes. The fluorescence intensity (at 338.4 nm) of the enzyme decreased distinctly with increasing CPC concentrations, and a new little fluorescence emission peak appeared near 372 nm. The inactivation of the enzyme by CPC had first been studied by using the kinetic method of the substrate reaction described by Tsou. The results showed that the enzyme was inactivated by a complex mechanism that had not been previously identified. The enzyme first quickly binds with CPC reversibly and then undergoes a slow irreversible inactivation. The inactivation reaction is a single molecule reaction and the apparent inactivation rate constant is a saturated trend being independent of CPC concentration if the concentration is sufficiently high. The micro rate constants of inactivation and the association constant were determined.     

Single Turnover Kinetics of Methylation by T4 DNA-(N6-Adenine)-Methyltransferase

Interaction of T4 DNA-(N6-adenine)-methyltransferase was studied with a variety of synthetic oligonucleotide substrates containing the native recognition site GATC or its modified variants. The data obtained in the decisecond and second intervals of the reaction course allowed for the first time the substrate methylation rates to be compared with the parameters of the steady-state reaction. It was established that the substrate reaction proceeds in two stages. Because it is shown that in steady-state conditions T4 MTase forms a dimeric structure, the following sequence of events is assumed. Upon collision of a T4 MTase monomer with an oligonucleotide duplex, an asymmetrical complex forms in which the enzyme randomly oriented relative to one of the strands of the specific recognition site catalyzes a fast transfer of the methyl group from S-adenosylmethionine to the adenosine residue (k ₁ = 0.21 s^–1). Simultaneously, a second T4 MTase subunit is added to the complex, providing for the continuation of the reaction. In the course of a second stage, which is by an order of magnitude slower (k ₂ = 0.023 s^–1 for duplex with the native site), the dimeric T4 MTase switches over to the second strand and the methylation of the second residue, target. The rate of the methyl group transfer from donor, S-adenosylmethionine, to DNA is much higher than the overall rate of the T4 MTase-catalyzed steady-state reaction, although this difference is considerably less than that shown for EcoRI MTase. Base substitutions and deletions in the recognition site affect the substrate parameters in different fashions. When the GAT sequence is disrupted, the proportion of the initial productive enzyme–substrate complexes is usually sharply reduced. The flipping of the adenosine residue to be modified in the recognition site upon interaction with the enzyme, revealed by fluorescence titration, supports the existing notions about the involvement of such a DNA deformation in reactions catalyzed by various DNA-MTases.     

An algebraic model for the kinetics of covalent enzyme inhibition at low substrate concentrations

This article describes an integrated rate equation for the time course of covalent enzyme inhibition under the conditions where the substrate concentration is significantly lower than the corresponding Michaelis constant, for example, in the Omnia assays of epidermal growth factor receptor (EGFR) kinase. The newly described method is applicable to experimental conditions where the enzyme concentration is significantly lower than the dissociation constant of the initially formed reversible enzyme–inhibitor complex (no “tight binding”). A detailed comparison with the traditionally used rate equation for covalent inhibition is presented. The two methods produce approximately identical values of the first-order inactivation rate constant (k_inact). However, the inhibition constant (K_i), and therefore also the second-order inactivation rate constant k_inact/K_i, is underestimated by the traditional method by up to an order of magnitude.