Quantifying the inactivation rate constants for the molecular species comprising the catalytic cycle of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase

When an unstable enzyme is incubated with its substrate(s), catalysis may cease before chemical equilibrium is attained. The residual substrate concentrations depend on their initial concentrations, the initial enzymic activity, and the inactivation rate constants for each molecular species that comprise the catalytic cycle. The underlying theory has been elaborated previously for single-substrate reactions and here it is extended to bi-substrate reactions. The theory is illustrated by application to glucose 6-phosphate dehydrogenase, which is unstable when exposed to a low concentration of sodium dodecyl sulphate. It is shown that the ternary complex containing both substrates is resistant to inactivation while each of the remaining complexes undergoes first-order decay. Rate constants for the inactivation of each complex are calculated.     

Quantifying the inactivation rate constants for the molecular species comprising the catalytic cycle of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase

When an unstable enzyme is incubated with its substrate(s), catalysis may cease before chemical equilibrium is attained. The residual substrate concentrations depend on their initial concentrations, the initial enzymic activity, and the inactivation rate constants for each molecular species that comprise the catalytic cycle. The underlying theory has been elaborated previously for single-substrate reactions and here it is extended to bi-substrate reactions. The theory is illustrated by application to glucose 6-phosphate dehydrogenase, which is unstable when exposed to a low concentration of sodium dodecyl sulphate. It is shown that the ternary complex containing both substrates is resistant to inactivation while each of the remaining complexes undergoes first-order decay. Rate constants for the inactivation of each complex are calculated.     

Chemical modification studies on alkaline phosphatase from pearl oyster (Pinctada fucata): a substrate reaction course analysis and involvement of essential arginine and lysine residues at the active site

Alkaline phosphatases (ALP, EC 3.1.3.1) are ubiquitous enzymes found in most species. ALP from a pearl oyster, Pinctada fucata (PALP), is presumably involved in nacreous biomineralization processes. Here, chemical modification was used to investigate the involvement of basic residues in the catalytic activity of PALP. The Tsou's plot analysis indicated that the inactivation of PALP by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and phenylglyoxal (PG) is dependent upon modification of one essential lysine and one essential arginine residue, respectively. Substrate reaction course analysis showed that the TNBS and PG inactivation of PALP followed pseudo-first-order kinetics and the second-order inactivation constants for the enzyme with or without substrate binding were determined. It was found that binding substrate slowed the PG inactivation whereas had little effect on TNBS inactivation. Protection experiments showed that substrates and competitive inhibitors provided significant protection against PG inactivation, and the modified enzyme lost its ability to bind the specific affinity column. However, the TNBS-induced inactivation could not be prevented in presence of substrates or competitive inhibitors, and the modified enzyme retained the ability to bind the affinity column. In a conclusion, an arginine residue involved in substrate binding and a lysine residue involved in catalysis were present at the active site of PALP. This study will facilitate to illustrate the role ALP plays in pearl formation and the mechanism involved.     

Mechanism-based inactivation of leukotriene A4 hydrolase/aminopeptidase by leukotriene A4. Mass spectrometric and kinetic characterization.

"Suicide" inactivation of leukotriene (LT) A4 hydrolase/aminopeptidase occurs via an irreversible mechanism-based process which is saturable, of pseudo firstorder, and dependent upon catalysis. Data obtained with either recombinant enzyme or enzyme purified from human leukocytes were similar. Apparent binding constants and inactivation rate constants are equivalent, compatible with a single type of substrate-enzyme complex which partitions between two fates, turnover and inactivation. Both catalytic functions are inactivated, consistent with an overlapping active site for this bifunctional enzyme. The partition ratio (turnover/inactivation) for the LTA4-enzyme complex is 129 +/- 16 for LTA4 hydrolase activity and 124 +/- 10 for aminopeptidase activity. The pH dependence for turnover and inactivation are indistinguishable with a maximum at pH 8. L-Proline p-nitroanilide, a weak substrate with a high Km for the aminopeptidase affords only partial protection against inactivation by LTA4. However, two potent competitive inhibitors, bestatin and captopril, protect both catalytic processes from inactivation, consistent with an active-site specificity for the suicide event. Electrospray ionization mass spectrometry indicates that the molecular weight of pure recombinant enzyme is 69,399 +/- 4 and that covalent modification accompanies catalysis, producing an LTA4:enzyme adduct with a molecular weight 69,717 +/- 4 and a 1:1 stoichiometry. In agreement with kinetic data, electrospray ionization mass spectrometry shows that bestatin inhibits the covalent modification of enzyme by LTA4 and that the extent of modification is proportional to the loss of enzymatic activity.     

Complete kinetic and thermodynamic characterization of the unisite catalytic pathway of Escherichia coli F1-ATPase. Comparison with mitochondrial F1-ATPase and application to the study of mutant enzymes

A complete analysis is presented of the component rate constants of the "unisite" reaction pathway in normal Escherichia coli F1-ATPase. Gibbs free energy profiles of the unisite reaction pathway were constructed for both normal E. coli F1 and bovine-heart mitochondrial F1, and comparison indicated that E. coli F1 is an ancestral form of the mitochondrial enzyme. Similar kinetic and thermodynamic analyses of the unisite reaction pathway were done for mutant beta-Asn-242 and beta-Val-242 E. coli F1-ATPases. Both mutations affected unisite binding and hydrolysis of MgATP but had little effect on release of products or binding of MgADP. It was apparent that a primary effect of the mutations was on the interaction between the catalytic nucleotide-binding domain and the substrate MgATP. The catalytic transition state [F1-ATP]++ was the most destabilized step in the reaction sequence. Measurements of delta delta G[F1.ATP]++ and linear free energy plots for the catalytic step were consistent with the view that, in normal enzyme, residue beta-Asp-242 accepts an H-bond from the transition-state substrate in order to facilitate catalysis. Both mutations impaired positive catalytic cooperativity. This was caused by energetic destabilization of the catalytic transition state and was an indirect effect, not a direct effect on signal transmission per se between catalytic nucleotide-binding domains on beta-subunits. Therefore, impairment of unisite catalysis and of positive catalytic cooperativity appeared to be linked. This may provide a unifying explanation as to why a series of other, widely separated mis-sense mutations within the catalytic nucleotide-binding domain on F1-beta-subunit, which have been reported to affect unisite catalysis, also impair positive catalytic cooperativity. Linear free energy plots for the ATP-binding step of unisite catalysis demonstrated that beta-Asn-242 and beta-Val-242 mutant enzymes did not suffer any gross disruptive change in structure of the catalytic nucleotide-binding domain, reinforcing the view that impairment of catalysis was due to a localized effect. Such analyses confirmed that six other F1-beta-subunit mutants, previously generated and characterized in this laboratory and thought to have inhibitory side-chain substitutions in the catalytic nucleotide-binding domain, are also devoid of gross structural disruption.     

Evolutionary optimization of the catalytic efficiency of enzymes.

1. The rate equation for a generalized Michaelian type of enzymic reaction mechanism has been analyzed in order to establish how the mechanism should be kinetically designed in order to optimize the catalytic efficiency of the enzyme for a given average magnitude of true and apparent first-order rate constants in the mechanism at given concentrations of enzyme, substrate and product. 2. As long as on-velocity constants for substrate and product binding to the enzyme have not reached the limiting value for a diffusion-controlled association process, the optimal state of enzyme operation will be characterized by forward (true and apparent) first-order rate constants of equal magnitude and reverse rate constants of equal magnitude. The drop in free energy driving the catalysed reaction will occur to an equal extent for each reaction step in the mechanism. All internal equilibrium constants will be of equal magnitude and reflect only the closeness of the catalysed reaction to equilibrium conditions. 3. When magnitudes of on-velocity constants for substrate and product binding have reached their upper limits, the optimal kinetic design of the reaction mechanism becomes more complex and has to be established by numerical methods. Numerical solutions, calculated for triosephosphate isomerase, indicate that this particular enzyme may or may not be considered to exhibit close to maximal efficiency, depending on what value is assigned to the upper limit for a ligand association rate constant. 4. Arguments are presented to show that no useful information on the evolutionary optimization of the catalytic efficiency of enzymes can be obtained by previously taken approaches that are based on the application of linear free-energy relationships for rate and equilibrium constants in the reaction mechanism.     

An acetylenic mechanism-based inhibitor of dopamine beta-hydroxylase

The catalytic action of dopamine beta-hydroxylase on 1-phenyl-1-propyne results in concomitant loss of enzyme activity. At pH 5.5 and 25 degrees C, 1-phenyl-1-propyne inactivates dopamine beta-hydroxylase in a mechanism-based fashion. The inactivation rate is first-order, follows saturation kinetics, and is strictly dependent on catalysis (oxygen and ascorbate are essential). The inactivation rate of saturating 1-phenyl-1-propyne (kinact) increases from 0.08 to 0.22 min-1 when the oxygen saturation increases from 21 to 100%, respectively. Inactivation also requires a copper-containing catalytically competent enzyme. Tyramine and norepinephrine (respectively, substrate and product of the normal catalytic reaction) protect against inactivation, and no regain of enzyme activity occurs after prolonged dialysis. Experiments with ether-extracted incubation solutions (+/- enzyme) showed no difference in their gas chromatography-mass spectral patterns implying that inactivation of dopamine beta-hydroxylase by 1-phenyl-1-propyne occurs through a kinetic process with a partition ratio (kcat/kinact) equal to or near 1. Thus, this acetylenic substrate analog appears to be a very efficient mechanism-based inhibitor of dopamine beta-hydroxylase. We propose that inactivation of this enzyme by 1-phenyl-1-propyne proceeds by formation of a reactive intermediate that occurs prior to product formation and that alkylates an amino acid residue at the active site of the enzyme.     

Functional role of histidine residues of alpha-ketoglutarate dehydrogenase

The protective effect of alpha-ketoglutarate dehydrogenase substrate and its analogs on the enzyme inactivation by diethylpyrocarbonate was studied. The values of true rate constants for diethylpyrocarbonate-induced inactivation and the Kd values for the enzyme complexes with ligands were determined. A comparison of Kd values for a number of ligands suggests that the histidine residue of the enzyme active center interacts with the alpha-keto group of the substrate. A mechanism of this histidine residue involvement in the catalytic act is proposed. According to this mechanism, the imidazole ring of histidine which is responsible for the substrate activation causes a simultaneous formation of a catalytically active form of the coenzyme--thiamine pyrophosphate ilide. It is assumed that the lower (as compared with the enzyme-substrate complexes) values of rate constants of inactivation by diethylpyrocarbonate for alpha-ketoglutarate dehydrogenase complexes with succinate, glutarate, and oxaloacetate are due to additional protonation of the histidine residue, eventually resulting in the blocking of the analogs interaction with the coenzyme.     

The effect of potassium ferricyanide and copper (II) ions on the properties of immobilized galactose oxidase

Preparations of galactosooxidase (EC 1.1.3.9) immobilized by activated aminorganosilica have been used to study potassium ferricyanide and bivalent copper ions on the enzyme activity and stability in continuous reactor under pulse conditions. Introduction of potassium ferricyanide is shown to activate the enzyme and inconsiderably affecting its stability with the substrate absent and inducing inactivation of galactosooxidase in the process of catalytic reaction. Cu2+ ions, exerting no effect on the activity of immobilized galactosooxidase, evoke the enzyme inactivation in the process of catalysis.     

Kinetics of Thermal Inactivation of Lactate Dehydrogenase from Rabbit Muscle

The kinetics of thermal inactivation of rabbit muscle lactate dehydrogenase at different temperatures has been studied using the kinetic method for the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [Adv. Enzymol. Relat. Areas Mol. Biol. (1988), 61, 381–436]. The results show that thermal inactivation of the enzyme is an irreversible reaction. Microscopic rate constants were determined for thermal inactivation of the free enzyme and the enzyme–substrate complex. The inactivation rate constant of the free enzyme is much larger than the rate constant of the enzyme–substrate complex. The results suggest that the presence of the substrate has a certain protective effect against thermal inactivation of the enzyme.     

Thermal inactivation of immobilized penicillin acylase in the presence of substrate and products

Inactivation of immobilized penicillin acylase has been studied in the presence of substrate (penicillin G) and products (phenylacetic acid and 6-aminopenicillanic acid), under the hypothesis that substances which interact with the enzyme molecule during catalysis will have an effect on enzyme stability. The kinetics of immobilized penicillin acylase inactivation was a multistage process, decay constants being evaluated for the free-enzyme and enzyme complexes, from whose values modulation factors were determined for the effectors in each enzyme complex at each stage. 6-Aminopenicillanic acid and penicillin G stabilized the enzyme in the first stage of decay. Modulation factors in that stage were 0.96 for penicillin G and 0.98 for 6-aminopenicillanic acid. Phenylacetic acid increased the rate of inactivation in both stages, modulating factors being -2.31 and -2.23, respectively. Modulation factors influence enzyme performance in a reactor and are useful parameters for a proper evaluation. (c) 1996 John Wiley & Sons, Inc.     

Stability and catalytic activity of alpha-amylase from barley malt at different pressure-temperature conditions

The impact of high hydrostatic pressure and temperature on the stability and catalytic activity of alpha-amylase from barley malt has been investigated. Inactivation experiments with alpha-amylase in the presence and absence of calcium ions have been carried out under combined pressure-temperature treatments in the range of 0.1-800 MPa and 30-75 degrees C. A stabilizing effect of Ca(2+) ions on the enzyme was found at all pressure-temperature combinations investigated. Kinetic analysis showed deviations of simple first-order reactions which were attributed to the presence of isoenzyme fractions. Polynomial models were used to describe the pressure-temperature dependence of the inactivation rate constants. Derived from that, pressure-temperature isokinetic diagrams were constructed, indicating synergistic and antagonistic effects of pressure and temperature on the inactivation of alpha-amylase. Pressure up to 200 MPa significantly stabilized the enzyme against temperature-induced inactivation. On the other hand, pressure also hampers the catalytic activity of alpha-amylase and a progressive deceleration of the conversion rate was detected at all temperatures investigated. However, for the overall reaction of blocked p-nitrophenyl maltoheptaoside cleavage and simultaneous occurring enzyme inactivation in ACES buffer (0.1 M, pH 5.6, 3.8 mM CaCl(2)), a maximum of substrate cleavage was identified at 152 MPa and 64 degrees C, yielding approximately 25% higher substrate conversion after 30 min, as compared to the maximum at ambient pressure and 59 degrees C.     

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.     

Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S-transferase, 1-1.

Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a KI of 0.78 mM and kmax of 0.011 min-1. S-Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S-methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pKa, which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pKa of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S-transferase, but is not required for enzyme activity.     

Suicide-peroxide inactivation of horseradish peroxidase in the presence of sodium n-dodecyl sulphate: a study of the enzyme deactivation kinetics

In the presence of the anionic surfactant sodium n-dodecyl sulphate (SDS), horseradish peroxidase (HRP) undergoes a deactivation process. Suicide inactivation of horseradish peroxidase by hydrogen peroxide(3 mM) was monitored by the absorbance change in product formation in the catalytic reaction cycle. The progress curve of the catalytic reaction cycle was obtained at 27degrees C and phosphate buffer 2.5 mM (pH = 7.0). The corresponding kinetic parameters i.e., intact enzyme activity (alpha i); the apparent rate constant of suicide inactivation by peroxide (ki); and the apparent rate constants of enzyme deactivation by surfactant (kd) were evaluated from the obtained kinetic equations. The experimental data are accounted for by the equations used in this investigation. Addition of SDS to the reaction mixture intensified the inactivation process. The deactivation ability of denaturant could be resolved from the observed inactivation effect of the suicide substrate by applying the proposed model. The results indicate that the deactivation and the inactivation processes are independent of each other.     

Interfacial catalysis by phospholipase A2: dissociation constants for calcium, substrate, products, and competitive inhibitors.

Interpretation of the kinetics of interfacial catalysis in the scooting mode as developed in the first paper of this series [Berg et al. (1991) Biochemistry 30 (first paper of six in this issue)], was based on the binding equilibrium for a ligand to the catalytic site of phospholipase A2. In this paper, we describe direct methods to determine the value of the Michaelis-Menten constant (KMS) for the substrate, as well as the equilibrium dissociation constants for ligands (KL) such as inhibitors (KI), products (KP), calcium (KCa), and substrate analogues (KS) bound to the catalytic site of phospholipase A2 at the interface. The KL values were obtained by monitoring the susceptibility to alkylation of His-48 at the catalytic site of pig pancreatic PLA2 bound to micellar dispersions of the neutral diluent 2-hexadecyl-sn-glycero-3-phosphocholine. The binding of the enzyme to dispersions of this amphiphile alone had little effect on the inactivation rate. The half-time for inactivation of the enzyme bound to micelles of the neutral diluent depended not only on the nature of the alkylating agent but also on the structure and the mole fraction of other ligands at the interface. The KL values for ligands obtained from the protection studies were in excellent accord with those obtained by monitoring the activation or inhibition of hydrolysis of vesicles of 1,2-dimyristoyl-sn-glycerophosphomethanol. Since only calcium, competitive inhibitors, and substrate analogues protected phospholipase A2 from alkylation, this protocol offered an unequivocal method to discern active-site-directed inhibitors from nonspecific inhibitors of PLA2, such as local anesthetics, phenothiazines, mepacrine, peptides related to lipocortin, 7,7-dimethyleicosadienoic acid, quinacrine, and aristolochic acid, all of which did not have any effect on the kinetics of alkylation nor did they inhibit the catalysis in the scooting mode.     

Effect of trypsin microenvironment on the rate constants of elementary stages of Nalpha-benzoyl-L-arginine ethyl ester hydrolysis

The hydrolysis reaction of Nalpha-benzoyl-L-arginine ethyl ester catalyzed by trypsin from pig pancreas was comparatively studied in an aqueous buffer solution and in the system of reversed micelles of Aerosol OT in octane (pH 8.5) to determine the mechanisms of influence of the enzyme microenvironment on the rate constants of the elementary stages of the enzymatic reaction. The temperature dependences of the catalytic constant kcat and the rate constant of the second order kcat/Km (s, catalysis efficiency) allowed the determination of the rate constants and the activation energy of elementary stages of the enzymatic reaction. It was revealed that a decrease in the efficiency of catalytic action of trypsin in inverted mycelles in comparison with an aqueous solution is first of all determined by a decrease in the rate constant of formation of the enzyme-substrate complex k1. Possible mechanisms of the effect of the microenvironment on the elementary stages of catalytic action of the enzyme are discussed.     

Kinetics of intermolecular cleavage by hammerhead ribozymes.

The hammerhead catalytic RNA effects cleavage of the phosphodiester backbone of RNA through a transesterification mechanism that generates products with 2'-3'-cyclic phosphate and 5'-hydroxyl termini. A minimal kinetic mechanism for the intermolecular hammerhead cleavage reaction includes substrate binding, cleavage, and product release. Elemental rate constants for these steps were measured with six hammerhead sequences. Changes in substrate length and sequence had little effect on the rate of the cleavage step, but dramatic differences were observed in the substrate dissociation and product release steps that require helix-coil transitions. Rates of substrate binding and product dissociation correlated well with predictions based on the behavior of simple RNA duplexes, but substrate dissociation rates were significantly faster than expected. Ribozyme and substrate alterations that eliminated catalytic activity increased the stability of the hammerhead complex. These results suggest that substrate destabilization may play a role in hammerhead catalysis.     

Kinetic studies on beta-site amyloid precursor protein-cleaving enzyme (BACE). Confirmation of an iso mechanism

The steady-state kinetic mechanism of beta-amyloid precursor protein-cleaving enzyme (BACE)-catalyzed proteolytic cleavage was evaluated using product and statine- (Stat(V)) or hydroxyethylene-containing (OM99-2) peptide inhibition data, solvent kinetic isotope effects, and proton NMR spectroscopy. The noncompetitive inhibition pattern observed for both cleavage products, together with the independence of Stat(V) inhibition on substrate concentration, suggests a uni-bi-iso kinetic mechanism. According to this mechanism, the enzyme undergoes multiple conformation changes during the catalytic cycle. If any of these steps are rate-limiting to turnover, an enzyme form preceding the rate-limiting conformational change should accumulate. An insignificant solvent kinetic isotope effect (SKIE) on k(cat)/K(m), a large inverse solvent kinetic isotope effect on k(cat), and the absence of any SKIE on the inhibition onset by Stat(V) during catalysis together indicate that the rate-limiting iso-step occurs after formation of a tetrahedral intermediate. A moderately short and strong hydrogen bond (at delta 13.0 ppm and phi of 0.6) has been observed by NMR spectroscopy in the enzyme-hydroxyethylene peptide (OM99-2) complex that presumably mimics the tetrahedral intermediate of catalysis. Collapse of this intermediate, involving multiple steps and interconversion of enzyme forms, has been suggested to impose a rate limitation, which is manifested in a significant SKIE on k(cat). Multiple enzyme forms and their distribution during catalysis were evaluated by measuring the SKIE on the noncompetitive (mixed) inhibition constants for the C-terminal reaction product. Large, normal SKIE values were observed for these inhibition constants, suggesting that both kinetic and thermodynamic components contribute to the K(ii) and K(is) expressions, as has been suggested for other iso-mechanism featuring enzymes. We propose that a conformational change related to the reprotonation of aspartates during or after the bond-breaking event is the rate-limiting segment in the catalytic reaction of beta-amyloid precursor protein-cleaving enzyme, and ligands binding to other than the ground-state forms of the enzyme might provide inhibitors of greater pharmacological relevance.