Suicide-Peroxide inactivation of microperoxidase-11: A kinetic study

The kinetics of microperoxidase-11 (MP-11) in the oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied, taking into account the inactivation of enzyme during reaction by its suicide substrate, H₂O₂. Concentrations of substrates were so selected that: 1) the reaction was first-order in relation to benign substrate, AH and 2) high ratio of suicide substrate to the benign substrate, [H₂O₂]>>[AH]. Validation and reliability of the obtained kinetic equations were evaluated in various nonlinear and linear forms. Fitting of experimental data into the obtained integrated equation showed a close match between the kinetic model and the experimental results. Indeed, a similar mechanism to horseradish peroxidase was found for the suicide-peroxide inactivation of MP-11. Kinetic parameters of inactivation including the intact activity of MP-11, α_i, and the apparent inactivation rate constant, k_i, were obtained as 0.282 ± 0.006 min^{? 1} and 0.497 ± 0.013 min^{? 1} at [H₂O₂] = 1.0 mM, 27°C, phosphate buffer 5.0 mM, pH = 7.0. Results showed that inactivation of microperoxidase as a peroxidase model enzyme can occur even at low concentrations of hydrogen peroxide (0.4 mM).     

Suicide-peroxide inactivation of microperoxidase-11: a kinetic study

The kinetics of microperoxidase-11 (MP-11) in the oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied, taking into account the inactivation of enzyme during reaction by its suicide substrate, H2O2. Concentrations of substrates were so selected that: 1) the reaction was first-order in relation to benign substrate, AH and 2) high ratio of suicide substrate to the benign substrate, [H2O2] > [AH]. Validation and reliability of the obtained kinetic equations were evaluated in various nonlinear and linear forms. Fitting of experimental data into the obtained integrated equation showed a close match between the kinetic model and the experimental results. Indeed, a similar mechanism to horseradish peroxidase was found for the suicide-peroxide inactivation of MP-11. Kinetic parameters of inactivation including the intact activity of MP-11, alphai, and the apparent inactivation rate constant, ki, were obtained as 0.282 +/- 0.006 min(-1) and 0.497 +/- 0.013(-1) min at [H2O2] = 1.0 mM, 27 degrees C, phosphate buffer 5.0 mM, pH = 7.0. Results showed that inactivation of microperoxidase as a peroxidase model enzyme can occur even at low concentrations of hydrogen peroxide (0.4 mM).     

Kinetic analysis of zymogen autoactivation in the presence of a reversible inhibitor.

Limited proteolysis is a highly specific irreversible process, which can serve to initiate physiological function by converting a precursor protein into a biologically active form. When the activating enzyme and the activated enzyme coincide, the process is an autocatalytic zymogen activation (i.e. reactions in which the zymogens serves as a substrate for the corresponding active enzyme). The activity of proteases is frequently regulated by the binding of specific protease inhibitors. Thus, to understand the biological regulation of proteolysis, one must understand the role of protease inhibitors. In the present study, a detailed kinetic analysis of autocatalytic reaction modulated by a reversible inhibitor is represented. On the basis of the kinetic equation, a novel procedure is developed to evaluate the kinetic parameters of the reaction. As an example of the application of this method, effects of acetamidine, p-amidinobenzamidine and benzamidine on the autoactivation of trypsinogen by trypsin were studied.     

Kinetic behaviour of proenzymes activation in the presence of different inhibitors for both activating and activated enzymes

In the present paper, a kinetic analysis of a general model for proenzyme activation, where the activating enzyme and also the activated one are reversibly inhibited in two steps by two different inhibitors, has been performed. The cases in which both inhibitors are the same, or in which the inhibition is irreversible (only one or the two inhibition routes) are treated as particular cases of the general model. In addition, the kinetic behaviour of many other proenzyme activation systems involving inhibition, particular cases of the reaction scheme under study, can be obtained. The total number of particular cases for the general model under study is 370, so this approach offers to the scientific community working in limited proteolysis regulation for the first time a method based on general solutions which only needs to be specified to their concrete problem of zymogen activation. Finally, new adimensional parameters are introduced, allowing the knowledgement, in the case that any of the inhibition routes is irreversible, the relative weight of both activation and irreversible inhibition routes.     

Hemes and hemoproteins. 5: Kinetics of the peroxidatic activity of microperoxidase-8: model for the peroxidase enzymes

The peroxidatic activity of the heme octapeptide from cytochrome c, microperoxidase-8 (MP-8), was assayed at 25 degrees C under conditions where formation of Compound I is rate limiting. In the pH range 6-9, the reaction rate increased linearly with a slope close to unity. The active form of the substrate is the hydroperoxide anion, HO2-, and an extrapolated second-order rate constant was obtained for the reaction of aquoMP-8 with HO2- of 3.7 X 10(8) M-1 sec-1, which is close to the second-order rate constants reported for reaction of the peroxidase enzymes with H2O2. Comparison with published data shows that the Fe3+ ion of MP-8 reacts as expected with simple anions, electrons, and HO2-, while the analogous reactions of the enzymes all show a requirement for one H+. We conclude that the peroxidase enzymes activate H2O2 under physiological conditions through a pH-independent, H+-coupled binding of the required H2O2-. The peroxidase activity of MP-8 can be increased more than tenfold by the presence of the guanidinium ion, which is ascribed to formation of the ion-pair GuaH+HO2-; this suggests a role for the invariant distal Arg in the enzymes.     

Kinetic and molecular properties of citraconyl-aldolase. The reversible denaturation and hybridization of the native and modified enzymes

1. The preparation of enzymically active N-citraconyl derivatives of fructose diphosphate aldolase from rabbit muscle is described. Reaction is restricted to amino groups and the derivatives are not very heterogeneous with respect to the number of substituents. 2. Linear double-reciprocal plots of enzyme velocity against substrate concentration are found up to about 15% blocking of amino groups. With more than 15% blocking, there is a marked downward curvature in the double-reciprocal plots at high substrate concentrations. 3. Over the range 0-25% blocking of amino groups the apparent V(max.) for fructose diphosphate falls to 10% that of the native enzyme, and the apparent K(m) rises from 1 to 400mum. 4. Various pieces of evidence suggest that citraconyl-aldolase is slightly distorted in structure compared with the native enzyme. However, the kinetic properties and tetrameric structure of citraconyl-aldolase can be completely recovered after denaturation in 4m-guanidine hydrochloride. 5. After removal of the citraconyl groups in acid conditions the kinetic and molecular properties of native enzyme are restored. 6. Hybrid forms of aldolase can be constructed containing native and citraconylated subunits and the suitability of these derivatives for the study of subunit interactions in the enzyme is discussed. 7. The kinetic properties of hybridized aldolase containing native and citraconylated subunits are not exactly those predicted from the kinetic properties of the two parental forms. This result is interpreted in terms of conformational changes induced in the native and modified subunits when both are present in a hybrid molecule, evidently as a result of interactions in the tetramer.     

Kinetic cooperativity in the concerted model for allosteric enzymes.

The cooperativity of enzyme-substrate interactions is investigated in the concerted allosteric model of Monod, Wyman and Changeux. The general case of K-V systems is considered, in which the two protomer conformational states R and T postulated in the theory differ in catalytic and binding properties. An expression for the Hill coefficient nH defined with respect to the asymptotic velocity V infinity to is analyzed in conditions which exclude substrate inhibition. Kinetic cooperativity is always positive (nH greater than 1) in the case of a dimer enzyme, and in the case of an inactive T state. Slight kinetic negative cooperativity (nH less than 1) occurs under restrictive conditions for larger numbers of protomers when the substrate binds significantly to the less active state of the enzyme, but the phenomenon remains negligible for trimers and tetramers. These conclusions differ from those obtained [A. Goldbeter, J. Mol.Biol.90 (1974) 185] with the Hill coefficient based on the absolute maximum velocity, which may exceed the experimental value V infinity to in K-V systems. The results extend those of Paulus and DeRiel [J. Mol. Biol. 97 (1975) 667] and support the view that in most cases, negative cooperativity is not compatible with a mechanism based on a concerted and conservative allosteric transition. The Hill coefficients for binding and catalysis are compared in K-V systems.     

Kinetic study of the thermal inactivation of cholinesterase enzymes immobilized in solid matrices

The thermal inactivation of immobilized cholinesterase enzymes (ChE) in solid matrices where the protein unfolding is blocked was studied, thus enabling investigation of the kinetics of the inactivation process directly from the native structure to the inactivated state. The thermal inactivation of butyrylcholinesterase (BChE), recombinant human acetylcholinesterase (rHuAChE), and eel acetylcholinesterase (AChE) enzymes was studied in dry films composed of poly(vinyl pyrollidone) (PVP), bovine serum albumin (BSA) and trehalose at 60 degrees -120 degrees C. The kinetics follows a bi-exponential decay equation representing a combination of fast and slow processes. The activation enthalpy DeltaH(#) and the activation entropy DeltaS(#) for each of the three enzymes have been evaluated. The values of DeltaH(#) for the fast process and for the slow process of BChE are 33+/-3, and 28+/-2 kcal/mol, respectively, and the values of DeltaS(#) are 0.84+/-0.04, and -18.2+/-0.5 cal/deg, respectively. The appropriate value of DeltaH(#) for rHuAChE is 26+/-2 Kcal/mol, for both processes and the values of DeltaS(#) are -17.6+/-0.9, and -23.0+/-0.9 cal/deg, respectively. Similarly, the values of DeltaH(#) for eelAChE are 30+/-3, 31+/-1 kcal/mol, and the values of DeltaS(#) are -6.7+/-0.5, -9.1+/-0.2 cal/deg respectively.     

Kinetic and structural analysis of bisubstrate inhibition of the Salmonella enterica aminoglycoside 6'-N-acetyltransferase

Aminoglycosides are antibacterial compounds that act by binding to the A site of the small 30S bacterial ribosomal subunit and inhibiting protein translation. Clinical resistance to aminoglycosides is generally the result of the expression of enzymes that covalently modify the antibiotic, including phosphorylation, adenylylation, and acetylation. Bisubstrate analogs for the aminoglycoside N-acetyltransferases are nanomolar inhibitors of Enterococcus faecium AAC(6')-Ii. However, in the case of the Salmonella enterica aac(6')-Iy-encoded aminoglycoside N-acetyltransferase, we demonstrate that a series of bisubstrate analogs are only micromolar inhibitors. In contrast to studies with AAC(6')-Ii, the inhibition constants toward AAC(6')-Iy are essentially independent of both the identity of the aminoglycoside component of the bisubstrate and the number of carbon atoms that are used to link the CoA and aminoglycoside components. The patterns of inhibition suggest that the CoA portion of the bisubstrate analog can bind to the enzyme-aminoglycoside substrate complex and that the aminoglycoside portion can bind to the enzyme-CoA product complex. However, at the high concentrations of bisubstrate analog used in crystallization experiments, we could crystallize and solve the three-dimensional structure of the enzyme-bisubstrate complex. The structure reveals that both the CoA and aminoglycoside portions bind in essentially the same positions as those previously observed for the enzyme-CoA-ribostamycin complex, with only a modest adjustment to accommodate the "linker". These results are compared to previous studies of the interaction of similar bisubstrate analogs with other aminoglycoside N-acetyltransferases.     

Kinetic analysis of enzyme systems with suicide substrate in the presence of a reversible, uncompetitive inhibitor.

We present a general kinetic analysis of enzyme catalyzed reactions evolving according to a Michaelis-Menten mechanism, in which an uncompetitive, reversible inhibitor acts. Simultaneously, enzyme inactivation is induced by an unstable suicide substrate, i.e. it is a Michaelis-Menten mechanism with double inhibition: one originating from the substrate and another originating from the reversible inhibitor. Rapid equilibrium of the reversible reaction steps involved is assumed and the time course equations for the reaction product have been derived under the assumption of limiting enzyme. The goodness of the analytical solutions has been tested by comparison with simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters from the time progress curve of the product is suggested.     

Partial reversible inactivation of enzymes due to binding to the human erythrocyte membrane

Summary Hypotonic human erythrocyte ghosts, devoid of the original glyceraldehyde-3-phosphate dehydrogenase content of the red cell, bind added glyceraldehyde-3-phosphate dehydrogenases, isolated from human erythrocytes, rabbit and pig muscle, as well as rabbit muscle aldolase. There are only slight differences in the affinities towards the various glyceraldehyde-3-phosphate dehydrogenases. On the other hand, glyceraldehyde-3-phosphate dehydrogenases are bound much stronger than aldolase; in an equimolar mixture the former can prevent the binding of the latter, or replace previously bound aldolase at the membrane surface. Binding is always accompanied by the partial inactivation of enzymes, which can be reverted by desorption. Unwashed ghosts rich in hemoglobin seem to have a more pronounced inactivating effect on bound glyceraldehyde-3-phosphate dehydrogenase. In isotonic media ghosts, whether white or unwashed, reseal and do not interact with the enzymes.     

Kinetic study of thermal inactivation for native and methoxypolyethylene glycol modified trypsin

Bovine pancreatic trypsin (Ti) has been modified with four kinds of methoxypolyethylene glycol (MPEG, molecular masses 350, 750, 2000 and 5000 Da) to enhance thermostability. The MPEG-modified Ti was more stable against temperature than the native form, the larger molecular mass moiety of MPEG showing higher thermostabillty. To investigate the mechanism of thermal inactivation, a new kinetic model, which has the ability of taking the thermal denaturation and autolysis effects of the proteases into account, has been used to analyze the thermal inactivation process of the native and modified Ti in detail. The kinetic analysis showed that the stabilization effect caused by MPEG modification was the result of a decrease in autolysis rate and a decrease in the rate of thermal denaturation. In addition, the possible mechanism of reduced autolysis and lower thermal denaturation rate were also discussed.     

Enzyme electrode for aromatic compounds exploiting the catalytic activities of microperoxidase-11

Microperoxidase-11 (MP-11) which has been immobilised in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode catalyzes the conversion of aromatic substances. This peroxide-dependent catalysis of microperoxidase has been applied in an enzyme electrode for the first time to indicate aromatic compounds such as aniline, 4-fluoroaniline, catechol and p-aminophenol. The electrode signal is generated by the cathodic reduction of the quinone or quinoneimine which is formed in the presence of both MP-11 and peroxide from the substrate. The same sensor principle will be extended to aromatic drugs.     

Discovering potent and selective reversible inhibitors of enzymes in complex proteomes

To realize the promise of genomics-based therapeutics, new methods are needed to accelerate the discovery of small molecules that selectively modulate protein activity. Toward this end, advances in combinatorial synthesis have provided unprecedented access to large compound libraries of considerable structural complexity and diversity, shifting the bottleneck in drug discovery to the development of efficient screens for protein targets. Screening for reversible enzyme inhibitors typically requires extensive target-specific work, including protein expression and purification, as well as the development of specific substrate assays. Here we report a proteomic method for the discovery of reversible enzyme inhibitors that avoids these steps. We show that competitive profiling of a library of candidate serine hydrolase inhibitors in complex proteomes with activity-based chemical probes identifies nanomolar reversible inhibitors of several enzymes simultaneously, including the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH), triacylglycerol hydrolase (TGH) and an uncharacterized membrane-associated hydrolase that lacks known substrates. The strategy tests inhibitors against numerous enzymes in parallel, assigning both potency and selectivity factors to each agent. In this way, promiscuous inhibitors were readily rejected in favor of equally potent compounds with 500-fold or greater selectivity for their targets.     

A general method for the analysis of random bisubstrate enzyme mechanisms

In the present communication, a general method for the kinetic analysis of random bisubstrate mechanisms is described. The method comprises a stepwise application of the following kinetic and ligand-binding experiments: determination of steady-state kinetic constants, product inhibition patterns, maximum rate relationships, application of alternate substrates, application of dead-end inhibitors, direct binding of substrates, kinetic isotope effects, and isotope exchange studies. This general method was applied to a practical example: a yeast alcohol dehydrogenase-catalyzed oxidation of 2-propanol by NAD⁺ at pH 7.0, 25°C. It was found that this fully reversible reaction proceeds by a steady-state random Bi-Bi mechanism, whereby both dead-end complexes are formed.     

On the role of reversible denaturation (unfolding) in the irreversible thermal inactivation of enzymes

The contribution of the reversible thermal unfolding of an enzyme toward the overall irreversible thermoinactivation process has been examined both theoretically and experimentally. Using bovine pancreatic ribonuclease as a model, we have studied the effect of such variables as pH and salts both on the equilibrium constant of reversible denaturation and on the rate constant of the overall irreversible process. It has been demonstrated that at temperatures where a significant fraction of the enzyme molecules are in the native conformation, there is a correlation between the enzyme thermostabilities with respect to the reversible and irreversible inactivations: greater stability against the former is accompanied by greater stability against the latter. On the other hand, at very high temperatures (where essentially all of the enzyme molecules are unfolded), such a correlation does not exist. These findings are considered in terms of a kinetic model for irreversible enzyme thermoinactivation, and the implications of the derived relationship are discussed.     

Kinetic analysis of a general model of activation of aspartic proteinase zymogens involving a reversible inhibitor. I. Kinetic analysis

Starting from a simple general reaction mechanism of activation of aspartic proteinases zymogens involving a uni- and a bimolecular simultaneous activation route and a reversible inhibition step, the time course equation of the zymogen, inhibitor and activated enzyme concentrations have been derived. Likewise, expressions for the time required for any reaction progress and the corresponding mean activation rates as well as the half-life of the global zymogen activation have been derived. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed.     

Novel bisubstrate analog inhibitors of serotonin N-acetyltransferase: the importance of being neutral

Linker modified novel bisubstrate analog inhibitors 4-7 for serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) have been designed and synthesized. Examination of these inhibitors with AANAT in vitro suggested that: (i) linker hydrogen bonding makes only modest contributions to the affinity of bisubstrate analog inhibitors studied; (ii) greater than or equal to four methylene groups between the indole and the coenzyme A (CoASH) moieties are required for a bisubstrate analog inhibitor to achieve strong AANAT inhibition; (iii) the AANAT active site appears not to accommodate positively charged linkers as well as neutral ones; and (iv) substrate amine pKa depression may constitute one strategy for AANAT substrate recognition and catalysis. The results reported here have enhanced our understanding of AANAT substrate recognition/catalysis, and are important for novel inhibitor design. Since AANAT belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily, our experimental strategies should find applications for other acetyltransferases.