Electrostatic environment at the active site of prolyl oligopeptidase is highly influential during substrate binding

The positive electrostatic environment of the active site of prolyl oligopeptidase was investigated by using substrates with glutamic acid at positions P2, P3, P4, and P5, respectively. The different substrates gave various pH rate profiles. The pKa values extracted from the curves are apparent parameters, presumably affected by the nearby charged residues, and do not reflect the ionization of a simple catalytic histidine as found in the classic serine peptidases like chymotrypsin and subtilisin. The temperature dependence of kcat/Km did not produce linear Arrhenius plots, indicating different changes in the individual rate constants with the increase in temperature. This rendered it possible to calculate these constants, i.e. the formation (k1) and decomposition (k-1) of the enzyme-substrate complex and the acylation constant (k2), as well as the corresponding activation energies. The results have revealed the relationship between the complex Michaelis parameters and the individual rate constants. Structure determination of the enzyme-substrate complexes has shown that the different substrates display a uniform binding mode. None of the glutamic acids interacts with a charged group. We conclude that the specific rate constant is controlled by k1 rather than k2 and that the charged residues from the substrate and the enzyme can markedly affect the formation but not the structure of the enzyme-substrate complexes.     

A study of the complex-formation of phenylalanyl-tRNA-synthetase from Escherichia coli using the tRNA-Phe method of small-angle x-ray scattering

The method of small-angle X-ray scattering was employed to analyse the equilibrium enzyme-substrate complexes in solution. A new approach of analysis of the experimental data was developed. This type of analysis provides the determination of dissociation constants and structural parameters of enzyme-substrate complexes. The radius of gyration (Rg) and dimensions of half-axis of the equivalent prolonged ellipsoid (a, b) of E. coliphenylalanyl-tRNA synthetase and its complexes with one or two tRNA(Phe) molecules have been determined. The values of these parameters speak in favour of structural rearrangements due to the interaction of the enzyme with tRNA(Phe). The thermodynamic characteristics of phenylalanyl-tRNA synthetase complexes with tRNA(Phe) testify to the negative cooperativity in binding of two tRNA molecules with the enzyme.     

Stopped-flow studies on the mechanism of oxidation of N-methyl-4-phenyltetrahydropyridine by bovine liver monoamine oxidase B

The kinetic mechanism of monoamine oxidase B involves either a binary or a ternary complex, depending on the substrate. In this study, stopped-flow kinetic data provide direct evidence for ternary complexes not only of reduced enzyme, oxygen, and product but also of reduced enzyme, oxygen, and substrate, both for benzylamine and for the tertiary amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the mechanism for a given substrate is not exclusive but, rather, is determined by competition between the alternate pathways as a result of different rate constants for the oxidation of the reduced enzyme, the reduced enzyme-product complex, and the reduced enzyme-substrate complex, as well as the different dissociation constants for the complexes. Comparison of the rate constants obtained from the stopped-flow studies with steady-state data indicates that the overall rate of reaction for the oxidation of MPTP by monoamine oxidase is dominated by the reductive step, but for benzylamine the steady-state rate is determined by a complex function of the rates of both the reductive and oxidative half-reactions.     

Binding of E. coli DNA photolyase to a defined substrate containing a single T mean value of T dimer.

The E. coli DNA photolyase is a flavoprotein that catalyzes the photoreversal of pyrimidine dimers. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and repairs the dimer upon absorbing a photon in the 300-600 nm range. The rate and equilibrium constants for the light-independent reaction were determined before, using randomly modified substrates that contained T mean value of T, T mean value of C and C mean value of C dimers in random sequence surrounding. In this paper we have determined these constants for a defined substrate (a 43 bp oligomer containing a T mean value of T dimer) using the gel retardation assay. We find that: the equilibrium constant and the off rate obtained with this substrate by this technique are similar to those obtained with randomly modified DNA using filter binding and flash photolysis techniques. the off rate with the defined substrate is heterogeneous indicating heterogeneity in the enzyme population or in the enzyme-substrate complexes, and the enzyme has 7.5 X 10(4)-fold higher affinity for pyrimidine dimer compared to non-dimer DNA nucleotides.     

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.     

Application of the small-angle x-ray scattering technique for the study of two-step equilibrium enzyme-substrate interactions

The small-angle x-ray scattering (SAXS) technique is used for the investigation of two-stage equilibrium macromolecular interactions of the enzyme-substrate type in solution. Experimental procedures and methods of analyzing the data obtained from SAXS have been elaborated. The algorithm for the data analysis allows one to determine the stoichiometric, equilibrium, and structural parameters of the enzyme-substrate complexes obtained. The thermodynamic characteristics for the formation of complexes of double-stranded oligonucleotide with Eco dam methyltransferase (MTase) have been determined and demonstrate a high cooperativity of MTase binding when the ternary complex containing the dimeric enzyme is formed. The structural parameters (R_g, R_c, semiaxes) have been determined for free enzyme and polynucleotides and of enzyme-substrate complexes, indicating structural rearrangements of the enzyme in the interaction with substrates. © 1996 John Wiley & Sons, Inc.     

Kinetics and thermodynamics of lactate dehydrogenases from beef heart, beef muscle, and flounder muscle.

The eight rate constants for a four-step ordered ternary-complex mechanism have been compared for lactate dehydrogenases (EC1.1.1.27) from three sources, beef heart, beef muscle, and flounder muscle. The rate constants were determined at temperatures ranging from 5 degrees C to 50 degrees C, and the corresponding activation parameters deltaG not equal to, deltaH not equal to, and deltaS not equal to were calculated. Significant differences are noted for the values for the three types of enzyme. The relative heights of the activation barriers are much the same in all three cases, differences in kinetic behavior resulting mainly from differences in the stable binary and ternary enzyme-substrate complexes. These complexes are, in general, at lower free-energy and enthalpy levels of the beef-heart and beef-muscle enzymes than for the flounder-muscle enzyme. A high degree of compensation is found between the enthalpies and entropies of activation, resulting in relatively small differences between the free energies (and rates) for homologous steps with different enzymes. Analysis of the results, on the assumption that the compensation effect is due to weak-bonding effects, suggests that there are fewer weak bonds in the stable complexes of the muscle enzymes.     

Isotopic probes yield microscopic constants: separation of binding energy from catalytic efficiency in the bovine plasma amine oxidase reaction

Bovine plasma amine oxidase catalyzes the oxidative deamination of primary amines. The reaction can be viewed as two half-reactions: enzyme reduction by substrate followed by enzyme reoxidation by dioxygen. Anaerobic stopped-flow kinetic measurements of the first half-reaction indicate very large deuterium isotope effects for benzylamine, m-tyramine, and dopamine, Dk = 13.5 +/- 1.3, which are ascribed to an intrinsic isotope effect. From the insensitivity of these isotope effects to amine concentration, stopped-flow data provide substrate dissociation constants, K1, and rate constants for the C-H bond cleavage step, k3, directly. Steady-state isotope effects have also been measured for benzylamine and six ring-substituted phenethylamines. Whereas a small range of values for kcat, 0.38-1.2 s-1, and Dkcat, 5.4-8.8, is observed, kcat/Km = 1.3 X 10(2) to 3.8 X 10(4) M-1 S-1 and D(kcat/Km) = 5.6-16.1 indicate a marked effect of ring substituent. As described earlier [Miller, S., & Klinman, J.P. (1982) Methods Enzymol. 87, 711], the availability of an intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic constants from steady-state data. By employment of a minimal mechanism for bovine plasma amine oxidase involving a single precatalytic and multiple postcatalytic enzyme-substrate complexes, equations have been derived that allow calculation of k3 and K1 when DKeq congruent to 1 less than Dk. Unexpectedly, in the case of K1, we have shown that this parameter can be calculated from steady-state parameters without the requirement for an intrinsic isotope effect. This result should have general application to both ping-pong and sequential ternary-complex enzyme mechanisms. Of significance for future applications of steady-state isotope effects to the calculation of microscopic constants, values for K1 and k3 derived from steady-state parameters and single turnover measurements indicate excellent agreement. Compilation of parameters among six ring-substituted phenethylamines reveals alteration in delta G for enzyme-substrate complex formation by 2.8 kcal/mol, together with an essentially invariant rate constant for C-H bond activation. A detailed discussion of the relevance of these findings to the interrelationship of binding energy and catalytic efficiency in enzyme reactions is presented.     

The kinetics of amylolysis : Part III. Effect of self-inhibition on the kinetic constants of beta-amylolysis

For beta-amylolysis, the number of bonds split per effective enzyme-substrate encounter has been determined with amyloses having various degrees of polymerization (d.p.), labelled with ¹⁴C. The degradation is shifted in the direction of the “single chain mechanism” with increasing d.p. By taking into account the “inactive, non-reacting” collisions leading to “self-inhibition”, the number of bonds split per “reactive” and “non-reactive” enzyme-substrate encounter was established. From these data, and from the inhibition constant of the inner D-glucose residues of the amylose chain, in conjunction with the Michaelis constant extrapolated to d.p.  4, the actual rate constants for the formation and dissociation of the “reactive” enzyme-substrate complexes and for the formation and dissociation of the “non-reactive” enzyme-substrate complexes were determined. It was found that the rate constants for the formation of the “reactive” complexes decrease slowly with the increase of “self-inhibition”, whereas those for the dissociation of these complexes decrease to a greater extent.     

Study of penicillin amidase from E. coli. pH-dependence of kinetic parameters of enzymatic hydrolysis of benzylpenicillin]

The authors studies pH-dependencies of the kinetic parameters (Vm, KM, Vm/KM) and constants of competitive inhibition by phenylacetic acid of penicillinamidase-catalyzed hydrolysis of benzylpenicillin. The experimental data are in agreement with the assumption according to which there are 3 equilibrium ionogenic forms of the enzyme and enzyme-substrate (or enzyme-inhibitor) complexes, i.e. acidic, neutral and alkaline, the neutral form being the only active form of the Michaelis complex. Values of pK in the ionogenic groups controlling interconversions of both the free enzyme (pK1 6.1 and pK2 7.6) and of the enzyme-substrate complex (pKa 6.1 and pK2 10.2 or the enzyzme-inhibitor complex (pK'1 6.1 and pK'2 9.5) were determined. From this and the previously published results it was concluded that the group with pK 6.1 was involved in the catalysis and the group with pK 10.2 in the maintenance of the active conformation of the active centre of penicillinamidase. The ionogenic group with pK 7.6 was apparently involved in the enzyme-substrate binding.     

Study of bacteriophage T4-encoded Dam DNA (adenine-N6)-methyltransferase binding with substrates by rapid laser UV cross-linking

DNA methyltransferases of the Dam family (including bacteriophage T4-encoded Dam DNA (adenine-N(6))-methyltransferase (T4Dam)) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. In this study, we describe the application of direct (i.e. no exogenous cross-linking reagents) laser UV cross-linking as a universal non-perturbing approach for studying the characteristics of T4Dam binding with substrates in the equilibrium and transient modes of interaction. UV irradiation of the enzyme.substrate complexes using an Nd(3+):yttrium aluminum garnet laser at 266 nm resulted in up to 3 and >15% yields of direct T4Dam cross-linking to DNA and AdoMet, respectively. Consequently, we were able to measure equilibrium constants and dissociation rates for enzyme.substrate complexes. In particular, we demonstrate that both reaction substrates, specific DNA and AdoMet (or product AdoHcy), stabilized the ternary complex. The improved substrate affinity for the enzyme in the ternary complex significantly reduced dissociation rates (up to 2 orders of magnitude). Several of the parameters obtained (such as dissociation rate constants for the binary T4Dam.AdoMet complex and for enzyme complexes with a nonfluorescent hemimethylated DNA duplex) were previously inaccessible by other means. However, where possible, the results of laser UV cross-linking were compared with those of fluorescence analysis. Our study suggests that rapid laser UV cross-linking efficiently complements standard DNA methyltransferase-related tools and is a method of choice to probe enzyme-substrate interactions in cases in which data cannot be acquired by other means.     

Kinetics and thermodynamics of catalysis by the inorganic pyrophosphatase of Escherichia coli in both directions

Combined evidence obtained from the measurements of pyrophosphate hydrolysis and synthesis, oxygen exchange between phosphate and water, enzyme-bound pyrophosphate formation and Mg2+ binding enabled us to deduce the overall scheme of catalysis by Escherichia coli inorganic pyrophosphatase in the presence of Mg2+. We determined the equilibrium constants for Mg2+ binding to various enzyme species and forward and reverse rate constants for the four steps of the catalytic reaction, namely, binding/release of PPi, hydrolysis/synthesis of PPi and successive binding/release of two Pi molecules. Catalysis by the E. coli enzyme in both directions, in contrast to baker's yeast pyrophosphatase, occurs via a single pathway, which requires the binding of Mg2+ to the sites of four types. Three of them can be filled in the absence of the substrates, and the affinity of one of them to Mg2+ is increased by two orders of magnitude in the enzyme-substrate complexes. The distribution of 18O-labelled phosphate isotopomers during the exchange indicated that hydrolysis of pyrophosphate in the active site is appreciably reversible. The equilibrium constant for this process estimated from direct measurements is 5.0. The ratio of the maximal velocities of pyrophosphate hydrolysis and synthesis is 69. The rate of the synthesis is almost entirely determined by the rate of the release of pyrophosphate from the enzyme. In the hydrolytic reaction, enzyme-bound pyrophosphate hydrolysis and successive release of two phosphate molecules proceed with nearly equal rate constants.     

Beta-lactamases as fully efficient enzymes. Determination of all the rate constants in the acyl-enzyme mechanism.

The rate constants for both acylation and deacylation of beta-lactamase PC1 from Staphylococcus aureus and the RTEM beta-lactamase from Escherichia coli were determined by the acid-quench method [Martin & Waley (1988) Biochem. J. 254, 923-925] with several good substrates, and, for a wider range of substrates, of beta-lactamase I from Bacillus cereus. The values of the acylation and deacylation rate constants for benzylpenicillin were approximately the same (i.e. differing by no more than 2-fold) for each enzyme. The variation of kcat./Km for benzylpenicillin with the viscosity of the medium was used to obtain values for all four rate constants in the acyl-enzyme mechanism for all three enzymes. The reaction is partly diffusion-controlled, and the rate constant for the dissociation of the enzyme-substrate complex has approximately the same value as the rate constants for acylation and deacylation. Thus all three first-order rate constants have comparable values. Here there is no single rate-determining step for beta-lactamase action. This is taken to be a sign of a fully efficient enzyme.     

Regulation of stromal sedoheptulose 1,7-bisphosphatase activity by pH and Mg2+ concentration

A scheme is proposed for the regulation of stromal sedoheptulose 1,7-bisphosphatase activity which enlarges upon a previously elaborated mechanism (Woodrow, I.E., and Walker, D.A. (1983) Biochim. Biophys. Acta 722, 508-516). The latter involves oxidized (inactive) and reduced (active) enzyme forms. Both the free enzymes and the enzyme-substrate complexes undergo slow oxidation/reduction. This study examines the behavior of the system under pH and Mg2+ concentration regimes that are likely to occur in the chloroplast stroma. The control of enzyme activity by pH can be described in terms of each free enzyme and enzyme-substrate complex existing in protonated and nonprotonated forms. The molecular dissociation constants for each protonation reaction were calculated from kinetic data. Mg2+ concentration changes modulate these constants. Under conditions that are likely to obtain in the stroma in the dark, the model predicts that approximately 99.1% of the enzyme will be in the inactive forms. Such inactivation is important since it would prevent the reductive pentose phosphate pathway from operating in darkness.     

Evidence of essential arginyl residues in chicken liver mevalonate-5-pyrophosphate decarboxylase

Chicken liver mevalonate-5-pyrophosphate decarboxylase (ATP:5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33.) is inactivated by phenylglyoxal in triethanolamine buffer at pH 8.15. The reaction follows pseudo-first-order kinetics with a second-order rate constant of 108 M-1 min-1. Appropriate treatment of the kinetic data for the inactivation reaction indicates that the reaction of a single phenylglyoxal molecule per active unit of the enzyme is enough to completely inactivate the protein. The partially inactivated enzyme shows unaltered Km but decreased V as compared to native mevalonate-5-pyrophosphate decarboxylase. The dissociation constants for the enzyme-substrate complexes were estimated from inactivation reactions at different concentrations of substrates. From the data it is concluded that the modified amino acid is important for the binding of both substrates.     

A comparison of lactate dehydrogenase from an ectothermic and an endothermic animal.

Adaptation to environmental temperature is examined in beef heart, beef muscle, and flounder muscle lactate dehydrogenases (EC 1.1.1.27). Low temperature adaptation in the ectothermic (flounder) enzyme is indicated by a reduced enthalpy of activation for kcat (enzyme turnover number, s-1) and increased catalytic efficiency. Also, the reaction rate at low substrate concentrations has a maximum at a lower temperature than in the endothermic enzymes. This is a result of altered bonding in the enzyme-substrate complexes. Adaptation to higher temperatures in the endothermic (beef) enzymes is suggested by a decreased sensitivity to heat denaturation, especially in the presence of substrates. A direct correlation is found between the degree of bonding in the enzyme-substrate complexes and the decrease in rate of heat denaturation caused by addition of substrates.     

Steady state kinetics of consecutive enzyme catalysed reactions involving single substrates: procedures for the interpretation of coupled assays

Sets of differential rate equations are written describing a linear sequence of reactions occurring in solution each catalysed by a control enzyme or one of the Michaelis-Menten type. It is shown that the solutions of these equations may be formulated as a set of Maclaurin polynomials, expressing the concentration of each reactant and of final product as a function of time. From arrays of such polynomials, general expressions are induced for the first non-zero term of the series. These are used to formulate a procedure (illustrated with an example simulated by numerical integration) by which results of coupled enzymic assays may be analysed in terms of maximal velocities and apparent Michaelis constants: correlation is made with other established methods for conducting coupled assays. The present procedure assumes a steady state of enzyme-substrate complexes but not of intermediate reactants.     

Fluoride inhibition of inorganic pyrophosphatase. I. Kinetic studies in a Mg2+-PPi system using a new continuous enzyme assay.

Reversible inhibition of bakers' yeast inorganic pyrophosphatase (EC 3.6.1.1) by fluoride has been studied as a function of substrate, metal-ion activator and inhibitor concentrations and pH using a new continuous enzyme assay with an automatic phosphate analyzer. The inhibition was shown to be the result of tight binding of fluoride by two catalytically active enzyme-substrate complexes. The reaction between pyrophosphatase and fluoride is relatively slow, so that the rate constants for the binding and release of the inhibitor were derived from phosphate formation curves measured on the time scale of enzyme assays. The pH-dependence of the inhibition reaction in the alkaline medium indicates that both the fluoride-enzyme interaction and the catalytic step of the pyrophosphatase reaction are controlled by the same group on the protein. In the acidic medium, the inhibition is considerably enhanced, presumably because of the protonation of another enzyme group.     

Inhibition kinetics of green crab (Scylla serrata) alkaline phosphatase activity by dithiothreitol or 2-mercaptoethanol

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme which catalyzes the nonspecific hydrolysis of phosphate monoesters. Some pollutants in seawater affect the enzyme activity causing loss of the biological function of the enzyme, which affects the exuviating crab-shell and threatens the survival of the animal. The present paper studies the effects of thiohydroxyal compounds on the activity of green crab alkaline phosphatase. The results show that thiohydroxyal compounds can lead to reversible inhibition. The equilibrium constants have been determined for dithiothreitol (DTT) and mercaptoethanol (ME) binding with the enzyme and/or the enzyme-substrate complexes. The results show that both DTT and ME are non-competitive inhibitors. The kinetics of enzyme inactivation by ME at low concentrations has been studied using the kinetic method of the substrate reaction. The results suggest that at pH 10.0, the action of ME on green crab ALP is first quick equilibrium binding and then slow inactivation. The microscopic rate constants were determined for inactivation and reactivation. The rate constant of the forward inactivation (k(+0)) is much larger than that of the reverse reactivation (k(-0)). Therefore, when the ME concentration is sufficiently large, the enzyme is completely inactivated.     

Quantitative description of absorption spectra of a pyridoxal phosphate-dependent enzyme using lognormal distribution curves

Ultraviolet-visible absorption spectra of cytosolic aspartate aminotransferase of pig hearts have been analyzed by resolution with lognormal distribution curves. These have been compared with spectra of reference Schiff bases of pyridoxal 5'-phosphate. Spectra of the free enzyme in two different states of protonation and of complexes with monoanions, dicarboxylates, the substrates L-glutamate, L-aspartate, and L-erythro-3-hydroxyaspartate, and the quasi-substrate 2-methylaspartate have been analyzed. Relative amounts of three tautomeric species have been estimated, as have amounts of various enzyme-substrate intermediates. Bandshape parameters which can be used as a guide to analysis of spectra of other pyridoxal phosphate-dependent enzymes are tabulated. Some formation constants and pKa values, which were evaluated at the same time as the spectra of the complexes, are also reported.