Conserved cysteine 126 in triosephosphate isomerase is required not for enzymatic activity but for proper folding and stability

In triosephosphate isomerase, Cys126 is a conserved residue located close to the catalytic glutamate, Glu165. Although it has been mentioned that Cys126 and other nearby residues are required to maintain the active site geometry optimal for catalysis, no evidence supporting this idea has been reported to date. In this work, we studied the catalytic and stability properties of mutants C126A and C126S of Saccharomyces cerevisiae TIM (wtTIM). None of these amino acid replacements induced significant changes in the folding of wtTIM, as indicated by spectroscopic studies. C126S and C126A have K(M) and k(cat) values that are concomitantly reduced by only 4-fold and 1.5-fold, respectively, compared to those of wtTIM; in either case, however, the catalytic efficiency (k(cat)/K(M)) of the enzyme is barely affected. The affinity of mutated TIMs for the competitive inhibitor 2-phosphoglycolate augmented also slightly. In contrast, greater susceptibility to thermal denaturation resulted from mutation of Cys126, especially when it was changed to Ser. By using values of the rate constants for unfolding and refolding, we estimated that, at 25 degrees C, C126A and C126S are less stable than wtTIM by about 5.0 and 9.0 kcal mol(-)(1), respectively. Moreover, either of these mutations slows down the folding rate by a factor of 10 and decreases the recovery of the active enzyme after thermal unfolding. Thus, Cys126 is required for proper stability and efficient folding of TIM rather than for enzymatic catalysis.     

Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power

The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rigid dimer interface. At this interface a critical hydrogen bond is formed between the main chain oxygen atom of the catalytic residue Lys13 and the completely buried side chain of Gln65 (of the same subunit). The sequence of Leishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68% sequence identity), shows that this highly conserved glutamine has been replaced by a glutamate. Therefore, the 1.8 A crystal structure of leishmania TIM (at pH 5.9) was determined. The comparison with the structure of trypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13. Ionization of this glutamic acid side chain causes a pH-dependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent the conserved hydrogen bond network, as seen in all other TIMs. Restoration of the hydrogen bonding network by its mutation to glutamine in the E65Q variant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26 degrees C (57 degrees C for leishmania TIM to 83 degrees C for the E65Q variant). This mutation does not affect the kinetic properties, showing that even point mutations can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power at the mesophilic temperature.     

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.     

Linear and non-linear pressure dependence of enzyme catalytic parameters

The pressure dependence of enzyme catalytic parameters allows volume changes associated with substrate binding and activation volumes for the chemical steps to be determined. Because catalytic constants are composite parameters, elementary volume change contributions can be calculated from the pressure differentiation of kinetic constants. Linear and non-linear pressure-dependence of single-step enzyme reactions and steady-state catalytic parameters can be observed. Non-linearity can be interpreted either in terms of interdependence between the pressure and other environmental parameters (i.e., temperature, solvent composition, pH), pressure-induced enzyme unfolding, compressibility changes and pressure-induced rate limiting changes. These different situations are illustrated with several examples.     

Differential effects on enzyme stability and kinetic parameters of mutants related to human triosephosphate isomerase deficiency

Human triosephosphate isomerase (TIM) deficiency is a very rare disease, but there are several mutations reported to be causing the illness. In this work, we produced nine recombinant human triosephosphate isomerases which have the mutations reported to produce TIM deficiency. These enzymes were characterized biophysically and biochemically to determine their kinetic and stability parameters, and also to substitute TIM activity in supporting the growth of an Escherichia coli strain lacking the tim gene. Our results allowed us to rate the deleteriousness of the human TIM mutants based on the type and severity of the alterations observed, to classify four “unknown severity mutants” with altered residues in positions 62, 72, 122 and 154 and to explain in structural terms the mutation V231M, the most affected mutant from the kinetic point of view and the only homozygous mutation reported besides E104D.     

Kinetic mechanism of human apurinic/apyrimidinic endonuclease action in nucleotide incision repair

Human major apurinic/apyrimidinic endonuclease (APE1) is a multifunctional enzyme that plays a central role in DNA repair through the base excision repair (BER) pathway. Besides BER, APE1 is involved in an alternative nucleotide incision repair (NIR) pathway that bypasses glycosylases. We have analyzed the conformational dynamics and the kinetic mechanism of APE1 action in the NIR pathway. For this purpose we recorded changes in the intensity of fluorescence of 2-aminopurine located in two different positions in a substrate containing dihydrouridine (DHU) during the interaction of the substrate with the enzyme. The enzyme was found to change its conformation within the complex with substrate and also within the complex with the reaction product, and the release of the enzyme from the complex with the product seemed to be the limiting stage of the enzymatic process. The rate constants of the catalytic cleavage of DHU-containing substrates by APE1 were comparable with the appropriate rate constants for substrates containing apurinic/apyrimidinic site or tetrahydrofuran residue, which suggests that NIR is a biologically important process.     

Purine nucleoside phosphorylase. Kinetic mechanism of the enzyme from calf spleen.

Ribose 1-phosphate, phosphate, and acyclovir diphosphate quenched the fluorescence of purine nucleoside phosphorylase at pH 7.1 and 25 degrees C. The fluorescence of enzyme-bound guanine was similar to that of anionic guanine in ethanol. Guanine and ribose 1-phosphate bound to free enzyme, whereas inosine and guanosine were not bound to free enzyme in the absence of phosphate. Thus, synthesis proceeded by a random mechanism, and phosphorolysis proceeded by an ordered mechanism. Steady-state kinetic data for the phosphorolysis of 100 microM guanosine were fitted to a bifunctional kinetic model with catalytic rate constants of 22 and 1.3 s-1. The dissociation rate constants for guanine from the enzyme-guanine complex at high and low phosphate concentrations were similar to the catalytic rate constants. Fluorescence changes of the enzyme during phosphorolysis suggested that ribose 1-phosphate dissociated from the enzyme ribose 1-phosphate-guanine complex rapidly and that guanine dissociated from the enzyme-guanine complex slowly. The association and dissociation rate constants for acyclovir diphosphate, a potent inhibitor of the enzyme (Tuttle, J. V., and Krenitsky, T. A. (1984) J. Biol. Chem. 259, 4065-4069), were also dependent on phosphate concentration. The effects of phosphate are discussed in terms of a dual functional binding site for phosphate.     

Multicriteria optimization of biochemical systems by linear programming: application to production of ethanol by Saccharomyces cerevisiae

In this study we present a method for simultaneous optimization of several metabolic responses of biochemical pathways. The method, based on the use of the power law formalism to obtain a linear system in logarithmic coordinates, is applied to ethanol production by Saccharomyces cerevisiae. Starting from an experimentally based kinetic model, we translated it to its power law equivalent. With this new model representation, we then applied the multiobjective optimization method. Our intent was to maximize ethanol production and minimize each of the internal metabolite concentrations. To ensure cell viability, all optimizations were carried out under imposed constraints. The different solutions obtained, which correspond to alternative patterns of enzyme overexpression, were implemented in the original model. We discovered few discrepancies between the S-system-optimized steady state and the corresponding optimized state in the original kinetic model, thus demonstrating the suitability of the S-system representation as the basis for the optimization procedure. In all optimized solutions, the ATP level reached its maximum and any increase in its activity positively affected the optimization process. This work illustrates that in any optimization study no single criteria is of general application being the multiobjective and constrained task the proper way to address it. It is concluded that the proposed multiobjective method can serve to carry out, in a single study, the general pattern of behavior of a given metabolic system with regard to its control and optimization.     

Directed coevolution of stability and catalytic activity in calcium-free subtilisin

We have coevolved high activity and hyperstability in subtilisin by sequentially randomizing 12 amino acid positions in calcium-free subtilisin. The optimal amino acid for each randomized site was chosen based on stability and catalytic properties and became the parent clone for the next round of mutagenesis. Together, the 12 selected mutations increased the half-life of calcium-free subtilisin at elevated temperature by 15,000-fold. The catalytic properties of the mutants were examined against a range of substrates. In general, only mutations occurring at or near the substrate-binding surface have measurable effects on catalytic constants. No direct influence of stability on catalytic properties was observed. A high-stability mutant, Sbt140, was a more efficient enzyme in terms of k(cat)/K(m) than a commercial version of subtilisin across a range of substrates but had a lower k(cat) against tight-binding substrates. The reason for this behavior was discerned by examining microscopic rate constants for the hydrolysis of a tight-binding peptide substrate. Burst kinetics were observed for this substrate, indicating that acylation is not rate-limiting. Although acylation occurs at the rate of substrate binding, k(cat) is attenuated by the slow release of the N-terminal product. Natural evolution appears to have optimized catalytic activity against a range of sequences by achieving a balance between substrate binding and the rate of release of the N-terminal product.     

Compensational phenomena in reactivation of dimethyl- and diethylphosphoryl butyrylcholinesterases.

The thermodynamic and kinetic parameters for spontaneous and oxime reactivation of dimethyl- and diethylphosphoryl butyrylcholinesterases (acylcholine acyl-hydrolase, EC 3.1.1.8) are reported. The enthalpy and entropy changes in both the binding (deltaH0 and deltaS0) and the dephosphorylation steps (deltaH* and deltaS*) were found to be coupled, resulting in a minor variation in free energy changes (deltaG0 and deltaG*). While neither enthalpies nor entropies alone bore any relationship with the kinetic parameters KD and kR, the changes of free energies (deltaG0 and deltaG*) correlated linearly with the logarithmic values of the dissociation constants (KD) and bimolecular rate constants (kR/KD), respectively. Compensation plots of entropies versus enthalpies gave straight lines with compensation temperatures of 275 K for the binding 260 K for the dephosphorylation. Spontaneous reactivation of dimethyl phosphoryl butyrylcholinesterase was investigated at various pH values and three temperatures. It implicated two catalytic sites with values of pKi of 9.4 and 7.5, and heats of ionisation of 5.3 and 9.6 kcal - mol-1, respectively. Possible conformational alteration of the inhibited enzyme arising from the binding of oximes is discussed.     

A pathogenic linked mutation in the catalytic core of human cystathionine beta-synthase disrupts allosteric regulation and allows kinetic characterization of a full-length dimer

Cystathionine beta-synthase catalyzes the condensation of serine and homocysteine to yield cystathionine and is the single most common locus of mutations associated with homocystinuria. In this study, we have examined the kinetic consequences of a pair of linked patient mutations, P78R/K102N, that are housed in the catalytic core of the protein and compared it to the effects of the corresponding single mutations. The P78R mutation affords purification of a mixture of higher order oligomers, P78R-I, which resembles the mixed quaternary state associated with wild-type enzyme. However, unlike wild-type enzyme, P78R-I converts over time to P78R-II, which exists predominantly as a full-length dimer. The specific activities of the K102N, P78R-I, and P78R-II mutants in the absence of AdoMet are approximately 3-, 9-, and 3-fold lower than of wild-type enzyme and are stimulated 2.9-, 2.5-, and 1.4-fold respectively by AdoMet. However, when linked, the specific activity of the resulting double mutant is comparable to that of wild-type enzyme but it is unresponsive to AdoMet, revealing that interactions between the two sites modulate the phenotype of the enzyme. Steady-state kinetic analysis for the double mutant reveals a sigmoidal dependence on homocysteine that is not observed with wild-type enzyme, which is ascribed to the mutation at the K102 locus and indicates changes in subunit interactions. Hydrogen-deuterium mass spectrometric analysis reveals that, even in the absence of AdoMet, the double mutant is locked in an activated conformation that is observed for wild-type enzyme in the presence of AdoMet, providing a structural rationale for loss of this allosteric regulation. To our knowledge, this is the first example of mutations in the catalytic core of cystathionine beta-synthase that result in failure of AdoMet-dependent regulation. Furthermore, analysis of individual single mutations has permitted, for the first time, partial kinetic characterization of a full-length dimeric form of human cystathionine beta-synthase.     

An investigation of the action of porcine pancreatic alpha-amylase on native and gelatinised starches

The action of pancreatic alpha-amylase (EC 3.2.1.1) on various starches has been studied in order to achieve better understanding of how starch structural properties influence enzyme kinetic parameters. Such studies are important in seeking explanations for the wide differences reported in postprandial glycaemic and insulinaemic indices associated with different starchy foodstuffs. Using starches from a number of different sources, in both native and gelatinised forms, as substrates for porcine alpha-amylase, we showed by enzyme kinetic studies that adsorption of amylase to starch is of kinetic importance in the reaction mechanism, so that the relationship between reaction velocity and enzyme concentration [E0] is logarithmic and described by the Freundlich equation. Estimations of catalytic efficiencies were derived from measurements of kcat/Km performed with constant enzyme concentration so that comparisons between different starches were not complicated by the logarithmic relationship between E0 and reaction velocity. Such studies reveal that native starches from normal and waxy rice are slightly better substrates than those from wheat and potato. After gelatinisation at 100 degrees C, kcat/Km values increased by 13-fold (waxy rice) to 239-fold (potato). Phosphate present in potato starch may aid the swelling process during heating of suspensions; this seems to produce a very favourable substrate for the enzyme. Investigation of pre-heat treatment effects on wheat starch shows that the relationship between treatment and kcat/Km is not a simple one. The value of kcat/Km rises to reach a maximum at a pre-treatment temperature of 75 degrees C and then falls sharply if the treatment is conducted at higher temperatures. It is known that amylose is leached from starch granules during heating and dissolves. On cooling, the dissolved starch is likely to retrograde and become resistant to amylolysis. Thus the catalytic efficiency tends to fall. In addition, we find that the catalytic efficiency on the different starches varies inversely with their solubility and we interpret this finding on the assumption that the greater the solubility, the greater is the likelihood of retrogradation. We conclude that although alpha-amylase is present in high activity in digestive fluid, the enzymic hydrolysis of starch may be a limiting factor in carbohydrate digestion because of factors related to the physico-chemical properties of starchy foods.     

Is the catalytic activity of triosephosphate isomerase fully optimized? An investigation based on maximization of entropy production

Triosephosphate isomerase (TIM) is often described as a fully evolved housekeeping enzyme with near-maximal possible reaction rate. The assumption that an enzyme is perfectly evolved has not been easy to confirm or refute. In this paper, we use maximization of entropy production within known constraints to examine this assumption by calculating steady-state cyclic flux, corresponding entropy production, and catalytic activity in a reversible four-state scheme of TIM functional states. The maximal entropy production (MaxEP) requirement for any of the first three transitions between TIM functional states leads to decreased total entropy production. Only the MaxEP requirement for the product (R-glyceraldehyde-3-phosphate) release step led to a 30% increase in enzyme activity, specificity constant k_cat/K_M, and overall entropy production. The product release step, due to the TIM molecular machine working in the physiological direction of glycolysis, has not been identified before as the rate-limiting step by using irreversible thermodynamics. Together with structural studies, our results open the possibility for finding amino acid substitutions leading to an increased frequency of loop six opening and product release.     

A simple method for the determination of individual rate constants for substrate hydrolysis by serine proteases

A simple method is presented for the determination of individual rate constants for substrate hydrolysis by serine proteases and other enzymes with similar catalytic mechanism. The method does not require solvent perturbation like viscosity changes, or solvent isotope effects, that often compromise nonspecifically the activity of substrate and enzyme. The rates of substrate diffusion into the active site (k1), substrate dissociation (k-1), acylation (k2), and deacylation (k3) in the accepted mechanism of substrate hydrolysis by serine proteases are derived from the temperature dependence of the Michaelis-Menten parameters kcat/Km and kcat. The method also yields the activation energies for these molecular events. Application to wild-type and mutant thrombins reveals how the various steps of the catalytic mechanism are affected by Na+-binding and site-directed mutations of the important residues Y225 in the Na+ binding environment and L99 in the S2 specificity site. Extension of this method to other proteases should enable the derivation of detailed information on the kinetic and energetic determinants of protease function.     

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.     

Effect of nucleotide substrate binding on the pKa of catalytic residues in barnase

A thermodynamic cycle is used to describe barnase catalysis, which considers explicitly the presence of different ionic states of the catalytic residues Glu-73 and His-102 in barnase during the enzyme-substrate recognition process. Reinterpretation of published experimental data using rate equations derived from this cycle provides estimates of the ionization constants of these catalytic side chains, in the free enzyme and in the barnase-GpA complex. In addition, the electrostatic properties of the barnase-d(CGAC) crystal complex and of a barnase-5′3′(AAGAAp)-O-methyl ester modeled complex are investigated by means of a continuum approach to account for solvent polarization effects. Taking GpA as a reference substrate, it is shown that Increasing the length of the bound nucleotide induces pK_a shifts in the catalytic side chains, which modulate the fraction of enzyme in the correct ionic form for achieving the transesterification reaction. The computed results are in good agreement with the experimental variation of the optimum pH of barnase activity. The present analysis underscores the influence of pH effects on the k_cat and K_M kinetic constants of barnase and provides the basic formalism for linking the effective kinetic parameters, which usually depend on the pH, to the theoretical estimates of the true kinetic constants. © 1996 Wiley-Liss, Inc.     

Evolution of regulatory enzymes towards functional simplicity

The potential kinetic complexity of polymeric regulatory enzymes does not seem to be often expressed in nature. Most of these enzymes exhibit in fact a rather simple kinetic behaviour. This functional simplicity is probably the consequence of constraints between rate constants or of blocking of some reaction steps. Functional simplicity is believed to have emerged in the course of neo-Darwinian evolution as a consequence of a trend towards an improved functional efficiency. Functional efficiency may be reached, in polymeric regulatory enzymes, when either of the two sets of conditions are met. The first set of conditions implies the occurrence of the unicity of enzyme conformation in any transition state, a loose coupling between subunits and an exact balance of the driving forces exerted by the enzyme in the forward and backward directions of the catalytic step. This situation results in constraints between rate constants which allow degenerescence of the steady state rate equation. The second set of conditions involves again the unicity of enzyme conformation in any of the transition states, associated with a tight coupling of subunits, and a driving force exerted by the enzyme much strongly in the forward than in the backward direction of the catalytic step. These conditions imply blocking of some reaction steps and again degenerescence of the corresponding rate equation. The most frequent types of quaternary structure and subunit interactions, namely loose coupling between subunits, and tight coupling associated with conservation of at least one symmetry axis, have probably emerged as molecular organizations, which precisely allow both functional efficiency and simplicity to occur. Indeed these situations probably represent the term of two different evolutionary trends. Therefore enzymes that have not reached this state usually exhibit more complex kinetic behaviour. Wavy curves, “bumps” and turning points may be considered as manifestations of the ancestral character of an enzyme.