Structure-activity relationships in engineered proteins: analysis of use of binding energy by linear free energy relationships

The activity of mutant enzymes can be analyzed quantitatively by structure-activity relationships in a manner analogous to Br?nsted or Hammett plots for simple organic reactions. The slopes of such plots, the beta values, indicate for the enzymatic reactions the fraction of the overall binding energy used in stabilizing particular complexes. In particular, information can be derived about the interactions between the enzyme and the transition state. The activities of many mutant tyrosyl-tRNA synthetases fit well simple linear free energy relationships. The formation of enzyme-bound tyrosyl adenylate (E.Tyr-AMP) from enzyme-bound tyrosine and ATP (E.Tyr-ATP) results in an increase in binding energy between the enzyme and the side chain of tyrosine and the ribose ring of ATP. Linear free energy plots of enzymes mutated in these positions give the fraction of the binding energy change that occurs on formation of the transition state for the chemical reaction and the various complexes. It is shown that groups that specifically stabilize the transition state of the reaction are characterized by beta values much greater than 1. This is found for residues that bind the gamma-phosphate of ATP (Thr-40 and His-45) and have previously been postulated to be involved in transition-state stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Linear free energy relationships in enzyme binding interactions studied by protein engineering.

Experiments on mutants of tyrosyl-tRNA synthetase have shown that there can be linear free energy relationships (LFERs) between changes in activation free energies and changes in binding energies when groups are deleted that bind to non-reacting parts of the substrate (Fersht et al., 1986, 1987). It has now been proposed (Straub and Karplus, 1990) that such LFERs can occur for the mutation of hydrogen bonding groups only for the limiting examples of Br?nsted beta of 0, 1 or infinity, and that fractional values of beta are not permissible. The reasoning behind this is that the energy of a hydrogen bond is not linear with distance and the (false) premise that an LFER requires that there is a linear relationship between bond energy and distance. We show from a simple model how LFERs can arise for binding interactions and how they can give fractional values of beta, in accord with experimental evidence. An LFER occurs between binding and catalysis when a set of interactions exists in which each member contributes to the binding energy of the transition state the same fraction of the binding energy it contributes to the products (both relative to the ground state).     

Electrostatic and redox potential effects on the rat of electron-transfer reaction of nicotinamide adenine dinucleotides with 1-substituted 5-ethylphenazines

The effects of redox potential and electric charge on the rate of electron-transfer reaction by a two-electron process were investigated. For electron donors, beta-NADH, beta-NADPH and alpha-NADH were used; they have similar structures but different charges and different redox potentials. For electron acceptors, the following 5-ethylphenazine derivatives were used: 1-(3-carboxypropyloxy)-5-ethylphenazine, 1-(3-ethoxycarbonylpropyloxy)-5-ethylphenazine, and 1-[N-(2-aminoethyl)carbamoylpropyloxy]-5-ethylphenazine. They have similar structures and different charges. Using these donors and acceptors, the potential and the charge effects were estimated separately. In the potential effect, a linear free energy relationship was observed for the change in the redox potential of the donor with a Br?nsted slope of about unity. On the other hand, the slope for the change in the potential of the acceptor was about 0.5. These results show that the potential effect due to electron donors is different from that due to electron acceptors. A linear relationship was also observed between activation free energy and electrostatic force (or potential). The redox potential effect and the electrostatic effect are independent and additive. New theory for the mechanism of electron-transfer reactions is needed to explain these results.     

FTIR spectroscopy of NH3 on acidic and ionotropic alginate aerogels

The acidity of alginate aerogel films has been investigated by infrared spectroscopy of adsorbed NH(3). Supercritical drying of the alginate provided samples with a surface area of several hundred square meters per gram, in which the probe molecule could reach all acidic sites. Free carboxylic groups were studied on acid-gelled alginates and were found to behave as effective Br?nsted acid sites. Ionotropic alginate gels formed by alkaline earth cations presented only the Lewis acidity of the cations. Ionotropic gels formed by transition metal cations presented both Lewis and Br?nsted sites, because of the presence of a fraction of free carboxylic groups. The incomplete salification was correlated to the pH of the gelling solutions.     

Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase

A previously determined crystal structure of the ternary complex of trehalose-6-phosphate synthase identified a putative transition state-like arrangement based on validoxylamine A 6'-O-phosphate and uridine diphosphate in the active site. Here linear free energy relationships confirm that these inhibitors are synergistic transition state mimics, supporting front-face nucleophilic attack involving hydrogen bonding between leaving group and nucleophile. Kinetic isotope effects indicate a highly dissociative oxocarbenium ion-like transition state. Leaving group (18)O effects identified isotopically sensitive bond cleavages and support the existence of a hydrogen bond between the nucleophile and departing group. Br?nsted analysis of nucleophiles and Taft analysis highlight participation of the nucleophile in the transition state, also consistent with a front-face mechanism. Together, these comprehensive, quantitative data substantiate this unusual enzymatic reaction mechanism. Its discovery should prompt useful reassessment of many biocatalysts and their substrates and inhibitors.     

Chemical rescue, multiple ionizable groups, and general acid-base catalysis in the HDV genomic ribozyme

In the ribozyme from the hepatitis delta virus (HDV) genomic strand RNA, a cytosine side chain is proposed to facilitate proton transfer in the transition state of the reaction and, thus, act as a general acid-base catalyst. Mutation of this active-site cytosine (C75) reduced RNA cleavage rates by as much as one million-fold, but addition of exogenous cytosine and certain nucleobase or imidazole analogs can partially rescue activity in these mutants. However, pH-rate profiles for the rescued reactions were bell shaped, and only one leg of the pH-rate curve could be attributed to ionization of the exogenous nucleobase or buffer. When a second potential ionizable nucleobase (C41) was removed, one leg of the bell-shaped curve was eliminated in the chemical-rescue reaction. With this construct, the apparent pK(a) determined from the pH-rate profile correlated with the solution pK(a) of the buffer, and the contribution of the buffer to the rate enhancement could be directly evaluated in a free-energy or Br?nsted plot. The free-energy relationship between the acid dissociation constant of the buffer and the rate constant for cleavage (Br?nsted value, beta, = approximately 0.5) was consistent with a mechanism in which the buffer acted as a general acid-base catalyst. These data support the hypothesis that cytosine 75, in the intact ribozyme, acts as a general acid-base catalyst.     

Experimental charge measurement at leaving oxygen in the bovine ribonuclease A catalyzed cyclization of uridine 3'-phosphate aryl esters

The title esters are demonstrated to be specific substrates of bovine pancreatic ribonuclease A (EC 3.1.27.5). The Br?nsted dependence of kcat/Km at pH 7.50 for the enzyme-catalyzed cyclization versus the pKa of the leaving phenol exhibits two regression lines of almost identical slope for respectively 2-chlorophenols and 2,6-unsubstituted phenols: log kcat/Km = -0.20 pKa ArOH + 5.47 (n = 5, r = 0.957); log kcat/Km = -0.17 pKa ArOH + 5.79 (n = 4, r = 0.965). Comparison of the Br?nsted beta 1g's with that for the standard reaction where imidazole catalyzes the cyclization (beta 1g = -0.59) indicates considerably less development of negative charge on the leaving oxygen in the enzyme case, providing experimental evidence for the hypothesis that electrophilic assistance is involved in catalysis. The existence of two essentially parallel Br?nsted correlations is not reflected in the standard reaction of substrate with imidazole. Modeling studies indicate that the phenyl ring of the substrate can take up a range of positions away from the active site; the presence of ortho chloro substituents considerably restricts the motion of the phenyl leaving group.     

Predicting the rate enhancement of protein complex formation from the electrostatic energy of interaction

The rate of association of proteins is dictated by diffusion, but can be enhanced by favorable electrostatic forces. Here the relationship between the electrostatic energy of interaction, and the kinetics of protein-complex formation was analyzed for the protein pairs of: hirudin-thrombin, acetylcholinesterase-fasciculin and barnase-barstar, and for a panel of point mutants of these proteins. Electrostatic energies of interaction were calculated as the difference between the electrostatic energy of the complex and the sum of the energies of the two individual proteins, using the computer simulation package DelPhi. Calculated electrostatic energies of interaction were compared to experimentally determined rates of association. One kcal/mol of Coulombic interaction energy increased the rate of association by a factor of 2.8, independent of the protein-complex or mutant analyzed. Electrostatic energies of interaction were also determined from the salt dependence of the association rate constant, using the same basic equation as for the theoretical calculation. A Br?nsted analysis of the electrostatic energies of interactions plotted versus experimentally determined ln(rate)s of association shows a linear relation between the two, with a beta value close to 1. This is interpreted as the energy of the transition state varies according to the electrostatic interaction energy, fitting a two state model for the association reaction. Calculating electrostatic rate enhancement from the electrostatic interaction energy can be used as a powerful tool to design protein complexes with altered rates of association and affinities.     

Origin of rate-acceleration in ester hydrolysis with metalloprotease mimics

Mimics of carboxypeptidase A, a prototypical metalloprotease, were synthesized by linking macrocyclicpolyamines to the primary side of beta-cyclodextrin followed by complexing with Zn(II). These enzyme mimics exhibit saturation kinetics in hydrolysis of p-nitrophenyl acetate (PNPA) and enhance the rate of hydrolysis reaction by almost 300-fold. The effective molarities (EM) of the mimics range from 0.2 to 1.9 M. Origin of the rate acceleration was examined: the reactivity of Zn(II) complexes of [12]aneN3 [12]aneN4, and [14]aneN4 for hydrolyzing PNPA increases with increase in basicity of the zinc bound hydroxides [Zn(II)-OH], yielding a linear Br?nsted plot. Free hydroxide fits well on this plot. A similar plot was obtained with the enzyme mimics. The Br?nsted relationships indicate that the Zn(II)-OH in the catalytic systems hydrolyzes the ester by direct nucleophilic attack on the ester carbonyl of cyclodextrin-bound but not Zn(II)-coordinated PNPA.     

Br?nsted-Evans-Polanyi relationships for C–C bond forming and C–C bond breaking reactions in thiamine-catalyzed decarboxylation of 2-keto acids using density functional theory

The concept of generalized enzyme reactions suggests that a wide variety of substrates can undergo enzymatic transformations, including those whose biotransformation has not yet been realized. The use of quantum chemistry to evaluate kinetic feasibility is an attractive approach to identify enzymes for the proposed transformation. However, the sheer number of novel transformations that can be generated makes this impractical as a screening approach. Therefore, it is essential to develop structure/activity relationships based on quantities that are more efficient to calculate. In this work, we propose a structure/activity relationship based on the free energy of binding or reaction of non-native substrates to evaluate the catalysis relative to that of native substrates. While Br?nsted-Evans-Polanyi (BEP) relationships such as that proposed here have found broad application in heterogeneous catalysis, their extension to enzymatic catalysis is limited. We report here on density functional theory (DFT) studies for C–C bond formation and C–C bond cleavage associated with the decarboxylation of six 2-keto acids by a thiamine-containing enzyme (EC 1.2.7.1) and demonstrate a linear relationship between the free energy of reaction and the activation barrier. We then applied this relationship to predict the activation barriers of 17 chemically similar novel reactions. These calculations reveal that there is a clear correlation between the free energy of formation of the transition state and the free energy of the reaction, suggesting that this method can be further extended to predict the kinetics of novel reactions through our computational framework for discovery of novel biochemical transformations.     

Formation and stability of the enolates of N-protonated proline methyl ester and proline zwitterion in aqueous solution: a nonenzymatic model for the first step in the racemization of proline catalyzed by proline racemase

Rate constants for the hydrolysis of L-proline methyl ester to form proline and methanol in D(2)O buffered at neutral pD and 25 degrees C and the deuterium enrichment of the proline product determined by electrospray ionization mass spectrometry are reported. The data give k(DO) = 5.3 +/- 0.5 M(-1) s(-1) as the second-order rate constant for carbon deprotonation of N-protonated proline methyl ester by deuterioxide ion in D(2)O at 25 degrees C and I = 1.0 (KCl). The data provide good estimates of carbon acidities of pK(a) = 21 for N-protonated proline methyl ester and pK(a) = 29 for proline zwitterion in water and of the second-order rate constant k(HO) = 4.5 x 10(-5) M(-1) s(-1) for carbon deprotonation of proline zwitterion by hydroxide ion at 25 degrees C. There is no detectable acceleration of the deprotonation of N-protonated proline methyl ester by the Br?nsted base 3-quinuclidinone in water, and it is not clear that such Br?nsted catalysis would make a significant contribution to the rate acceleration for deprotonation of bound proline at proline racemase. A comparison of the first-order rate constants k(HO)[HO(-)] = 4.5 x 10(-11) s(-1) for deprotonation of free proline zwitterion in water at pH 8 and k(cat) = 2600 s(-1) for deprotonation of proline bound to the active site of proline racemase at pH 8 shows that the enzymatic rate acceleration for proline racemase is ca. 10(13)-fold. This corresponds to a 19 kcal/mol stabilization of the transition state for deprotonation of the enzyme-bound carbon acid substrate by interaction with the protein catalyst. It is suggested that (1) much of the rate acceleration of the enzymatic over the nonenzymatic reaction in water may result from transfer of the substrate proline zwitterion from the polar solvent water to a nonpolar enzyme active site and (2) the use of thiol anions rather than oxygen anions as Br?nsted bases at this putative nonpolar enzyme active site may be favored, because of the smaller energetic price for desolvation of thiol anions than for desolvation of the more strongly solvated oxygen anions.     

Structural determinants influencing the reaction of cysteine-containing peptides with palmitoyl-coenzyme A and other thioesters

Non-enzymatic thioesterification of specific cysteinyl peptides with fatty acyl-CoA has been previously demonstrated in both liposomes and aqueous medium. To identify the molecular basis for the differential reactivity of polypeptides in aqueous solutions, 26 synthetic cysteinyl peptides encompassing the palmitoylation sites of well known proteins (protein zero, proteolipid protein, beta-adrenergic receptor, p21(K-ras), transferrin receptor, CD-4 and SNAP-25) and six small thiol compounds were incubated separately with [3H]palmitoyl-CoA, [14C]acetyl-CoA and p-nitrophenyl thioacetate (NPTA). For each peptide, both the observed reaction rate constant at pH 7.5 and the pH-independent rate constant (k(2)) were calculated, and reactivity of the attacking sulfhydryl group was characterized using the Br?nsted equation (log k(2)=beta(nuc) pK(a)+C). In general, peptides bearing basic and aromatic amino acid residues showed the lowest thiol pK(a)s, and consequently displayed the highest acylation rates. Reaction with palmitoyl-CoA was complicated to analyze because of the variable partition of peptides in the acyl chain donor/detergent micelles. In contrast, a linear Br?nsted relationship was found for the reaction of the peptides with the water-soluble acetyl-CoA (beta(nuc)=0.59). A similar beta(nuc) value was obtained with the neutral NPTA, indicating that electronic effects other than those responsible for the acid-base properties of the thiol are less important. Thus, the concentration of the thiolate anion appears to be the major factor influencing the rate of the nucleophilic substitution reaction. These findings and the fact that the acylation sites in most proteins are surrounded by basic amino acids may partially explain the specificity of non-enzymatic palmitoylation regarding the acceptor sequences.     

A nucleophilic catalysis step is involved in the hydrolysis of aryl phosphate monoesters by human CT acylphosphatase

Acylphosphatase, one of the smallest enzymes, is expressed in all organisms. It displays hydrolytic activity on acyl phosphates, nucleoside di- and triphosphates, aryl phosphate monoesters, and polynucleotides, with acyl phosphates being the most specific substrates in vitro. The mechanism of catalysis for human acylphosphatase (the organ-common type isoenzyme) was investigated using both aryl phosphate monoesters and acyl phosphates as substrates. The enzyme is able to catalyze phosphotransfer from p-nitrophenyl phosphate to glycerol (but not from benzoyl phosphate to glycerol), as well as the inorganic phosphate-H(2)18O oxygen exchange reaction in the absence of carboxylic acids or phenols. In short, our findings point to two different catalytic pathways for aryl phosphate monoesters and acyl phosphates. In particular, in the aryl phosphate monoester hydrolysis pathway, an enzyme-phosphate covalent intermediate is formed, whereas the hydrolysis of acyl phosphates seems a more simple process in which the Michaelis complex is attacked directly by a water molecule generating the reaction products. The formation of an enzyme-phosphate covalent complex is consistent with the experiments of isotope exchange and transphosphorylation from substrates to glycerol, as well as with the measurements of the Br?nsted free energy relationships using a panel of aryl phosphates with different structures. His-25 involvement in the formation of the enzyme-phosphate covalent complex during the hydrolysis of aryl phosphate monoesters finds significant confirmation in experiments performed with the H25Q mutated enzyme.     

Dehydration polycondensation of dicarboxylic acids and diols using sublimating strong brønsted acids

We investigated catalytic activities of strong br?nsted acids for dehydration polycondensations of dicarboxylic acids and diols, which were carried out at low temperature (<100 °C) under reduced pressure (0.3-3 mmHg). Strong Br?nsted acids, bis(perfluoroalkanesulfonyl)imide and perfluoroalkanesulfonic acid, showed higher activity than p-toluenesulfonic acid or rare-earth catalysts at 60 °C. In particular, bis(nonafluorobutanesulfonyl)imide (Nf(2)NH) showed the highest activity to synthesize not only aliphatic polyester (M(n) > 19000) but also aromatic polyester (M(n) > 7000). The used Nf(2)NH was sublimated from the reaction flask during polycondensation, and the sublimate, Nf(2)NH, was extra pure so that we can reuse the catalyst without loss of the activity in the dehydration polycondensations.     

Triflimide (HNTf2)-catalyzed aldehyde cross-aldol reaction using "super silyl" enol ethers

The synthesis of the acetaldehyde-derived tris(trimethylsilyl)silyl (super silyl) enol ether is described, as well as its use in the high-yielding aldehyde cross-aldol reaction. The super silyl enol ether shows unprecedented reactivity in giving the 1:1 adduct in very high yield. This reaction is catalyzed by 0.05 mol% of the Br?nsted acid triflimide (HNTf2) and is complete within 15 min, making the protocol very attractive for large-scale synthesis.     

Mutagenesis of the conserved active-site tyrosine changes a retaining sialidase into an inverting sialidase

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase changes the mechanism of catalysis from retention of anomeric configuration to an unprecedented inverting mechanism in which water efficiently functions as the nucleophile. Three mutants, Y370A, Y370D, and Y370G, were produced recombinantly in Escherichia coli, and all are catalytically active against the activated substrate 4-methylumbelliferyl alpha-D-N-acetylneuraminide. The Y370D mutant was also shown to catalyze the hydrolysis of natural substrate analogues such as 3'-sialyllactose. A comparison of the pH-rate profiles for the wild-type and the Y370D mutant sialidase reveals no major differences, although with respect to the kinetic term k(cat)/K(m), an ionized form of the aspartate-370 enzyme is catalytically compromised. For the wild-type enzyme, the value of the Br?nsted parameter beta(lg) on k(cat) is 0.02 +/- 0.03, while for the Y370D mutant sialidase beta(lg) = -0.55 +/- 0.03 for the substrates with bad leaving groups. Thus, for the wild-type enzyme, a nonchemical step(s) is rate-limiting, but for the tyrosine mutant cleavage of the glycosidic C-O bond is rate-determining. The Br?nsted slopes derived for the kinetic parameter k(cat)/K(m) display a similar trend (beta(lg) -0.30 +/- 0.04 and -0.74 +/- 0.04 for the wild-type and Y370D, respectively). These results reveal that the tyrosine residue lowers the activation free energy for cleavage of 6'-sialyllactose, a natural substrate analogue, by more than 24.9 kJ mol(-1). Evidence is presented that the mutant sialidases operate by a dissociative mechanism, and the wild-type enzyme operates by a concerted mechanism.     

Beta-glucosidase: substrate, solvent, and viscosity variation as probes of the rate-limiting steps

The second-order rate constants (kcat/Km) for the beta-glucosidase-catalyzed hydrolysis of aryl beta-D-glucopyranosides show a bell-shaped dependence of pH. The pKas that characterize this dependence are 4.4 (delta Hion approximately equal to 0) and 6.7 (delta Hion approximately equal to 0). In D2O these pKas are increased by 0.5 (+/- 0.1) unit, but there is no solvent isotope effect on the pH-independent second-order rate constant. Nath and Rydon [Nath, R. L., & Rydon, H. N. (1954) Biochem. J. 57, 1-10] examined the kinetics of the beta-glucosidase-catalyzed hydrolysis of a series of substituted phenyl glucosides. We have extended this study to include glucosides with phenol leaving groups of pKa less than 7. Br?nsted plots for this extended series were nonlinear for both kcat/Km and kcat. Br?nsted coefficients for those compounds with leaving groups of pKa greater than 7 (for kcat/Km) or pKa greater than 8.5 (for kcat) were nearly equal to -1.0, indicating substantial negative charge buildup on the leaving group in the transition state. The nonlinearity indicates an intermediate in the reaction. This was confirmed by partitioning experiments in the presence of methanol as a competing glucose acceptor. A constant product ratio, [methyl glucoside]/[glucose], was found with aryl glucoside substrates varying over 16,000-fold in reactivity (V/K), indicative of a common intermediate. Viscosity variation (in sucrose-containing buffers) was used to probe the extent to which the beta-glucosidase reactions are diffusion-controlled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Highly efficient Brønsted acidic ionic liquid-based catalysts for biodiesel synthesis from vegetable oils

Biodiesel has been produced by transesterification of canola oil with methanol in the presence of highly Br?nsted acidic ionic liquids based on 1-benzyl-1H-benzimidazole, and the effect of reaction temperature, type and amount of catalyst, molar ratio and reaction time investigated. The results show that the 4B ionic liquid has the highest catalytic activity and best recyclability under the optimised reaction conditions. Thus, this ionic liquid is able to catalyze the transesterification of canola oil to its methyl esters in 5 h with yields of more than 95%. Density functional calculations (B3LYP), using the 6-311G basis set, have been performed to have a better understanding on the reactivity of these catalysts. The catalytic activity of 4B for the transesterification of other vegetable oils and alcohols has also been studied.     

Mechanistic requirements for the efficient enzyme-catalyzed hydrolysis of thiosialosides

The sialidase from Micromonospora viridifaciens has been found to catalyze the hydrolysis of aryl 2-thio-alpha-D-sialosides with remarkable efficiency: the first- and second-order rate constants, kcat and kcat/Km, for the enzyme-catalyzed hydrolysis of PNP-S-NeuAc are 196 +/- 5 s(-1) and (6.7 +/- 0.7) x 10(5) M(-1) s(-1), respectively. A reagent panel of eight aryl 2-thio-alpha-D-sialosides was synthesized and used to probe the mechanism for the M. viridifaciens sialidase-catalyzed hydrolysis reaction. In the case of the wild-type enzyme, the derived Br?nsted parameters (beta(lg)) on kcat and kcat/Km are -0.83 +/- 0.11 and -1.27 +/- 0.17 for substrates with thiophenoxide leaving groups of pKa values > or = 4.5. For the general-acid mutant, D92G, the derived beta(lg) value on kcat for the same set of leaving groups is -0.82 +/- 0.12. When the conjugate acid of the departing thiophenol was < or = 4.5, the derived Br?nsted slopes for both the wild-type and the D92G mutant sialidase were close to zero. In contrast, the nucleophilic mutant, Y370G, did not display a similar break in the Br?nsted plots, and the corresponding values for beta(lg), for the three most reactive aryl 2-thiosialosides, on kcat and kcat/Km are -0.76 +/- 0.28 and -0.84 +/- 0.04, respectively. Thus, for the Y370G enzyme glycosidic C-S bond cleavage is rate-determining for both kcat and kcat/Km, whereas, for both the wild-type and D92G mutant enzymes, the presented data are consistent with a change in rate-determining step from glycosidic C-S bond cleavage for substrates in which the pKa of the conjugate acid of the leaving group is > or = 4.5, to either deglycosylation (kcat) or a conformational change that occurs prior to C-S bond cleavage (kcat/Km) for the most activated leaving groups. Thus, the enzyme-catalyzed hydrolysis of 2-thiosialosides is strongly catalyzed by the nucleophilic tyrosine residue, yet the C-S bond cleavage does not require the conserved aspartic acid residue (D92) to act as a general-acid catalyst.