Free energy perturbation (FEP) simulation on the transition states of cocaine hydrolysis catalyzed by human butyrylcholinesterase and its mutants

A novel computational protocol based on free energy perturbation (FEP) simulations on both the free enzyme and transition state structures has been developed and tested to predict the mutation-caused shift of the free energy change from the free enzyme to the rate-determining transition state for human butyrylcholinesterase (BChE)-catalyzed hydrolysis of (-)-cocaine. The calculated shift, denoted by DeltaDeltaG(1 --> 2), of such kind of free energy change determines the catalytic efficiency (kcat/KM) change caused by the simulated mutation transforming enzyme 1 to enzyme 2. By using the FEP-based computational protocol, the DeltaDeltaG(1 --> 2) values for the mutations A328W/Y332A --> A328W/Y332G and A328W/Y332G --> A328W/Y332G/A199S were calculated to be -0.22 and -1.94 kcal/mol, respectively. The calculated DeltaDeltaG(1 --> 2) values predict that the change from the A328W/Y332A mutant to the A328W/Y332G mutant should slightly improve the catalytic efficiency and that the change from the A328W/Y332G mutant to the A328W/Y332G/A199S mutant should significantly improve the catalytic efficiency of the enzyme for the (-)-cocaine hydrolysis. The predicted catalytic efficiency increases are supported by the experimental data showing that kcat/KM = 8.5 x 10(6), 1.4 x 10(7), and 7.2 x 10(7) min(-1) M(-1) for the A328W/Y332A, A328W/Y332G, and A328W/Y332G/A199S mutants, respectively. The qualitative agreement between the computational and experimental data suggests that the FEP simulations may provide a promising protocol for rational design of high-activity mutants of an enzyme. The general computational strategy of the FEP simulation on a transition state can be used to study the effects of a mutation on the activation free energy for any enzymatic reaction.     

A bioorganometallic approach for the electrochemical detection of proteins: a study on the interaction of ferrocene-peptide conjugates with papain in solution and on Au surfaces

In this paper, a new bioorganometallic approach for the detection of proteins using surface-bound ferrocene-peptide conjugates is presented. Specifically, a series of peptide conjugates of 1'-aminoferrocene-1-carboxylic acid (ferrocene amino acid, Fca) is synthesized: Boc-Fca-Gly-Gly-Tyr(Bzl)-Arg(NO2)-OMe (2), Thc-Fca-Gly-Gly-Tyr(Bzl)-Arg(NO2)-OMe (3), Thc-Fca-Gly-Gly-Tyr(Bzl)-Arg(NO2)-OH (4), Boc-Fca-Gly-Gly-Arg(Mtr)-Tyr-OMe (7), Thc-Fca-Gly-Gly-Arg(Mtr)-Tyr-OMe (8), Thc-Fca-Gly-Gly-Arg(Mtr)-Tyr-OH (9), Thc-Fca-Gly-Gly-Arg-Tyr-OH (10). The peptide is conjugated to the C-terminal side of Fca and compounds 4, 7-10 possess a thiostic acid linked to the N-terminal side of Fca in order to facilitate formation of thin films on gold substrates. Competitive inhibition towards papain was determined for Thc-Fca-Gly-Gly-Tyr(Bzl)-Arg(NO2)-OH (4), Thc-Fca-Gly-Gly-Arg(Mtr)-Tyr-OH (9) and Thc-Fca-Gly-Gly-Arg-Tyr-OH (10). The binding interaction between the peptide modified substrates and papain enzyme was studied using real-time surface plasmon resonance (SPR) imaging. Peptide 10 showed the strongest binding affinity to papain. Adsorption/desorption rate constants were ka = 1.75+/-0.05 x 10(5) M(-1) s(-1) and kd = 2.90 +/- 0.05 x 10(-2) s(-1). Interactions of papain with Fca-peptide 10 were investigated by cyclic voltammetry. The interaction results were also verified by measuring the electrochemical response of the peptide-papain interaction as function of increasing enzyme concentration. A linear relationship was observed for papain concentration of up to 80 nM. Shifting to higher potentials as well as decrease in the overall signal intensity was observed. The detection limit was 4 x 10(-9) M.     

Remarkable supramolecular catalysis of glycoside hydrolysis by a cyclodextrin cyanohydrin

(6AR,6DR)-6A,6D-di-C-cyano-beta-cyclodextrin (3) was synthesized and shown to catalyze hydrolysis of nitrophenyl glycosides with the reaction following Michaelis-Menten kinetics. At pH 7.4 and 25 degrees C, hydrolysis of 4-nitrophenyl-beta-glucopyranoside (2) was catalyzed with KM = 15 mM, kcat = 8.2 x 10-6 s-1, and kcat/kuncat = 1217. Catalysis was observed with concentration of 3 as low as 10 muM. Hydrolysis of the corresponding alpha-glucoside, alpha-galactoside, alpha-mannoside, and 2-nitrophenyl-beta-galactoside was also catalyzed by 3, with kcat/kuncat ranging from 283 to 2147. A series of analogues of 3 was prepared and investigated for catalysis of the hydrolysis of 2: (6AR,6DR)-6A,6D-di-C-propyl-beta-cyclodextrin (9) was not catalytic, while 6A,6D-di-C-cyano-6A,6D-dideoxy-beta-cyclodextrin (12) had a low catalytic activity (kcat/kuncat = 4). A kcat/kuncat = 48 was found for 6A,6D-dialdehydo-beta-cyclodextrin dihydrate (11). It was proposed that 3 acts by general acid catalysis on the bound substrate.     

A peptide dendrimer enzyme model with a single catalytic site at the core

Catalytic esterase peptide dendrimers with a core active site were discovered by functional screening of a 65,536-member combinatorial library of third-generation peptide dendrimers using fluorogenic 1-acyloxypyrene-3,6,8-trisulfonates as substrates. In the best catalyst, RMG3, ((AcTyrThr)(8)(DapTrpGly)(4)(DapArgSerGly)(2)DapHisSerNH2), ester hydrolysis is catalyzed by a single catalytic histidine residue at the dendrimer core. A pair of arginine residues in the first-generation branch assists substrate binding. The catalytic proficiency of dendrimer RMG3 (kcat/KM = 860 M(-1) min(-1) at pH 6.9) per catalytic site is comparable to that of the multivalent esterase dendrimer A3 ((AcHisSer)(8)(DapHisSer)(4)(DapHisSer)2DapHisSerNH2) which has fifteen histidines and five catalytic sites (Delort, E. et al. J. Am. Chem. Soc. 2004, 126, 15642-15643). Remarkably, catalysis in the single site dendrimer RMG3 is enhanced by the outer dendritic branches consisting of aromatic amino acids. These interactions take place in a relatively compact conformation similar to a molten globule protein as demonstrated by diffusion NMR. In another dendrimer, HG3 ((AcIlePro)(8)(DapIleThr)(4)(DapHisAla)(2)DapHisLeuNH2) by contrast, catalysis by a core of three histidine residues is unaffected by the outer dendritic layers. Dendrimer HG3 or its core HG1 exhibit comparable activity to the first-generation dendrimer A1 ((AcHisSer)(2)DapHisSerNH2). The compactness of dendrimer HG3 in solution is close to that a denatured peptide. These experiments document the first esterase peptide dendrimer enzyme models with a single catalytic site and suggest a possible relationship between packing and catalysis in these systems.     

Four orders of magnitude rate increase in artificial enzyme-catalyzed aryl glycoside hydrolysis

[reaction: see text] (6AR,6DR)-6A,6D-Di-C-cyano-beta-cyclodextrin (1) and 6A,6D-di-C-cyano-alpha-cyclodextrin (2) were synthesized and shown to catalyze hydrolysis of aryl glycosides into glucose and phenol with a reaction following Michaelis-Menten kinetics. At pH 8.0 and 59 degrees C hydrolysis of 4-nitrophenyl alpha-glucopyranoside was catalyzed by 1 with KM = 10.5 +/- 1.5 mM, kcat = 1.42(+/-0.09) x 10(-4) s(-1), and kcat/kuncat = 7922. Catalysis was observed with a concentration of 1 as low as 10 microM. Hydrolysis of the other aryl glycosides containing stereochemical variation in the sugar-moiety and 4-nitro-, 2-nitro-, 2-aldehydo-, and 2,4-dinitro- were also catalyzed by 1 and 2 with kcat/kuncat ranging from 4 to 7100. Hydrolysis of a phenyl beta-d-glucoside or the thioglycoside tolylthio beta-D-glucoside was also catalyzed. From a series of prepared analogues of 1 it was found that the catalysis was associated with the hydroxyl groups alpha to the nitril groups. The monocyanohydrin 6-C-cyano-beta-cyclodextrin (3) was also found to catalyze the hydrolysis of 4-nitrophenyl beta-glucopyranoside with kcat/kuncat = 1356. It was proposed that the cyclodextrin cyanohydrins 1-3 catalyze the hydrolysis by general acid catalysis on the bound substrate.     

Combinatorial Biomimetic Chemistry: Parallel Synthesis of a Small Library of β‐Hairpin Mimetics Based on Loop III from Human Platelet‐Derived Growth Factor B

Combinatorial diversity in hypervariable β‐hairpin loops is exploited by the immune system to select binding sites on antibodies for a wide variety of different protein antigens. In a first step towards mimicking this strategy in vitro, for the selection of novel protein ligands, an approach is described here for the parallel synthesis of small libraries of conformationally defined β‐hairpin protein epitope mimetics. Starting from a protruding hairpin loop in platelet‐derived growth factor B (PDGF‐B), 8 and 12 residues were first transplanted from the protein to a D ‐Pro‐L ‐Pro template, to afford the cyclic peptide‐loop mimetics 1 and 2 , respectively. NMR and MD studies in aqueous solution show that both mimetics populate conformations which closely mimic the β‐hairpin in the crystal structure of the native protein (Fig. 5). Based on 1 as a scaffold, a library of 24 mimetics was synthesized in which the four residues at the tip of the loop (VRKK) were held constant, and flanking residues at positions 1, 2, 7, and 8 in the hairpin were varied (Fig. 7). The library was prepared by parallel synthesis in a two‐stage solid‐phase assembly/solution‐phase cyclization process. The products were analyzed by MS, NMR, and CD. 2D‐NOESY revealed for most library members characteristic long‐range NOEs that show that the hairpin conformation is stably maintained. The results suggest that this approach may be useful for the synthesis of much larger libraries of peptide and protein mimetics based on a β‐hairpin scaffold.     

Reevaluation of the kinetics of polynuclear mimics for manganese catalases

Considerable effort has been expended in order to understand the mechanism of manganese catalases and to develop functional mimics for these enzymes. For many years, the most efficient reactivity mimic was [MnIVsalpn(mu-O)]2 [H2salpn = 1,3-bis(salicylideneiminato)propane], a compound that cycles between the MnIV2 and MnIII2 oxidation levels instead of the MnII2 and MnIII2 oxidation states used by the enzyme, with kcat = 250 s(-1) and kcat/KM = 1000 M(-1) s(-1). Recently, a truly exceptional high value of kcat was reported for the complex [Mn(bpia)(mu-OAc)]22+ [bpia = bis(picolyl)(N-methylimidazol-2-yl)amine]. On the basis of a calculated kcat value of 1100 s(-1) and an efficiency kcat/KM of 34 000 M(-1) s(-1), this complex has been suggested to represent a significant breakthrough in catalytic efficiencies of manganese catalase mimics. However, a plot of ri/[cat]T vs [H2O2]0, where the saturation value approaches 1.5 s(-1), is inconsistent with the 1100 s(-1) value tabulated for kcat. Similar discrepancies are observed for two other families of manganese complexes containing either a Mn2(mu-OPh)22+ core and different substituted tripodal ligands or complexes of methyl and ethyl salicylimidate, with an Mn2(mu-OPh)24+ core. Reevaluation of the kinetic parameters for these three systems reveals that the originally reported values were overestimated by a factor of approximately 1000 for both kcat and kcat/KM. We discuss the origin of the discrepancy between the previously published kinetic parameters and the newly derived values. Furthermore, we provide a short analysis of the existing manganese catalase mimics in an effort to provide sound directions for future investigations in this field.     

Manganese-substituted carbonic anhydrase as a new peroxidase

Carbonic anhydrase is a zinc metalloenzyme that catalyzes the hydration of carbon dioxide to bicarbonate. Replacing the active-site zinc with manganese yielded manganese-substituted carbonic anhydrase (CA[Mn]), which shows peroxidase activity with a bicarbonate-dependent mechanism. In the presence of bicarbonate and hydrogen peroxide, (CA[Mn]) catalyzed the efficient oxidation of o-dianisidine with kcat/KM=1.4 x 10(6) m(-1) s(-1), which is comparable to that for horseradish peroxidase, kcat/KM=57 x 10(6) m(-1) s(-1). CA[Mn] also catalyzed the moderately enantioselective epoxidation of olefins to epoxides (E=5 for p-chlorostyrene) in the presence of an amino-alcohol buffer, such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES). This enantioselectivity is similar to that for natural heme-based peroxidases, but has the advantage that CA[Mn] avoids the formation of aldehyde side products. CA[Mn] degrades during the epoxidation limiting the yield of the epoxidations to <12 %. Replacement of active-site residues Asn62, His64, Asn67, Gln92, or Thr200 with alanine by site-directed mutagenesis decreased the enantioselectivity demonstrating that the active site controls the enantioselectivity of the epoxidation.     

Epothilone C macrolactonization and hydrolysis are catalyzed by the isolated thioesterase domain of epothilone polyketide synthase

Epothilone C is produced by the combined action of one nonribosomal peptide synthetase (NRPS) and nine polyketide synthase (PKS) modules in a multienzyme system. The final step in the biosynthesis is the thioesterase (TE)-catalyzed cyclorelease of epothilone from the EpoF protein. It has been unclear whether isolated PKS TE domains could exhibit macrolactonization activity. Here we demonstrate that the excised epothilone TE domain can catalyze the efficient cyclization of the N-acetylcysteamine thioester of seco-epothilone C to generate epothilone C (kcat/KM = 0.41 +/- 0.03 min-1 mM-1). The TE domain also catalyzes the hydrolysis of both the N-acetylcysteamine thioester of seco-epothilone C (kcat = 0.087 +/- 0.005 min-1, KM = 291 +/- 53 muM) and that of the epothilone C (kcat = 0.67 +/- 0.01 min-1, KM = 117 +/- 5 muM) to form seco-epothilone C.     

A gate mechanism indicated in the selectivity filter of the potassium channel KscA

Classical molecular dynamics (MD) and non-equilibrium steered molecular dynamics (SMD) simulations were performed on the molecular structure of the potassium channel KcsA using the GROMOS 87 force fields. Our simulations focused on mechanistic and dynamic properties of the permeation of potassium ions through the selectivity filter of the channel. According to the SMD simulations a concerted movement of ions inside the selectivity filter from the cavity to extracellular side depends on the conformation of the peptide linkage between Val76 and Gly77 residues in one subunit of the channel. In SMD simulations, if the carbonyl oxygen of Val76 is positioned toward the ion bound at the S3 site (gate-opened conformation) the net flux of ions through the filter is observed. When the carbonyl oxygen leaped out from the filter (gate-closed conformation), ions were blocked at the S3 site and no flux occurred. A reorientation of the Thr75-Val76 linkage indicated by the CHARMM-based MD simulations performed Berneche and Roux [(2005) Structure 13:591–600; (2000) Biophys J 78:2900–2917] as a concomitant process of the Val76-Gly77 conformational interconversion was not observed in our GROMOS-based MD simulations.     

Substrate specificity of prostate-specific antigen (PSA)

Background: The serine protease prostate-specific antigen (PSA) is a useful clinical marker for prostatic malignancy. PSA is a member of the kallikrein subgroup of the (chymo)trypsin serine protease family, but differs from the prototypical member of this subgroup, tissue kallikrein, in possessing a specificity more similar to that of chymotrypsin than trypsin. We report the use of two strategies, substrate phage display and iterative optimization of natural cleavage sites, to identify labile sequences for PSA cleavage.Results: Iterative optimization and substrate phage display converged on the amino-acid sequence SS(Y/F)YIS(G/S) as preferred subsite occupancy for PSA. These sequences were cleaved by PSA with catalytic efficiencies as high as 220–3100 M⁻¹ s⁻¹, compared with values of 2–46 M⁻¹ s⁻¹ for peptides containing likely physiological target sequences of PSA from the protein semenogelin. Substrate residues that bind to secondary (non-S1) subsites have a critical role in defining labile substrates and can even cause otherwise disfavored amino acids to bind in the primary specificity (S1) pocket.Conclusions: The importance of secondary subsites in defining both the specificity and efficiency of cleavage suggests that substrate recognition by PSA is mediated by an extended binding site. Elucidation of preferred subsite occupancy allowed refinement of the structural model of PSA and should facilitate the development of more sensitive activity-based assays and the design of potent inhibitors.     

Simultaneous analysis of ultrafast fluorescence decays of FMN binding protein and its mutated proteins by molecular dynamic simulation and electron transfer theory

Ultrafast fluorescence decays of FMN binding proteins (FBP) from Desulfovibrio vulgaris (Miyazaki F) were analyzed with an electron transfer (ET) theory by Kakitani and Mataga (KM theory). Time-dependent distances among isoalloxazine (Iso) and Trp-32, Tyr-35, and Trp-106 in wild-type FBP (WT), among Iso and Tyr-32, Tyr-35, and Trp-106 in W32Y (Trp-32 was replaced by Tyr-32), and among Iso and Tyr-35 and Trp-106 in W32A (Trp-32 was replaced by Ala-32) were determined by molecular dynamic simulation (MD). Electrostatic energies between Iso anion and all other ionic groups, between Trp-32 cation and all other ionic groups, and between Tyr-32 cation and all other ionic groups were calculated in WT, W32Y, and W32A, from the MD coordinates. ET parameters contained in KM theory, such as frequency (nu 0), a coefficient of the ET process (beta), a critical distance of the ET process ( R 0), standard free energy related to the electron affinity of the excited Iso ( G Iso (0)), and the static dielectric constant in FBP species (epsilon 0), were determined with and without inclusion of the electrostatic energy, so as to fit the calculated fluorescence decays with the observed decays of all FBP species, by a nonlinear least-squares method according to the Marquardt algorithm. In the analyses the parameters, nu 0, beta, and R 0 were determined separately between Trp residues and Tyr residues among all FBP species. Calculated fluorescence intensities with the inclusion of the electrostatic energy fit quite well with the observed ones of all WT, W32Y, and W32A.     

Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst

The self-sufficient cytochrome P450 BM-3 enzyme from Bacillus megaterium catalyzes subterminal hydroxylation of saturated long-chain fatty acids and structurally related compounds. Since the primary structure of P450 BM-3 is homologous to that of mammalian P450 type II, it represents an excellent model for this family of enzymes. During studies on the directed evolution of P450 BM-3 into a medium-chain fatty-acid hydroxylase, several mutants, in particular the triple mutant Phe87Val, Leu188Gln, Ala74Gly, were observed to hydroxylate indole, producing indigo and indirubin at a catalytic efficiency of 1365 M(-1)s(-1) (kcat=2.73 s(-1) and Km=2.0 mM). Both products were unequivocally characterized by NMR and MS analysis. Wild-type P450 BM-3 is incapable to hydroxylate indole. These results demonstrate that an enzyme can be engineered to catalyze the transformation of substrates with structures widely divergent from those of its native substrate.     

Synthesis and activity of histidine-containing catalytic peptide dendrimers

Peptide dendrimers built by iteration of the diamino acid dendron Dap-His-Ser (His = histidine, Ser = Serine, Dap = diamino propionic acid) display a strong positive dendritic effect for the catalytic hydrolysis of 8-acyloxypyrene 1,3,6-trisulfonates, which proceeds with enzyme-like kinetics in aqueous medium (Delort, E.; Darbre, T.; Reymond, J.-L. J. Am. Chem. Soc. 2004, 126, 15642-3). Thirty-two mutants of the original third generation dendrimer A3 ((Ac-His-Ser)8(Dap-His-Ser)4(Dap-His-Ser)2Dap-His-Ser-NH2) were prepared by manual synthesis or by automated synthesis with use of a Chemspeed PSW1100 peptide synthesizer. Dendrimer catalysis was specific for 8-acyloxypyrene 1,3,6-trisulfonates, and there was no activity with other types of esters. While dendrimers with hydrophobic residues at the core and histidine residues at the surface only showed weak activity, exchanging serine residues in dendrimer A3 against alanine (A3A), beta-alanine (A3B), or threonine (A3C) improved catalytic efficiency. Substrate binding was correlated with the total number of histidines per dendrimer, with an average of three histidines per substrate binding site. The catalytic rate constant kcat depended on the placement of histidines within the dendrimers and the nature of the other amino acid residues. The fastest catalyst was the threonine mutant A3C ((Ac-His-Thr)8(Dap-His-Thr)4(Dap-His-Thr)2Dap-His-Thr-NH2), with kcat = 1.3 min(-1), kcat/k(uncat) = 90'000, KM = 160 microM for 8-bytyryloxypyrene 1,3,6-trisulfonate, corresponding to a rate acceleration of 18'000 per catalytic site and a 5-fold improvement over the original sequence A3.     

Using trimethylamine dehydrogenase in an enzyme linked amperometric electrode. Part 1. Wild-type enzyme redox mediation

An amperometric enzyme electrode was studied based on the wild-type protein trimethylamine dehydrogenase (TMADH), which catalyses the oxidative N-demethylation of trimethylamine to produce dimethylamine and formaldehyde. Ferrocene derivatives were investigated electrochemically, as free diffusing electron acceptors for recycling of the prosthetic groups of the immobilised enzyme. Ferricinium had the highest rates but, inhibited the enzyme, possibly as a result of a conformational change initiated at the Val-344 residue where it binds close to the 4Fe-4S cluster, interrupting the electron transfer between flavin mononucleotide (FMN) and 4Fe-4S by changing the redox potential of one or both of the prosthetic groups. (Dimethylamino)methylene ferrocene (DMAMFe) (k(s) = 0.93 x 10(5) M(-1) s(-1)) did not show inhibition and was used as a comparison for steady-state characterisation. The sensor response was studied over the pH range 6.0-1.0. Plots of kcat/KM revealed two ionisations with pKa values of 7.5 and 10. The pKa of 10 was attributed to the ionisation of the secondary amine in DMAMFe, whereas the pKa of 7.5 was thought to reflect the ionisations of the intramolecular electron pathway. A TMADH/DMAMFe amperometric enzyme electrode was successfully used for the determination of TMA in different fish samples (detection limit: 2 mg TMA-N per lOOg wet fish muscle). The obtained results compared well with a reference method based on picric acid.     

Role of Tyr-435 of <Emphasis Type="Italic">Vibrio harveyi</Emphasis> Chitinase A in Chitin Utilization

Vibrio harveyi chitinase A or VhChiA (EC.3.2.1.14) is a member of GH-18 chitinases that catalyzes chitin degradation from marine biomaterials. Our earlier structural data of VhChiA suggested that Tyr-435 marks the ending of subsite +2 and may influence binding of the interacting substrate at the aglycone binding sites. This study reports the effects of Tyr-435 using site-directed mutagenesis technique. Mutation of Tyr-435 to Ala (mutant Y435A) enhanced both binding and catalytic efficiency of VhChiA, whereas substitution of Tyr-435 to Trp (mutant Y435W) lessened the ability of the enzyme to bind and hydrolyze chitin substrates. The increased activity of Y435A can be explained by partial removal of a steric clash around subsite (+2), thereby allowing a chitin chain to move beyond or to access the enzyme’s active site from the aglycone side more straightforwardly.     

Mechanistic insight into the activity of tyrosinase from variable-temperature studies in an aqueous/organic solvent

The activity of mushroom tyrosinase towards a representative series of phenolic and diphenolic substrates structurally related to tyrosine has been investigated in a mixed solvent of 34.4% methanol-glycerol (7:1, v/v) and 65.6% (v/v) aqueous 50 mM Hepes buffer at pH 6.8 at various temperatures. The kinetic activation parameters controlling the enzymatic reactions and the thermodynamic parameters associated with the process of substrate binding to the enzyme active species have been deduced from the temperature variation of the kcat and KM parameters. The activation free energy is dominated by the enthalpic term, the value of which lies in the relatively narrow range of 61+/-9 kJ mol(-1) irrespective of substrate or reaction type (monophenolase or diphenolase). The activation entropies are small and generally negative and contribute no more than 10% to the activation free energy. The substrate binding parameters are characterized by large and negative enthalpy and entropy contributions, which are typically dictated by polar protein-substrate interactions. The substrate 4-hydroxyphenylpropionic acid exhibits a strikingly anomalous temperature dependence of the enzymatic oxidation rate, with deltaH(double dagger) approximately = 150 kJ mol(-1) and deltaS(double dagger) approximately = 280 J K(-1) mol(-1), due to the fact that it can competitively bind to the enzyme through the phenol group, like the other substrates, or the carboxylate group, like carboxylic acid inhibitors. A kinetic model that takes into account the dual substrate/inhibitor nature of this compound enables rationalization of this anomalous behavior.     

Easy oxidation and nitration of human myoglobin by nitrite and hydrogen peroxide

The modification of human myoglobin (HMb) by reaction with nitrite and hydrogen peroxide has been investigated. This reaction is important because NO(2) (-) and H(2)O(2) are formed in vivo under conditions of oxidative and nitrative stress, where protein derivatization has been often observed. The abundance of HMb in tissues and in the heart makes it a potential source and target of reactive species generated in the body. The oxidant and nitrating species produced by HMb/H(2)O(2)/NO(2) (-) are nitrogen dioxide and peroxynitrite, which can react with exogenous substrates and endogenous protein residues. Tandem mass analysis of HMb modified by stoichiometric amounts of H(2)O(2) and NO(2) (-) indicated the presence of two endogenous derivatizations: oxidation of C110 to sulfinic acid (76 %) and nitration of Y103 to 3-nitrotyrosine (44 %). When higher concentrations of NO(2) (-) and H(2)O(2) were used, nitration of Y146 and of the heme were also observed. The two-dimensional gel-electrophoretic analysis of the modified HMbs showed spots more acidic than that of wild-type HMb, a result in agreement with the formation of sulfinic acid and nitrotyrosine residues. By contrast, the reaction showed no evidence for the formation of protein homodimers, as observed in the reaction of HMb with H(2)O(2) alone. Both HMb and the modified HMb are active in the H(2)O(2)/NO(2) (-)-dependent nitration of exogenous phenols. Their catalytic activity is quite similar and the endogenous modifications of HMb therefore have little effect on the reactivity of the protein intermediates.     

Mechanistic study of protein phosphatase-1 (PP1), a catalytically promiscuous enzyme

The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for kcat/KM indicative of catalysis by both acidic and basic residues, with kinetic pKa values of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Br?nsted beta(lg) of -0.32, considerably less negative than that of the uncatalyzed hydrolysis of monoester dianions (-1.23). Kinetic isotope effects in the leaving group with the substrate 4NPP are (18)(V/K) bridge = 1.0170 and (15)(V/K) = 1.0010, which, compared against other enzymatic KIEs with and without general acid catalysis, are consistent with a loose transition state with partial neutralization of the leaving group. PP1 also efficiently catalyzes the hydrolysis of 4-nitrophenyl methylphosphonate (4NPMP). The enzymatic hydrolysis of a series of aryl methylphosphonate substrates yields a Br?nsted beta(lg) of -0.30, smaller than the alkaline hydrolysis (-0.69) and similar to the beta(lg) measured for monoester substrates, indicative of similar transition states. The KIEs and the beta(lg) data point to a transition state for the alkaline hydrolysis of 4NPMP that is similar to that of diesters with the same leaving group. For the enzymatic reaction of 4NPMP, the KIEs are indicative of a transition state that is somewhat looser than the alkaline hydrolysis reaction and similar to the PP1-catalyzed monoester reaction. The data cumulatively point to enzymatic transition states for aryl phosphate monoester and aryl methylphosphonate hydrolysis reactions that are much more similar to one another than the nonenzymatic hydrolysis reactions of the two substrates.     

Screening combinatorial libraries for optimal enzyme substrates by mass spectrometry.

A method has been developed for the rapid identification of optimal enzyme substrates from combinatorial libraries. This methodology was validated by screening a 361-member N-terminally formylated tripeptide library, f-XXR (X = 19 different amino acids), for optimal substrates of Escherichia coli peptide deformylase (PDF). The library was synthesized on a solid phase via the split-pool synthesis method. The N-terminal formyl group was added by treating the resin with a 1:1 (mol/mol) mixture of HCO(2)H and DCO(2)D in the presence of dicyclohexylcarbodiimide. In a mass spectrum, each member of the library produced a doublet peak (separated by 1.0063 Da). Limited treatment of this library with E. coli PDF resulted in the deformylation of those peptides that are the most efficient substrates of the enzyme. The deformylated products, due to loss of the mass-degenerate formyl group, each generated a singlet peak in the mass spectrum. Thus, the PDF product peaks were readily identified and sequenced via tandem mass spectrometry. The results showed that PDF strongly prefers a norleucine and, to a lesser extent, a phenylalanine as the N-terminal residue, whereas it has little selectivity at the penultimate position. This result is in excellent agreement with the literature data and therefore demonstrates the methodology as an effective approach to the identification of optimal enzyme substrates. This method should be generally applicable to other enzymes as well as synthetic catalysts.