pH-independent Rate Constants Research Articles

Acidity of persulfides and its modulation by the protein environments in sulfide quinone oxidoreductase and thiosulfate sulfurtransferase

Persulfides (RSSH/RSS–) participate in sulfur metabolism and are proposed to transduce hydrogen sulfide (H2S) signaling. Their biochemical properties are poorly understood. Herein, we studied the acidity and nucleophilicity of several low molecular weight persulfides using the alkylating agent, monobromobimane. The different persulfides presented similar pKa values (4.6-6.3) and pH-independent rate constants (3.2-9.0 × 103 M–1 s–1), indicating that the substituents in persulfides affect properties to a lesser extent than in thiols because of the larger distance to the outer sulfur. The persulfides had higher reactivity with monobromobimane than analogous thiols and putative thiols with the same pKa, providing evidence for the alpha effect (enhanced nucleophilicity by the presence of a contiguous atom with high electron density). Additionally, we investigated two enzymes from the human mitochondrial H2S oxidation pathway that form catalytic persulfide intermediates, sulfide quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST, rhodanese). The pH dependence of the activities of both enzymes were measured using sulfite and/or cyanide as sulfur acceptors. The TST half-reactions were also studied by stopped-flow fluorescence spectroscopy. Both persulfidated enzymes relied on protonated groups for reaction with the acceptors. Persulfidated SQOR appeared to have a pKa of 7.8 ± 0.2. Persulfidated TST presented a pKa of 9.38 ± 0.04, probably due to a critical active site residue rather than the persulfide itself. The apparent pKa for the TST thiol was 6.5 ± 0.1 and it reacted in the anionic state with thiosulfate. Overall, our study contributes to a fundamental understanding of persulfide properties and their modulation by protein environments.

Mobile Molecules: Reactivity Profiling Guides Faster Movement on a Cysteine Track.

We previously reported a molecular hopper, which makes sub-nanometer steps by thiol-disulfide interchange along a track with cysteine footholds within a protein nanopore. Here we optimize the hopping rate (ca. 0.1 s-1 in the previous work) with a view towards rapid enzymeless biopolymer characterization during translocation within nanopores. We first took a single-molecule approach to obtain the reactivity profiles of individual footholds. The pKa values of cysteine thiols within a pore ranged from 9.17 to 9.85, and the pH-independent rate constants of the thiolates with a small-molecule disulfide varied by up to 20-fold. Through site-specific mutagenesis and a pH increase from 8.5 to 9.5, the overall hopping rate of a DNA cargo along a five-cysteine track was accelerated 4-fold, and the rate-limiting step 21-fold.

Study of base-catalyzed isomerization of d-glucose with a focus on reaction kinetics

We explored the isomerization of d-glucose into d-fructose using the simplest possible base catalyst, aqueous NaOH, to maintain a constant pH value during the reaction. Under the applied mild conditions (T 50–90 °C, pH 9.5–11.5), yields of d-fructose of up to 31% were observed. Selectivity-conversion plots were not significantly influenced by variation of the temperature, pH value or substrate concentration. A reaction network for kinetic modelling includes d-glucose-d-fructose interconversion, co-production of d-mannose and d-allulose (also known as d-psicose) as well as decomposition paths after deprotonation of the hexoses. All four hexoses were employed as substrates in the isomerization. Thermodynamic ionization constants of the saccharides were measured by means of potentiometric titration. In the kinetic studies, pH-independent rate constants as well as activation energies were determined. The obtained kinetic and thermodynamic results as well as selectivity-conversion correlations present a useful benchmark for soluble and solid base catalysts.

Rate and Mechanism of Electrochemical Formation of Surface-Bound Hydrogen on Pt(111) Single Crystals.

The formation of surface-bound hydrogen from one proton and one electron plays an enabling role in renewable hydrogen production. Quantifying the surface-bound hydrogen formation, however, requires decoupling the delicate interplay of numerous processes. We study cyclic voltammetry (CV) at fast scan rates to characterize the rate constant for the surface-bound hydrogen formation (also known as underpotential deposition hydrogen, UPD Had). We find that the formation of Had on Pt(111) single crystals is ∼100× faster in acid than in base. Reaction-order analysis indicates that the formation of Had occurs as a standard proton-coupled electron transfer (PCET) reaction in acid, whereas in base, it displays a pH-independent rate constant, indicating the presence of a chemical step such as the reorganization of interfacial water. Our results provide a methodology for quantifying the interfacial PCET kinetics and reveal the mechanistic nature of the UPD Had formation as the reason the hydrogen evolution electrocatalysis on Pt is faster in acid than in base.

Physical heterogeneity control on effective mineral dissolution rates

Hydrologic heterogeneity may be an important factor contributing to the discrepancy in laboratory and field measured dissolution rates, but the governing factors influencing mineral dissolution rates among various representations of physical heterogeneity remain poorly understood. Here, we present multiple reactive transport simulations of anorthite dissolution in 2D latticed random permeability fields and link the information from local grid scale (1 cm or 4 m) dissolution rates to domain-scale (1m or 400 m) effective dissolution rates measured by the flux-weighted average of an ensemble of flow paths. We compare results of homogeneous models to heterogeneous models with different structure and layered permeability distributions within the model domain. Chemistry is simplified to a single dissolving primary mineral (anorthite) distributed homogeneously throughout the domain and a single secondary mineral (kaolinite) that is allowed to dissolve or precipitate. Results show that increasing size in correlation structure (i.e. long integral scales) and high variance in permeability distribution are two important factors inducing a reduction in effective mineral dissolution rates compared to homogeneous permeability domains. Larger correlation structures produce larger zones of low permeability where diffusion is an important transport mechanism. Due to the increased residence time under slow diffusive transport, the saturation state of a solute with respect to a reacting mineral approaches equilibrium and reduces the reaction rate. High variance in permeability distribution favorably develops large low permeability zones that intensifies the reduction in mixing and effective dissolution rate. However, the degree of reduction in effective dissolution rate observed in 1 m × 1 m domains is too small (<1% reduction from the corresponding homogeneous case) to explain several orders of magnitude reduction observed in many field studies. When multimodality in permeability distribution is approximated by high permeability variance in 400 m × 400 m domains, the reduction in effective dissolution rate increases due to the effect of long diffusion length scales through zones with very slow reaction rates. The observed scale dependence becomes complicated when pH dependent kinetics are compared to the results from pH independent rate constants. In small domains where the entire domain is reactive, faster anorthite dissolution rates and slower kaolinite precipitation rates relative to pH independent rates at far-from-equilibrium conditions reduce the effective dissolution rate by increasing the saturation state. However, in large domains where less- or non-reactive zones develop, higher kaolinite precipitation rates in less reactive zones increase the effective anorthite dissolution rates relative to the rates observed in pH independent cases.

Determination of pH-independent rate constants of thiolate–disulfide redox transitions

Real-time evolution of qNMR spectra combined with detailed protonation constant data affords kinetic constants pertaining to thiolate–disulfide reactions with specific chemical species.

Ultrafast Photoinduced Interfacial Proton Coupled Electron Transfer from CdSe Quantum Dots to 4,4'-Bipyridine.

Pyridine and derivatives have been reported as efficient and selective catalysts for the electrochemical and photoelectrochemical reduction of CO2 to methanol. Although the catalytic mechanism remains a subject of considerable recent debate, most proposed models involve interfacial proton coupled electron transfer (PCET) to electrode-bound catalysts. We report a combined experimental and theoretical study of the photoreduction of 4,4'-bipyridium (bPYD) using CdSe quantum dots (QDs) as a model system for interfacial PCET. We observed ultrafast photoinduced PCET from CdSe QDs to form doubly protonated [bPYDH2](+•) radical cations at low pH (4-6). Through studies of the dependence of PCET rate on isotopic substitution, pH and bPYD concentration, the radical formation mechanism was identified to be a sequential interfacial electron and proton transfer (ET/PT) process with a rate-limiting pH independent electron transfer rate constant, kint, of 1.05 ± 0.13 × 10(10) s(-1) between a QD and an adsorbed singly protonated [bPYDH](+). Theoretical studies of the adsorption of [bPYDH](+) and methylviologen on QD surfaces revealed important effects of hydrogen bonding with the capping ligand (3-mercaptopropionic acid) on binding geometry and interfacial PCET. In the presence of sacrificial electron donors, this system was shown to be capable of generating [bPYDH2](+•) radical cations under continuous illumination at 405 nm with a steady-state photoreduction quantum yield of 1.1 ± 0.1% at pH 4. The mechanism of bPYD photoreduction reported in this work may provide useful insights into the catalytic roles of pyridine and pyridine derivatives in the electrochemical and photoelectrochemical reduction of CO2.

Hydrolysis of phosphodiester catalyzed by metallomicelles with histidine residue: Kinetics and mechanism

A new 2-amino-N-dodecyl-3-(1H-imidazole-4-yl)propanamide (L2) surfactant was synthesized and its metallomicelles made of La(III), Cu(II), Co(II) and Zn(II) complexes were used as hydrolytic metalloenzyme mimics to catalyze the hydrolysis of phosphodiester bis(p-nitrophenyl) phosphate (BNPP) in buffer solution at 35°C. The effects of long alkyl ligand, metal ions, temperature and pH on the catalytic hydrolysis of phosphodiester were investigated. The ternary complex kinetic model for metallomicellar catalysis was suggested to analyze the experimental data. The kinetic and thermodynamic parameters k′N, KM, KT, Km and pKa were obtained. The effect of pH was especially analyzed. The pH-independent rate constant of the reaction catalyzed by metallomicelles LaL2, CuL2, CoL2 and ZnL2 was evaluated as 7.03×10−4, 6.51×10−5, 4.28×10−5 and 1.78×10−5s−1, respectively. The different association manners between metallomicelles and substrate were proposed. A hydroxide ion OH− associated metallomicelle, which was detected by ESI-MS spectral analysis, was suggested as the real active species to catalyze the hydrolysis of phosphodiester. Imidazole group might accelerate the hydrolysis by activating H2O associated the metal into metal-OH−. Effect of microenvironment of micelle on the catalysis hydrolysis of BNPP was also discussed.

Toward a Small Molecule, Biomimetic Carbonic Anhydrase Model: Theoretical and Experimental Investigations of a Panel of Zinc(II) Aza-Macrocyclic Catalysts

A panel of five zinc-chelated aza-macrocycle ligands and their ability to catalyze the hydration of carbon dioxide to bicarbonate, H(2)O + CO(2) → H(+) + HCO(3)(–), was investigated using quantum-mechanical methods and stopped-flow experiments. The key intermediates in the reaction coordinate were optimized using the M06-2X density functional with aug-cc-pVTZ basis set. Activation energies for the first step in the catalytic cycle, nucleophilic CO(2) addition, were calculated from gas-phase optimized transition-state geometries. The computationally derived trend in activation energies was found to not correspond with the experimentally observed rates. However, activation energies for the second, bicarbonate release step, which were estimated using calculated bond dissociation energies, provided good agreement with the observed trend in rate constants. Thus, the joint theoretical and experimental results provide evidence that bicarbonate release, not CO(2) addition, may be the rate-limiting step in CO(2) hydration by zinc complexes of aza-macrocyclic ligands. pH-independent rate constants were found to increase with decreasing Lewis acidity of the ligand-Zn complex, and the trend in rate constants was correlated with molecular properties of the ligands. It is suggested that tuning catalytic efficiency through the first coordination shell of Zn(2+) ligands is predominantly a balance between increasing charge-donating character of the ligand and maintaining the catalytically relevant pK(a) below the operating pH.

1,4,7,10-Tetraazacyclododecane Metal Complexes as Potent Promoters of Phosphodiester Hydrolysis under Physiological Conditions

Previously reported mono- and dinuclear Zn(II), Cu(II), and Ni(II) complexes of 1,4,7,10-tetrazacyclododecane ([12]aneN4 or cyclen) with different heterocyclic spacers (triazine, pyridine) of various lengths (bi- and tripyridine) or an azacrown-pendant have been tested for the hydrolysis of bis(4-nitrophenyl)phosphate (BNPP) under physiological conditions (pH 7-9, 25 degrees C). All Zn(II) complexes promote the hydrolysis of BNPP under physiological conditions, while those of Cu(II) and Ni(II) do not have a significant effect on the hydrolysis reaction. The hydrolysis kinetics in buffered solutions (0.05 M Bis/Tris, TRIS, HEPES, or CHES, I=0.1 M, NaCl) at 25 degrees C were determined by the initial slope method (product conversion<5%). Comparison of the second-order pH-independent rate constants (kBNPP, M(-1) s(-1)) for the mononuclear complexes ZnL1, ZnL3, and ZnL6, which are 6.1x10 (-5), 5.1x10(-5), and 5.7x10(-5), respectively, indicate that the heterocyclic moiety improves the rate of hydrolysis up to six times over the parent Zn([12]aneN4) complex (kBNPP=1.1x10(-5) M(-1) s(-1)). The reactive species is the Zn(II)-OH- complex, in which the Zn(II)-bound OH- acts as a nucleophile. For dinuclear complexes Zn2L2, Zn2L4, and Zn2L5, the rate of reaction is defined by the degree of cooperation between the metal centers, which is determined by the spacer length. Zn2L2 and Zn2L4 possessing shorter spacers are able to hydrolyze BNPP 1 to 2 orders of magnitudes faster than Zn2L5. The second-order rate constants k of Zn2L4 and Zn2L2 at pH 7, 8, and 9 are significantly higher than those of previously reported related complexes. The high BNPP hydrolytic activity may be related to pi-stacking and hydrophobic interactions between the aromatic spacer moieties and the substrate. Complexes Zn2L4 and Zn2L2 show hydrolytic activity at pH 7 and 8, which allows for the hydrolysis of activated phosphate esters under physiological conditions.

1,4,7,10-Tetraazacyclododecane Metal Complexes as Potent Promoters of Carboxyester Hydrolysis under Physiological Conditions

New 1,4,7,10-tetrazacyclododecane ([12]aneN4 or cyclen) ligands with different heterocyclic spacers (triazine and pyridine) of various lengths (bi- and tripyridine) or an azacrown pendant and their mono- and dinuclear Zn(II), Cu(II), and Ni(II) complexes have been synthesized and characterized. The pKa values of water molecules coordinated to the complexed metal ions were determined by potentiometric pH titrations and vary from 7.7 to 11.2, depending on the metal-ion and ligand properties. The X-ray structure of [Zn2L2]mu-OH(ClO4)3.CH3CN.H2O shows each Zn(II) ion in a tetrahedral geometry, binding to three N atoms of cyclen (the average distance of Zn-N = 2.1 A) and having a mu-OH bridge at the apical site linking the two metal ions (the average distance of Zn-O- = 1.9 A). The distance between the Zn(II) ion and the fourth N atom is 2.6 A. All Zn(II) complexes promote the hydrolysis of 4-nitrophenyl acetate (NA) under physiological conditions, while those of Cu(II) and Ni(II) do not have a significant effect on the hydrolysis reaction. The kinetic studies in buffered solutions (0.05 M Tris, HEPES, or CHES, I = 0.1 M, NaCl) at 25 degrees C in the pH range of 6-11 under pseudo-first-order reaction conditions (excess of the metal complex) were analyzed by applying the method of initial rates. Comparison of the second-order pH-independent rate constants (kNA, M-1 s-1) for the mononuclear complexes ZnL1, ZnL3, and ZnL8, which are 0.39, 0.27, and 0.38, respectively, indicates that the heterocyclic moiety improves the rate of hydrolysis up to 4 times over the parent Zn([12]aneN4) complex (kNA = 0.09 M-1 s-1). The reactive species is the Zn(II)-OH- complex, in which the Zn(II)-bound OH- acts as a nucleophile, which attacks intermolecularly the carbonyl group of the acetate ester. For dinuclear complexes Zn2L2, Zn2L4, Zn2L5, Zn2L6, and Zn2L7, the mechanism of the reaction is defined by the degree of cooperation between the metal centers, determined by the spacer length. For Zn2L7, having the longest triaryl spacer, the two metal centers act independently in the hydrolysis; therefore, the reaction rate is twice as high as the rate of the mononuclear analogue (kNA = 0.78 M-1 s-1). The complexes with a monoaryl spacer show saturation kinetics with the formation of a Michaelis-Menten adduct. Their hydrolysis rates are 40 times higher than that of the Zn[12]aneN4 system (kNA approximately 4 M-1 s-1). Zn2L6 is a hybrid between these two mechanisms; a clear saturation curve is not visible nor are the metal cores completely independent from one another. Some of the Zn(II) complexes show a higher hydrolytic activity under physiological conditions compared to other previously reported complexes of this type.

Characterization of Ester Hydrolysis in Terms of Microscopic Rate Constants

Hydroxide-catalyzed ester hydrolysis for molecules of coexisting species is quantitated in terms of microscopic rate constants, a new, species-specific physicochemical parameter. Relationships between the overall and component reactions, as well as the macroscopic and microscopic rate constants are deduced. Experimental techniques, evaluation methods, and feasibility are discussed. Species-specific, pH-independent rate constants of four coexisting, differently hydrolyzing microspecies are determined for the first time. Protonation of an alpha-amino and beta-imidazolyl site in amino acid esters has been found to accelerate the hydroxide-catalyzed hydrolysis by factors of 120 and 7.5, respectively, whereas they jointly exert a nearly 3000-fold acceleration. A total of 20 microscopic protonation equilibrium constants, as component parameters in the rate equations, have also been determined. The species-specific rate constants have been found to correlate with the site- and species-specific basicity of the leaving group and the NMR chemical shift of an adjacent proton. Individual contributions of the various microforms to the overall hydrolysis rate are depicted in microscopic reaction fraction diagrams.

Phosphate Triester Hydrolysis Promoted by an N2S(thiolate)Zn Complex: Mechanistic Implications for the Metal-Dependent Reactivity of Peptide Deformylase

The zinc(II) complex (PATH)ZnOH, where PATH is an N2S(thiolate) ligand, has been investigated for its ability to promote the hydrolysis of the phosphate triester tris(4-nitrophenyl) phosphate (TNP). The hydrolysis of TNP was examined as a function of PATH-zinc(II) complex concentration, substrate concentration, and pH in a water/ethanol mixture (66:33 v/v) at 25 degrees C. The reaction is first order in both zinc(II) complex and substrate, and the second-order rate constants were derived from linear plots of the observed pseudo-first-order rate constants versus zinc complex concentration at different pH values. A pH-rate profile yielded a kinetic pK(a) of 8.52(5) for the zinc-bound water molecule and a pH-independent rate constant of 16.1(7) M(-1) s(-1). Temperature-dependent studies showed linear Eyring behavior, yielding the activation parameters DeltaH++ = 36.9(1) kJ mol(-1) and DeltaS++ = -106.7(4) J mol(-1) K(-1). Interpretation of the kinetic data leads to the conclusion that hydrolysis of TNP takes place through a hybrid mechanism, in which the metal center plays a dual role of providing a nucleophilic hydroxide and activating the substrate through a Lewis acid effect. The synthesis and structural characterization of the related nickel(II) and iron(II) complexes [(PATH)2Ni2]Br2 (2) and (PATH)2Fe2Cl2 (3) are also described. Taken together, these data suggest a possible explanation for the low reactivity of the zinc(II) form of peptide deformylase as compared to the iron(II) form.

Synthesis and characterization of the aqueous solution chemistry of the food-derived carcinogen model N-acetoxy- N-(1-methyl-5 H-pyrido[4,5- b]indol-3-yl)acetamide and its N-pivaloyloxy analogue

We report the synthesis of N-acetoxy- N-(1-methyl-5 H-pyrido[4,5- b]indol-3-yl)acetamide, 7 , its N-pivaloyloxy analogue, 9 , and improved synthesis of indole-2-acetonitrile, 3 (70% in five steps from indole-2-carboxylic acid), the carcinogenic amine Trp-P-2, 4 (40% from 3 ), and the nitro compound, 5 (40% from 4 by oxidation with H 2O 2 using Mo(CO) 6 catalyst). In aqueous solution at neutral pH, 7 primarily undergoes C–O bond cleavage to yield the hydroxamic acid, 8 , but under the same conditions the sterically hindered 9 decomposes predominately by N–O bond cleavage with a pH independent rate constant that is 7.5-fold smaller than that for 7 . In the pH range 0.5–7.0 three different processes for the decomposition of 9 were detected by kinetics. Only the process that dominates at neutral pH generates a nitrenium species that can be trapped by N 3 −.

Absolute Aggregation Rate Constants of Hematite Particles in Aqueous Suspensions: A Comparison of Two Different Surface Morphologies

Measurements of absolute aggregation rate constants were performed for two samples of well-characterized spheroidal hematite particles with rather different surface morphology. At high ionic strength, the system is in the fast aggregation regime with pH independent rate constants around (1–4) × 10−18m3/s. At low ionic strengths, the rate constant is a function of pH and goes through a flat maximum around the point of zero charge (PZC) where fast aggregation conditions are reached. With increasing pH the rate constants increase gradually below the PZC while they decrease very rapidly above the PZC. Above the PZC the rate constants are rather well predicted by the classical DLVO theory. Below the PZC, however, it is necessary to assume a distribution in the surface potentials with a coefficient of variation around 30% to account for the observed behavior. In spite of the pronounced differences in the surface roughness of both hematite samples, their aggregation rate constants are rather similar.

Effects of cyclodextrin on hydrolysis and the Smiles rearrangement of salicylic acid esters

The effect of β-cyclodextrin (β-CD) on “aqueous” hydrolysis of methyl,p-, andm-nitrophenyl salicylates as well as on the Smiles rearrangement ofp-nitrophenyl salicylate was studied. No effect of β-CD on the pH-independent rate constant of “aqueous” hydrolysis of methyl ester was observed, while β-CD accelerated “aqueous” hydrolysis of nitrophenyl esters byca. 10 times. The inclusion of these esters into the cavity of β-CD is accompanied by a change in the mechanism of hydrolysis: free ester in the deprotonated form undergoes hydrolysis through the mechanism of intramolecular general base catalysis, while the ester bound to cyclodextrin is hydrolyzed due to the nucleophilic attack of the deprotonated hydroxyl group of β-CD at a neutral substrate molecule. The effects of cyclodextrin on the rate constant of borate-catalyzed hydrolysis were interpreted by assuming that the substrate bound to β-CD undergoes borate-assisted attack at the deprotonated cyclodextrin hydroxyl group. The Smiles rearrangement, which is an intramolecular nucleophilic substitution reaction, is accelerated in the presence of β-CD.

Kinetics and mechanism of covalent addition of S(IV) species to the acridinium cation

The reaction of acridine with S(IV) species (SO2•H2O, HSO3−, and SO32−) to form the adduct acridine–S(IV) has been studied spectrophotometrically throughout the pH range 2.6–8 in aqueous solutions. The observed pseudo-first-order rate constants, kobs, were determined at 25 °C and ionic strength I = 0.11 M, and the pH profile of the rate reached a maximum at pH ≈ 6.1. At constant pH the kobs values were a linear function of the total S(IV) concentration with slopes that increased significantly with pH. These data are consistent with the rate-determining attack of SO3H− and SO32− upon the C-9 of the acridinium cation. A nonlinear least-squares fitting of the experimental values to the model equation, within the overall pH region studied, yields the pH-independent rate constants k1 = 3.7 ± 0.1 and k2 = (6.24 ± 0.04) × 104 M−1 s−1 for the attack of these two species, respectively. The experimental results agree very well with the kinetic model. Due to the experimental conditions used we did not detect any possible pseudobase formation in the pH range studied. The reactivity of the S(IV) species with acridine follows the order: [Formula: see text] The value obtained for the ratio k1/k2 is similar to the results given for other addition reactions of S(IV) species to the double bond of carbonyl compounds such as benzaldehyde and formaldehyde. Key words: covalent addition, acridine, acridine – S(IV) adducts, kinetics and mechanism.

Evaluation of hydrogen-bonding and enantiomeric P2-S2 hydrophobic contacts in dynamic aspects of molecular recognition by papain.

1. 2-(N'-Acetyl-D-phenylalanyl)hydroxyethyl 2'-pyridyl disulphide (compound IV) (m.p. 59 degrees C; [alpha]D20 -6.6 degrees (c 1.2 in methanol)) was synthesized. 2. The results of a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group (Cys-25) of papain (EC 3.4.22.2) together with analogous kinetic data for the reactions of related time-dependent inhibitors, notably the L-enantiomer of compound (IV) (compound III) and the L- and D-enantiomers of 2-(N'-acetylphenylalanylamino)ethyl 2'-pyridyl disulphide (compounds I and II respectively), were used to assess the contributions of the (P1)-NH ... O = C < (Asp-158) and (P2) > C = O ... H-N-(Gly-66) hydrogen bonds and enantiomeric P2-S2 hydrophobic contacts in two manifestations of dynamic molecular recognition in papain-ligand association: (a) signalling to the catalytic-site region to provide for a (His-159)-IM(+)-H-assisted transition state and (b) the dependence of P2-S2 stereoselectivity on hydrogen-bonding interactions outside the S2 subsite. The analysis involved determination of the reactivities of individual ionization states of the reactions (pH-independent rate constants, k) and associated macroscopic pKa values and difference kinetic specificity energies (delta delta GKS = -RT1n(k1/k2), where k1 is the pH-independent second-order rate constant for reaction with one inhibitor and k2 is the analogous rate constant in the same ionization state for reaction with another inhibitor so that, when the structural change provides that k2 > k1, delta delta GKS is positive. 3. The kinetic data further illuminate the nature of the interdependence of binding interactions in papain first noted by Kowlessur, Topham, Thomas, O'Driscoll, Templeton & Brocklehurst [(1989) Biochem. J. 258, 755-764] in the S2 subsite, S1-S2 intersubsite and catalytic-site regions. Of particular note is the apparent dependence of the binding of the N-Ac-D-Phe moiety on the binding of the leaving group to (His-159)-Im+H and the fact that the resulting rate enhancement is more effective when (P1)-N-H is absent than when it is present. This result revealed by kinetic analysis goes beyond the conclusion suggested by model building that it is possible to make all of the binding contacts in complexes involving the D-enantiomers [(II) and (IV)] as in those involving the L-enantiomers [(I) and (III)].(ABSTRACT TRUNCATED AT 400 WORDS)

An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.

Kinetics of reaction of aquacobalamin with azide ion and hydrazoic acid: pH profile and activation parameters

The kinetics of reaction of aquacobalamin (vitamin B12a) with azide anion and hydrazoic acid, producing azidocobalamin, has been investigated as a function of ligand concentration, pH, temperature, and pressure, by stoped-flow spectrophotometry. The pH-independent rate constants (25 °C, Ionic strength I= 1.0 mol dm–3), and activation parameters are k1= 525 ± 14 dm3 mol–1 s–1, ΔH1= 65 ± 1 kJ mol–1, ΔS1= 26 ± 2 J K–1 mol–1, and ΔV1= 6.4 ± 0.1 cm3 mol–1 for the reaction with N3–, and k2= 103 ± 13 dm3 mol–1 s–1, ΔH2= 65 ± 3 kJ mol–1, ΔS2= 13 ± 8 J K–1 mol–1, and ΔV2= 5.5 ± 0.3 cm3 mol–1 for the reaction with HN3. The experimental data are considered in terms of both a limiting dissociative and a dissociative interchange mechanism and it is concluded that they cannot be used to distinguish between the two possible mechanistic pathways.