Kinetics of formation of the primary compound (compound I) from hydrogen peroxide and turnip peroxidases.

A kinetic study of the reaction of two turnip peroxidases (P1 and P7) with hydrogen peroxide to form the primary oxidized compound (compound I) has been carried out over the pH range from 2.4 to 10.8. In the neutral and acidic pH regions, the rates depend linearly on hydrogen peroxide concentration whereas at alkaline pH values the rates display saturation kinetics. A compound is made with the cyanide binding reaction to peroxidases since the two reactions are influenced in the same manner by ionization of groups on the native enzymes. Two different ionization processes of peroxidase P1 with pKa values of 3.9 and 10 are required to explain the rate pH profile for the reaction with H2O2. Protonation of the former group and ionization of the latter causes a decrease in the rate of reaction of the enzyme with H2O2. In the case of peroxidase P7 a minimum model involves three ionizable groups with pKa values of 2.5, 4 and 9. Protonation of the former two groups and ionization of the latter lowers the reaction rate. In the pH-independent region, the rate of formation of compound I was measured as a function of temperature. From the Arhenius plots the activation energy for the reaction was calculated to be 2.9 +/- 0.1 kcal/mol for P1 and 5.4 +/- 0.3 kcal/mol for P7. However, the rates are independent of viscosity in glycerol-water mixtures up to 30% glycerol.     

Kinetic studies on human cysteinyl-tRNA synthetase.

The detailed pH and temperature kinetics of human term placenta cysteinyl-tRNA synthetase (EC 6.1.1.16) were studied. The ATP-PPi exchange reaction catalyzed by the cysteinyl-tRNA synthetase was highly dependent on temperature, pH, and ionic strength. The Arrhenius plot at temperatures between 5 degrees and 40 degrees was linear, giving an activation energy of 19 +/- 2.5 Kcal/mol. The pH dependence of the kinetic parameters Km and Vmax was investigated. Apparent pKa value of 6.4 was observed in the pH-dependence of Vmax/Km plot. The pH versus Vmax plot showed two apparent pKa values of about 5.8 and 7.8. Van't Hoff's enthalpies were used to differentiate the nature of the possible groups responsible for the ionization. These results are valuable for the selection of chemical modifying reagents in characterizing the amino acid residues involved in substrate (nucleotide) binding or catalysis.     

The aminopeptidase from Aeromonas proteolytica can function as an esterase

The aminopeptidase from Aeromonas proteolytica (AAP) can catalyze the hydrolysis of L-leucine ethyl ester ( L-Leu-OEt) with a rate of 96 +/- 5 s-1 and a Km of 700 microM. The observed turnover number for L-Leu-OEt hydrolysis by AAP is similar to that observed for peptide hydrolysis, which is 67 +/- 5 s-1. The k(cat) values for the hydrolysis of L-Leu-OEt and L-leucine- p-nitroanilide ( L- pNA) catalyzed by AAP were determined at different pH values under saturating substrate concentrations. Construction of an Arrhenius plot from the temperature dependence of AAP-catalyzed ester hydrolysis indicates that the rate-limiting step does not change as a function of temperature and is product formation. The activation energy ( Ea) for the activated ES ester complex is 13.7 kJ mol-1, while the enthalpy and entropy of activation at 25 degrees C calculated over the temperature range 298-338 K are 11.2 kJ mol-1 and -175 J K-1 mol-1, respectively. The free energy of activation at 25 degrees C was found to be 63.4 kJ mol-1. The enthalpy of ionization was also measured and was found to be very similar for both peptide and ester substrates, yielding values of 20 kJ mol-1 for L-Leu-OEt and 25 kJ mol-1 for L- pNA. For peptide and L-amino acid ester cleavage reactions catalyzed by AAP, and 6.07, respectively. Proton inventory data suggest that two protons are transferred in the rate-limiting step of ester hydrolysis while only one is transferred in peptide hydrolysis. The combination of these data with the available X-ray crystallographic, kinetic, spectroscopic, and thermodynamic data for AAP provides new insight into the catalytic mechanism of AAP.     

Some peculiarities of the reaction kinetics of ATP hydrolysis by Ca2+-dependent ATPase of chloroplasts]

The dependence of the reaction rate of ATP hydrolysis by CF1-ATPase of chloroplasts on the substrate (CaATP) concentration is of complex nature. It is assumed that such dependence may be due to either conformational changes of the enzyme during the increase in CaATP content in the reaction medium, or consecutive binding of the substrate to its active sites, differing in their affinity to the substrate of the reaction.     

Horseradish peroxidase. XXXII. pH dependence of the oxidation of L-(-)-tyrosine by compound I.

The rate of oxidation of L-(-)-tyrosine by horseradish peroxidase compound 1 has been studied as a function of pH at 25 degrees C and ionic strength 0.11. Over the pH range of 3.20--11.23 major effects of three ionizations were observed. The pKa values of the phenolic (pKa = 10.10) and amino (pKa = 9.21) dissociations of tyrosine and a single enzyme ionization (pKa = 5.42) were determined from nonlinear least squares analysis of the log rate versus pH profile. It was noted that the less acidic form of the enzyme was most reactive; hence, the reaction is described as base catalyzed. The rate of tyrosine oxidation falls rapidly with the deprotonation of the phenolic group.     

pH dependence of the kinetic parameters of L-asparaginase.

The concentration dependence of the rate of hydrolysis of L-asparagine by Escherichia coli L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been measured over the range pH 4.5 to pH 9.1 by a direct spectrophotometric assay at 220 nm and by a coupled assay utilizing glutamate dehydrogenase to detect the ammonia produced. The velocity of the hydrolysis reaction at saturating levels of substrate is independent of pH over this interval. The plot of V/km over the same interval is bell-shaped, being dependent on pKa values of 6.58 and 8.69. The higher pKa is attributed to the amino group of asparagine. The lower pKa is associated with the enzyme active site and is probably due to an imidazole group.     

The kinetics of the alkaline dissociation of myosin.

The rates of two processes in alkaline (pH 10.5–11.5) myosin solutions at 0 °C have been investigated: production of ionized tyrosine residues and production of light subunits. The progressive absorbance change is shown to result from a first-order irrevocable exposure to solvent and subsequent ionization of 40% of the tyrosine residues. Extrapolation to zero time gives the spectrophotometric ionization curve for native myosin; the pK of the abnormal tyrosines exceeds 12. Similarly, extrapolation to infinite time gives the curve for denatured myosin; the pK of the normal tyrosines (and of all tyrosines after denaturation) is 11.0–11.6. From the pH dependence of the rate, it is found that activation requires ionization of six residues and that their pK is much greater than 11.3. The rate of production of subunits was determined by fractionating the reaction mixture and determining the weight of light subunits produced. The process is also first order. Within experimental error, the rate constants for these two processes are equal. We conclude that they have the same rate-determining step. The data are consistent with either of two simple possible mechanisms. These are a rapid conformation change, followed by rate-determining subunit dissociation, followed by a rapid, irrevocable conformation change; or, a rapid conformation change, followed by a rate-determining, irrevocable conformation change, followed by rapid subunit dissociation.     

The pH dependence of tyrosine cyanuration in proteins

The pH, temperature, and reagent concentration dependences of the cyanuration of tyrosines within proteins are compared with those of the free amino acid. The results indicate that the mechanism of the cyanuration reaction is a nucleophilic aromatic substitution of the SN 2 type, where the tyrosine oxygen acts as a nucleophile and the displacement takes places at a carbon atom which is part of the six-membered ring of the s-triazene. This reaction must compete with the hydrolysis of cyanuric fluoride in aqueous media. These competing reactions generate curves for the pH dependence of the reactivity of tyrosine with cyanuric fluoride whihc vary in a predictable manner with the extent of ionization of tyrosine and the concentration of cyanuric fluoride. In the case of proteins, the dependence of the degree of tyrosine reactivity on pH and temperature within the pH range of full reactivity of the free amino acid reflects constraints imposed by the microenvironment of these groups within the three-dimensional structure of the macromolecule.     

The effect of pH and temperature on the reaction of fully reduced and mixed-valence cytochrome c oxidase with dioxygen

The reaction of fully reduced and mixed-valence cytochrome oxidase with O2 has been followed in flow-flash experiments, starting from the CO complexes, at 428, 445, 605 and 830 nm between pH 5.8b and 9.0 in the temperature range of 2-40 degrees C. With the fully reduced enzyme, four kinetic phase with rate constants at pH 7.4 and 25 degrees C of 9 x 10(4), 2.5 x 10(4), 1.0 x 10(4) and 800 s(-1), respectively, are observed. The rates of the three last phases display a very small temperature dependence, corresponding to activation energies in the range 13-54 kJ x mol(-1). The rates of the third and fourth phases decrease at high pH due to the deprotonation of groups with pKa values of 8.3 and 8.8, respectively, but also the second phase appears to have a small pH dependence. In the reaction of the mixed-valence enzyme, three kinetic phases with rate constants at pH 7.4 and 25 degrees C of 9 x 10(4), 6000 and 150 s(-1), respectively, are observed. The third phase only has a small temperature dependence, corresponding to an activation energy of 20 kJ x mol(-1). No pH dependence could be detected for any phase. Reaction schemes consistent with the experimental observations are presented. The pH dependencies of the rates of the two final phase in the reaction of the fully reduced enzyme are proposed to be related to the involvement of protons in the reduction of a peroxide intermediate. The temperature dependence data suggest that the reorganization energies and driving forces are closely matched in all electron transfer steps with both enzyme forms. It is suggested that the slowest step in the reaction of the mixed-valence enzyme is a conformation change involved in the reaction cycle of cytochrome oxidase as a proton pump.     

Quenching of the intrinsic fluorescence of liver alcohol dehydrogenase by the alkaline transition and by coenzyme binding

The fluorescence of alcohol dehydrogenase is quenched by the acid dissociation of some group on the protein having an apparent pKa of 9.6 at 25 degrees C. The pKa of this alkaline quenching transition is unchanged by the binding of trifluoroethanol or pyrazole to the enzyme or by the selective removal of the active site of Zn2+ ion. This indicates that the ionization of a zinc-bound water molecule is not responsible for the quenching. The binding of NAD+ to the enzyme causes a drop in protein fluorescence and an apparent shift in the alkaline quenching transition to lower pH. In the ternary complex formed with NAD+ and trifluoroethanol the alkaline transition is difficult to discern between pH 6 and pH 11. In the NAD+-pyrazole ternary complex, however, a small but noticeable fluorescence transition is observed with a pKa(app) approximately 9.5. We propose that the alkaline transition centered at pH 9.6 is not shifted to lower pH upon binding NAD+. Instead, the amplitude of the alkaline quenching effect is decreased to the point that it is difficult to detect when NAD+ is bound. We present a model that describes the dependence of the fluorescence of the protein on pH and NAD+ concentration in terms of two independently operating, dynamic quenching mechanisms. Our data and model cast serious doubt on the identification, made previously in the literature, between the alkaline quenching pKa and the pKa of the group whose ionization is coupled to NAD+ binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic effects on modification of charged groups in the active site cleft of subtilisin by protein engineering

The dielectric constant in the active site cleft of subtilisin from Bacillus amyloliquefaciens has been probed by mutating charged residues on the rim and measuring the effect on the pKa value of the active site histidine (His64) by kinetics. Mutation of a negatively charged surface residue, which is 12 to 13 A from His64, to an uncharged one Asp----Ser99) lowers the pKa of the histidine by up to 0.4 unit at low ionic strength (0.005 to 0.01 M). This corresponds to an apparent dielectric constant of about 40 to 50 between Asp99 and His64. The mutation is in an external loop that is known to tolerate a serine at position 99 from homologies with subtilisins from other bacilli. The environment between His64 and Asp99 is predominantly protein. Another charged residue that is at a similar distance from His64 (14 to 15 A) and is also in an external loop that is known to tolerate a serine residue is Glu156, at the opposite side of the active site. There is only water in a direct line between His64 and Glu156. Mutation of Glu----Ser156 also lowers the pKa of His64 by up to 0.4 unit at low ionic strength. This change again corresponds to an apparent dielectric constant of about 40 to 50. The pKa values were determined from the pH dependence of kcat/KM for the hydrolysis of peptide substrates, with a precision of typically +/- 0.02 unit. The following suggests that the changes in pKa are real and not artefacts of experimental conditions: Hill plots of the data for pKa determination have gradients (h) of -1.00(+/- 0.02), showing that there are negligible systematic deviations from theoretical ionization curves involving a monobasic acid: the pH dependence for the hydrolysis of two different substrates (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl p-nitroanilide and benzoyl-L-valyl-L-glycyl-L-arginyl p-nitroanilide) gives identical results so that the pKa is independent of substrate; the pH dependence is unaffected by changing the concentration of enzyme, so that aggregation is not affecting the results; the shift in pKa is masked by high ionic strength, as expected qualitatively for ionic shielding of electrostatic interactions.     

Enzymatic lipid removal from surfaces--lipid desorption by a pH-induced "electrostatic explosion"

Removal of lipidic molecules from surfaces can be accomplished using detergents containing lipases. Surface cleaning is usually performed under alkaline conditions due to increased solubility of the hydrolysis products, especially free fatty acids. This paper shows that removal of a triacylglycerol film from a surface can be dramatically enhanced in a sequential system where pH is shifted to alkaline conditions after an initial lipolytic reaction period at or below neutral pH. Data from three different biophysical techniques, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), quartz crystal microbalance with dissipation monitoring (QCM-D), and total internal reflection fluorescence spectroscopy (TIRF) clearly show the effects of such cleaning procedure. Initially the reaction is carried out at pH below the pKa value of the fatty acids formed upon triacylglycerol hydrolysis, and the protonated fatty acids accumulate in the film. The mechanism of lipid removal, induced by increasing pH to a value above the fatty acid pKa, is explained by a burst caused by electrostatic repulsion between rapidly ionised fatty acids, i.e. by an "electrostatic explosion". Performing the initial hydrolysis at pH 6 and the subsequent rinse at pH 10, using triolein as model substrate, lipid removal from surfaces by both commercial detergent lipases and non-commercial lipases was significantly improved compared to a reaction at constant pH 10.     

A probe of the active site acidity of carboxypeptidase A

The substrate analogue 2-(1-carboxy-2-phenylethyl)-4-phenylazophenol is a potent competitive inhibitor of carboxypeptidase A. Upon ligation to the active site, the azophenol moiety undergoes a shift of pKa from a value of 8.76 to a value of 4.9; this provides an index of the Lewis acidity of the active site zinc ion. Examination of the pH dependence of Ki for the inhibitor shows maximum effectiveness in neutral solution (limiting Ki = 7.6 X 10(-7) M), with an increase in Ki in acid (pK1 = 6.16) and in alkaline solution (pK2 = 9.71, pK3 = 8.76). It is concluded that a proton-accepting enzymic functional group with the lower pKa (6.2) controls inhibitor binding, that ionization of this group is also manifested in the hydrolysis of peptide substrates (kcat/Km), and that the identity of this group is the water molecule that binds to the active site metal ion in the uncomplexed enzyme (H2OZn2+L3). Reverse protonation state inhibition is demonstrated, and conventional concepts regarding the mechanism of peptide hydrolysis by the enzyme are brought into question.     

Implications for an ionized alkyl-enzyme intermediate during StEH1-catalyzed trans-stilbene oxide hydrolysis

The catalytic mechanism of epoxide hydrolase (EC 3.3.2.3) involves acid-assisted ring opening of the oxirane during the alkylation half-reaction of hydrolysis. Two tyrosyl residues in the active site of epoxide hydrolases have been shown to contribute to the catalysis of enzyme alkylation, but their mechanism of action has not been fully described. We have investigated the involvement of the active site Tyr154 and Tyr235 during S,S-trans-stilbene oxide hydrolysis catalyzed by potato epoxide hydrolase StEH1. Tyr phenol ionizations of unliganded enzyme as well as under pre-steady-state conditions during catalysis were studied by direct absorption spectroscopy. A transient UV absorption, indicative of tyrosinate formation, was detected during the lifetime of the alkyl-enzyme intermediate. The apparent pKa of Tyr ionization was 7.3, a value more than 3 pH units below the estimated pKa of protein Tyr residues in the unliganded enzyme. In addition, the pH dependencies of microscopic kinetic rates of catalyzed S,S-trans-stilbene oxide hydrolysis were determined. The alkylation rate increased with pH and displayed a pKa value identical to that of Tyr ionization (7.3), whereas the reverse (epoxidation) reaction did not display any pH dependence. The rate of alkyl-enzyme hydrolysis was inversely dependent on tyrosinate formation, decreasing with its buildup in the active site. Since alkyl-enzyme hydrolysis is the rate-limiting step of the overall reaction, kcat displayed the same decrease with pH as the hydrolysis rate. The compiled results suggested that the role of the Tyr154/Tyr235 pair was not as ultimate proton donor to the alkoxide anion but to stabilize the negatively charged alkyl-enzyme through electrophilic catalysis via hydrogen bonding.     

Biochemical and kinetic analysis of the GH3 family beta-xylosidase from Aspergillus awamori X-100

The beta-xylosidase from Aspergillus awamori X-100 belonging to the family 3 glycoside hydrolase revealed a distinctive transglycosylating ability to produce xylooligosaccharides with degree of polymerization more than 7. In order to explain this fact, the enzyme has been subjected to the detailed biochemical study. The enzymatic hydrolysis of p-nitrophenyl beta-D-xylopyranoside was found to occur with overall retention of substrate anomeric configuration suggesting cleavage of xylosidic bonds through a double-displacement mechanism. Kinetic study with aryl beta-xylopyranosides substrates, in which leaving group pK(a)s were in the range of 3.96-10.32, revealed monotonic function of log(k(cat)) and no correlation of log(k(cat)/Km) versus pKa values indicating deglycosylation as a rate-limiting step for the enzymatic hydrolysis. The classical bell-shaped pH dependence of k(cat)/Km indicated two ionizable groups in the beta-xylosidase active site with apparent pKa values of 2.2 and 6.4. The kinetic parameters of hydrolysis, Km and k(cat), of p-nitrophenyl beta-D-1,4-xylooligosaccharides were very close to those for hydrolysis of p-nitrophenyl-beta-D-xylopyranoside. Increase of p-nitrophenyl-beta-D-xylopyranoside concentration up to 80 mM led to increasing of the reaction velocity resulting in k(cat)(app)=81 s(-1). Addition of alpha-methyl D-xylopyranoside to the reaction mixture at high concentration of p-nitrophenyl-beta-D-xylopyranoside (50 mM) caused an acceleration of the beta-xylosidase-catalyzed reactions and appearance of a new transglycosylation product, alpha-methyl D-xylopyranosyl-1,4-beta-D-xylopyranoside, that was identified by 1H NMR spectroscopy. The kinetic model suggested for the enzymatic reaction was consistent with the results obtained.     

Effect of enzyme and ligand protonation on the binding of folates to recombinant human dihydrofolate reductase: implications for the evolution of eukaryotic enzyme efficiency.

There is marked pH dependence of the rate constant (koff) for tetrahydrofolate (H4folate) dissociation from its ternary complex with human dihydrofolate reductase (hDHFR) and NADPH. Similar pH dependence of H4folate dissociation from the ternary complex of a variant of hDHFR with the substitution Phe31----Leu (F31L hDHFR) causes this dissociation to become rate limiting in the enzyme mechanism at pH approximately 5, and this accounts for the marked decrease in kcat for this variant as the pH is decreased from 7 to 5. This decreased kcat at low pH is not seen for most DHFRs. koff for dissociation of folate, dihydrofolate (H2folate), and H4folate from their binary complexes with hDHFR is similarly pH dependent. For all the complexes examined, the pH dependence of koff in the range pH 5-7 is well described by a pKa of about 6.2 and must be due to ionization of a group on the enzyme. In the higher pH range (7-10), koff increases further as the pH is raised, and this relation is governed by a second pKa which is close to the pKa for ionization of the amide group (HN3-C4O) of the respective ligands. Thus, ionization of the ligand amide group also increases koff. Evidence is presented that the dependence of pH on koff for hDHFR accounts for the shape of the kcat versus pH curve for both hDHFR as well as its F31L variant and contributes to the higher efficiency of hDHFR compared with bacterial DHFR.     

Kinetics of the oxidation of reduced nicotinamide adenine dinucleotide by horseradish peroxidase compounds I and II

The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 degrees C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I-NADH reaction can be explained in terms of a single ionization of pKa = 4.7 +/- 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-II-NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 +/- 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II-NADH reaction was observed. Over the pH range of 4-10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 X 10(5) to 5.6 X 10(2) M-1 s-1 and of HRP-II with NADH varied from 4.4 X 10(4) to 4.1 M-1 s-1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH.     

Sequence-specific Ni(II)-dependent peptide bond hydrolysis in a peptide containing threonine and histidine residues

Previously we demonstrated that Ni(II) complexes of Ac-Thr-Glu-Ser-His-His-Lys-NH2 hexapeptide, representing residues 120-125 of human histone H2A, and some of its analogs undergo E-S peptide bond hydrolysis. In this work we demonstrate a similar coordination and reactivity pattern in Ni(II) complexes of Ac-Thr-Glu-Thr-His-His-Lys-NH2, its threonine analogue, studied using potentiometry, electronic absorption spectroscopy and HPLC. For the first time we present the detailed temperature and pH dependence of such Ni(II)-dependent hydrolysis reactions. The temperature dependence of the rate of hydrolysis yielded activation energy E(a) = 92.0 kJ mol(-1) and activation entropy DeltaS# = 208 J mol(-1) K(-1). The pH profile of the reaction rate coincided with the formation of the four-nitrogen square-planar Ni(II) complex of Ac-Thr-Glu-Thr-His-His-Lys-NH2. These results expand the range of protein sequences susceptible to Ni(II) dependent cleavage by those containing threonine residues and permit predictions of the course of this reaction at various temperatures and pH values.     

Catalytic role of the metal ion of carboxypeptidase A in ester hydrolysis.

The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of cryoenzymologic methods. Kinetic studies of the hydrolysis of the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate have been carried out with both the native and the Co2+-substituted enzyme in the 25 to --45 degrees C temperature range. In the --25 to --45 degrees C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (ks) processes. In Arrhenius plots, ks extrapolates to kcat at 25 degrees C for both enzymes in aqueous solution, indicating that the same catalytic rate-limiting step is observed. The slow process is analyzed for both metal enzymes, as previously reported (Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc Natl. Acad. Sci. U. S. A. 73, 3882-3886), to involve the deacylation of a mixed anhydride acyl-enzyme intermediate. Near --60 degrees C the acyl-enzyme intermediate of both metal enzymes can be stabilized for spectral characterization. The pH and temperature dependence of ks reveals a catalytic ionizing group with a metal ion-dependent shift in pKa and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Co2+ enzyme. These parameters identify the ionizing catalytic group as the metal-bound water molecule. Extrapolation of the pKa data to 25 degrees C indicates that this ionization coincides with that observed in the acidic limb of the pH profile of log(kcat/Km(app)) for substrate hydrolysis under steady state conditions. The results indicate that in the esterolytic reaction of carboxypeptidase. A deacylation of the mixed anhydride intermediate is catalyzed by a metal-bound hydroxide group.