Phosphotransferase activity associated with rat osseous plate alkaline phosphatase: a possible role in biomineralization.

1. Alkaline phosphatase from rat osseous plate catalyzed the transfer of phosphate from p-nitrophenylphosphate to glycerol, ethanolamines, Tris, glucose and 1-amino-1-methyl-2-propanol, in a wide range of pH. Serine did not stimulate phosphotransferase activity of the enzyme. 2. The best phosphotransferase acceptors were diethanolamine and glycerol while glucose was the poorest phosphotransferase acceptor used. 3. Diethanolamine and glycerol affected both VM and KM of p-nitrophenylphosphate hydrolysis with activation constants (KA) of 0.25 and 0.85 M, respectively. 4. A kinetic model was proposed for the phosphotransferase reaction observed with alkaline phosphatase from rat osseous plates.     

Reactions of prostaglandin H synthase in the presence of the stabilizing agents diethyldithiocarbamate and glycerol

Both cyclooxygenase and peroxidase reactions of prostaglandin H synthase were studied in the presence and absence of diethyldithiocarbamate and glycerol at 4 degrees C in phosphate buffer (pH 8.0). Diethyldithiocarbamate reacts with the high oxidation state intermediates of prostaglandin H synthase; it protects the enzyme from bleaching and loss of activity by its ability to act as a reducing agent. For the reaction of diethyldithiocarbamate with compound I, the second-order rate constant k2,app, was found to fall within the range of 5.8 x 10(6) +/- 0.4 x 10(6) M-1.s-1 less than k2,app less than 1.8 x 10(7) +/- 0.1 x 10(7) M-1.s-1. The reaction of diethyldithiocarbamate with compound II showed saturation behavior suggesting enzyme-substrate complex formation, with kcat = 22 +/- 3 s-1, Km = 67 +/- 10 microM, and the second-order rate constant k3,app = 2.0 x 10(5) +/- 0.2 x 10(5) M-1.s-1. In the presence of both diethyldithiocarbamate and 30% glycerol, the parameters for compound II are kcat = 8.8 +/- 0.5 s-1, Km = 49 +/- 7 microM, and k3,app = 1.03 x 10(5) +/- 0.07 x 10(5) M-1.s-1. The spontaneous decay rate constants of compounds I and II (in the absence of diethyldithiocarbamate) are 83 +/- 5 and 0.52 +/- 0.05 s-1, respectively, in the absence of glycerol; in the presence of 30% glycerol they are 78 +/- 5 and 0.33 +/- 0.02 s-1, respectively. Neither cyclooxygenase activity nor the rate constant for compound I formation using 5-phenyl-4-pentenyl-1-hydroperoxide is altered by the presence of diethyldithiocarbamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alkaline phosphatase. 31P NMR probes of the mechanism

31P NMR signals from substrates and products of alkaline phosphatase have been adapted to measure the rates and product ratios for the hydrolysis and phosphotransferase reactions from pH 6 to 10. Below pH 8, glycerol is a poorer acceptor than H2O (glycerol phosphates:Pi = 0.5). Tris is a more effective acceptor below pH 8, showing a maximum acceptor efficiency at pH 8 (Tris phosphate:Pi = 2). Phosphotransferase efficiencies are in the order expected for the pKaS of the alcohol groups, Tris less than glycerol Cl, C3 less than glycerol C2. Tris and glycerol induce chemical shifts in 113Cd(II) present at the A site but not the B or C sites of the metal triad present at each active center of Cd(II)6 alkaline phosphatase, suggesting that the alcoxides of the acceptors coordinate the A site metal and become the nucleophiles attacking the phosphoseryl residue (E-P) in the second step of the mechanism. The interaction is through the oxygen of Tris. The transferase activity of the amino alcohol shows a bell-shaped pH dependency. Aliphatic alcohol acceptors show small increases in acceptor activity between pH 6 and 8, with 5-fold increases from pH 8 to 10 (at pH 10, glycerol phosphates:Pi = 2.5). 31P NMR inversion transfer has been used to measure the koff for Pi dissociation from the noncovalent enzyme complex (E . P). For the Zn(II)4 alkaline phosphatase koff is essentially pH independent at approximately 35 s-1. For Cd(II) or Mg(II) at the B site in place of Zn(II), koff less than or equal to 1 s-1 X Cl-ion, which appears to coordinate the A site metal ion, enhances koff, suggesting that both Cl- and HPO2-4 can coordinate the A site metal ion in a 5-coordinate intermediate. pH control of the alkaline phosphatase mechanism appears to reside in the stability of E-P and not the dissociation of E . P, compatible with the hypothesis that the activity-linked pKa is that of a H2O molecule coordinated to the A site metal, which in the hydroxide form becomes the nucleophile attacking the phosphoseryl group (E-P).     

Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The complete time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the low molecular weight (acid) phosphotyrosyl protein phosphatase from bovine heart was elucidated and analyzed in detail. Burst titration kinetics were demonstrated for the first time with this class of enzyme. At pH 7.0, 4.5 degrees C, a transient pre-steady-state "burst" of p-nitrophenol was formed with a rate constant of 48 s-1. The burst was effectively stoichiometric and corresponded to a single enzyme active site/molecule. The burst was followed by a slow steady-state turnover of the phosphoenzyme intermediate with a rate constant of 1.2 s-1. Product inhibition studies indicated an ordered uni-bi kinetic scheme for the hydrolysis. Partition experiments conducted for several substrates revealed a constant product ratio. Vmax was constant for these substrates, and the overall rate of hydrolysis was increased greatly in the presence of alcohol acceptors. An enzyme-catalyzed 18O exchange between inorganic phosphate and water was detected and occurred with kcat = 4.47 x 10(-3) s-1 at pH 5.0, 37 degrees C. These results were all consistent with the existence of a phosphoenzyme intermediate in the catalytic pathway and with the breakdown of the intermediate being the rate-limiting step. The true Michaelis binding constant Ks = 6.0 mM, the apparent Km = 0.38 mM, and the rate constants for phosphorylation (k2 = 540 s-1) and dephosphorylation (k3 = 36.5 s-1) were determined under steady-state conditions with p-nitrophenyl phosphate at pH 5.0 and 37 degrees C in the presence of phosphate acceptors. The energies of activation for the enzyme-catalyzed hydrolysis at pH 5.0 and 7.0 were 13.6 and 14.1 kcal/mol, respectively. The activation energy for the enzyme-catalyzed medium 18O exchange between phosphate and water was 20.2 kcal/mol. Using the available equilibrium and rate constants, an energetic diagram was constructed for the enzyme-catalyzed reaction.     

Mechanisms of acylation of chymotrypsin by phenyl esters of benzoic acid and acetic acid.

The kinetics of the acylation of alpha-chymotrypsin by a series of substituted phenyl p-nitrobenzoates have been studied by stopped flow and conventional spectrophotometry. Electron withdrawal in the leaving group accelerates the rate of acylation, and the p value obtained for eight esters is +1.96. The pH- and pD-independent acylation rate constants are, respectively, 1.40 X 10(4) M-1S-1 and 1.23 X 10(4) M-1S-1 for p-nitrophenyl p-nitrobenzoate, and, respectively, 2.19 X 10(3) M-1S-1 and 1968 X 10(3) M-1S-1 for p-nitrophenyl benzoate at 25 degrees. An analysis of structure-reactivity results and kinetic solvent isotope effects indicates a mechanism for acylation by phenylbenzoates in which initial reaction is a nucleophilic attack by an imidazole of the enzyme (His 57). Subsequently, there is rapid transfer of the acylating group to the serine 195 from the acylimidazole species. The kinetic solvent isotope effects for acylation by p-nitrophenyl phenyl acetate and p-nitrophenyl phenyl acetate and p-nitrophenyl hydrocinnamate, in 5%, v/v, acetonitrile, are 1.3 and 2.0, respectively. The latter ester is inhibited more than is p-nitrophenyl benzoate when 5%, v/v, dioxane is substituted for 5%, v/v, acetonitrile as co-solvent. In the presence of 5%, v/v, dioxane a change in the kinetic solvent isotope effect to 1.7 is found for p-nitrophenyl benzoate and p-nitrophenyl phenylacetate while that for the analogous hysdrocinnamate ester is unaffected. The results for the latter substrate are in accord with a general base-catalysed mechanism. Electron-withdrawal groups in the phenyl ring of phenyl acetates accelerate the enzyme acylation yielding a leaving group p of 2.05. The kinetic solvent isotope effects for acylation by p-nitrophenyl thiolacetate and by p-nitrophenyl acetate are close to 2.0. The mechanism of acylation of chymotrypsin by phenyl acetates is not unambiguously defined using these data.     

A pre-steady-state kinetic analysis of substrate binding to human recombinant deoxycytidine kinase: a model for nucleoside kinase action.

Deoxycytidine kinase (dCK) is an enzyme with broad substrate specificity which can phosphorylate pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. In this study, stopped-flow experiments were used to monitor intrinsic fluorescence changes induced upon binding of various phosphate donors (ATP, UTP, and the nonhydrolyzable analogue AMP-PNP) and the acceptor dCyd to recombinant dCK. Monophasic kinetics were observed throughout. The nucleotides as well as dCyd bound to the enzyme by a two-step mechanism, involving a rapid initial equilibrium step, followed by a protein conformational change that is responsible for the fluorescence change. The bimolecular association rate constants for nucleotide binding [(4-10) x 10(3) M-1 s-1] were 2-3 orders of magnitude lower than those for dCyd binding [(1.3-1.5 x 10(6) M-1 s-1]. This difference most likely is due predominantly to the large difference in the forward rate constants of the conformational changes (0.04-0.26 s-1 vs 560-710 s-1). Whereas the kinetics of the binding of ATP, UTP, and AMP-PNP to dCK showed some differences, UTP exhibiting the tightest binding, no significant differences were observed for the binding of dCyd to dCK in the presence or absence of phosphate donors. However, the binding of dCyd to dCK in the presence of ATP or UTP was accompanied by a 1.5- or 3-fold higher quenching amplitude as compared with dCyd alone or in the presence of AMP-PNP. We conclude that ATP and UTP induce a conformational change in the enzyme, thereby enabling efficient phosphoryl transfer.     

A kinetic study of protein-protein interactions.

Kinetic studies have been carried out of the monomer-dimer interaction of insulin, beta-lactoglobulin, and alpha-chymotrypsin using stopped-flow and temperature-jump techniques. The pH indicators bromothymol blue, bromophenol blue, and phenol red were used to monitor pH changes associated with the monomer-dimer interaction. In all three cases a kinetic process was observed which could be attributed to a simple monomer-dimer equilibrium, and association (k1) and dissociation (k-1) rate constants were determined. The results obtained are as follows: for insulin at 23 degrees C, pH 6.8, 0.125 M KNO3, k1 = 1.14 X 10(8) M-1 s-1, k-1 - 1.48 X 10(4)s(-1); for beta-lactoglobulin AB at 35 degrees C, pH 3.7, 0.025 M KNO3, d1 = 4.7 X 10(4) M-1 s-1, k-1 = 2.1 s-1; for alpha-chymotrypsin at 25 degreesC, pH 4.3, 0.05 M KNO3 k1 - 3.7 X 10(3) M-1 s-1, k-1 - 0.68 s-1. The kinetic behavior of the separated beta-lactoglobulin A and B was similar to that of the mixture. In the case of chymotrypsin, bromophenol blue was found to activate the enzyme catalyzed hydrolysis of p-nitrophenyl acetate, and a rate process was observed with the temperature jump which could be attributed to a conformational change of the indicator-protein complex. The association rate constant for dimer formation of insulin approaches the value expected for a diffusion-controlled process, while the values obtained for the other two proteins are below those expected for a diffusion-controlled reaction unless unusally large steric and electrostatic effects are present.     

Engineering E. coli Alkaline Phosphatase Yields Changes of Catalytic Activity,Thermal Stability and Phosphate Inhibition

To investigate the function of aspartic acid residue 101 and arginine residue 166 in the active site of Escherichia coli alkaline phosphatase (EAP), two single mutants D101S (Asp 101 &#77 Ser) and R166K (Arg 166 &#77 Lys) and a double mutant D101S/R166K of EAP were generated through site-directed mutagenesis based on over-lap PCR method. Their enzymatic kinetic properties, thermal stabilities and possible reaction mechanism were explored. In the presence of inorganic phosphate acceptor, 1 M diethanolamine buffer, the k cat for D101S mutant enzyme increased 10-fold compared to that of wild-type EAP. The mutant R166K has a 2-fold decrease of k cat relative to the wild-type EAP, but the double mutant D101S/R166K was in the middle of them, indicative of an additive effect of these two mutations. On the other hand, the catalytic efficiencies of mutant enzymes are all reduced because of a substantial increase of K m values. All three mutants were more resistant to phosphate inhibitor than the wild-type enzyme. The analysis of the kinetic data suggests that (1) the D101S mutant enzyme obtains a higher catalytic activity by allowing a faster release of the product; (2) the R166K mutant enzyme can reduce the binding of the substrate and phosphate competitive inhibitor; (3) the double mutant enzyme has characteristics of both quicker catalytic turnover number and decreased affinity for competitive inhibitor. Additionally, pre-steady-state kinetics of D101S and D101S/R166K mutants revealed a transient burst followed by a linear steady state phase, obviously different from that of wild-type EAP, suggesting that the rate-limiting step has partially change from the release of phosphate from non-covalent E-Pi complex to the hydrolysis of covalent E-Pi complex for these two mutants.     

Engineering E. coli Alkaline Phosphatase Yields Changes of Catalytic Activity, Thermal Stability and Phosphate Inhibition

To investigate the function of aspartic acid residue 101 and arginine residue 166 in the active site of Escherichia coli alkaline phosphatase (EAP), two single mutants D101S (Asp 101 →Ser) and R166K (Arg 166 →Lys) and a double mutant D101S/R166K of EAP were generated through site-directed mutagenesis based on over-lap PCR method. Their enzymatic kinetic properties, thermal stabilities and possible reaction mechanism were explored. In the presence of inorganic phosphate acceptor, 1 M diethanolamine buffer, the k cat for D101S mutant enzyme increased 10-fold compared to that of wild-type EAP. The mutant R166K has a 2-fold decrease of k cat relative to the wild-type EAP, but the double mutant D101S/R166K was in the middle of them, indicative of an additive effect of these two mutations. On the other hand, the catalytic efficiencies of mutant enzymes are all reduced because of a substantial increase of K m values. All three mutants were more resistant to phosphate inhibitor than the wild-type enzyme. The analysis of the kinetic data suggests that (1) the D101S mutant enzyme obtains a higher catalytic activity by allowing a faster release of the product; (2) the R166K mutant enzyme can reduce the binding of the substrate and phosphate competitive inhibitor; (3) the double mutant enzyme has characteristics of both quicker catalytic turnover number and decreased affinity for competitive inhibitor. Additionally, pre-steady-state kinetics of D101S and D101S/R166K mutants revealed a transient burst followed by a linear steady state phase, obviously different from that of wild-type EAP, suggesting that the rate-limiting step has partially change from the release of phosphate from non-covalent E-Pi complex to the hydrolysis of covalent E-Pi complex for these two mutants.     

Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity

The function of aspartic acid residue 101 in the active site of Escherichia coli alkaline phosphatase was investigated by site-specific mutagenesis. A mutant version of alkaline phosphatase was constructed with alanine in place of aspartic acid at position 101. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, pH 8.0, both the kcat and the Km for the mutant enzyme increase by approximately 2-fold, resulting in almost no change in the kcat/Km ratio. Under conditions of no external phosphate acceptor and pH 8.0, both the kcat and the Km for the mutant enzyme decrease by approximately 2-fold, again resulting in almost no change in the kcat/Km ratio. The kcat for the hydrolysis of 4-methyl-umbelliferyl phosphate and p-nitrophenyl phosphate are nearly identical for both the wild-type and mutant enzymes, as is the Ki for inorganic phosphate. The replacement of aspartic acid 101 by alanine does have a significant effect on the activity of the enzyme as a function of pH, especially in the presence of a phosphate acceptor. At pH 9.4 the mutant enzyme exhibits 3-fold higher activity than the wild-type. The mutant enzyme also exhibits a substantial decrease in thermal stability: it is half inactivated by treatment at 49 degrees C for 15 min compared to 71 degrees C for the wild-type enzyme. The data reported here suggest that this amino acid substitution alters the rates of steps after the formation of the phospho-enzyme intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amine inversion in proteins. A 13C-NMR study of proton exchange and nitrogen inversion rates in N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine,N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine methyl ester, and reductively methylated concanavalin A

Exchange rates were calculated as a function of pH from line widths of methylamine resonances in 13C-NMR spectra of N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine (TML) and N epsilon,N epsilon,N alpha,N alpha-tetramethyllysine methyl ester (TMLME). The pH dependence of the dimethyl alpha-amine exchange rate could be adequately described by assuming base-catalyzed chemical exchange between two diastereotopic methyl populations related by nitrogen inversion. Deprotonation of the alpha-amine was assumed to occur by proton transfer to (1) OH-, (2) water, (3) a deprotonated amine or (4) RCO2-. Microscopic rate constants characterizing each of these transfer processes (k1, k2, k3 and k4, respectively) were determined by fitting the rates calculated from line width analysis to a steady-state kinetic model. Using this procedure it was determined that for both TML and TMLME k2 approximately equal to 1-10 M-1 s-1, k3 approximately equal to 10(6) M-1 s-1 and ki, the rate constant for nitrogen inversion was about 10(8)-10(9) s-1. Upper limits of 10(12) and 10(3) M-1 s-1 could be determined for k1 and k4, respectively. A similar kinetic analysis was used to explain pH-dependent line-broadening effects observed for the N-terminal dimethylalanyl resonance in 13C-NMR spectra of concanavalin A, reductively methylated using 90% [13C]formaldehyde. From exchange data below pH 4 it could be determined that amine inversion was limited by the proton transfer rate to the solvent, with a rate constant estimated at 20 M-1 s-1. Above pH 4, exchange was limited by proton transfer to other titrating groups in the protein structure. Based upon their proximity, the carboxylate side chains of Asp-2 and Asp-218 appear to be likely candidates. The apparent first-order microscopic rate constant characterizing proton transfer to these groups was estimated to be about 1 X 10(4) s-1. Rate constants characterizing nitrogen inversion (ki), proton transfer to OH- (k1) and proton transfer to the solvent (k2) were estimated to be of the same order of magnitude as those determined for the model compounds. On the basis of our results, it is proposed that chemical exchange processes associated with base-catalyzed nitrogen inversion may contribute to 15N or 13C spin-lattice relaxation times in reductively methylated peptides or proteins.     

DNA strand transfer catalyzed by vaccinia topoisomerase: peroxidolysis and hydroxylaminolysis of the covalent protein-DNA intermediate

Vaccinia topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at sites containing the sequence 5'-CCCTT downward arrow. The covalently bound topoisomerase can religate the CCCTT strand to a 5'-OH-terminated polynucleotide or else transfer the strand to a non-DNA nucleophile such a water or glycerol. Here, we report that vaccinia topoisomerase also catalyzes strand transfer to hydrogen peroxide. The observed alkaline pH-dependence of peroxidolysis is consistent with enzyme-mediated attack by peroxide anion on the covalent intermediate. The reaction displays apparent first-order kinetics. From a double-reciprocal plot of k(obs) versus [H(2)O(2)] at pH 10, we determined a rate constant for peroxidolysis of 6.3 x 10(-)(3) s(-)(1). This rate is slower by a factor of 200 than the rate of topoisomerase-catalyzed strand transfer to a perfectly aligned 5'-OH DNA strand but is comparable to the rate of DNA strand transfer across a 1-nucleotide gap. Strand transfer to 2% hydrogen peroxide is 300 times faster than strand transfer to 20% glycerol and approximately 2000 times faster than topoisomerase-catalyzed hydrolysis of the covalent intermediate. Hydroxylamine is also an effective nucleophile in topoisomerase-mediated strand transfer (k(obs) = 6.4 x 10(-)(4) s(-)(1)). The rates of the peroxidolysis, hydroxylaminolysis, glycerololysis, and hydrolysis reactions catalyzed by the mutant enzyme H265A were reduced by factors of 100-700, in accordance with the 100- to 400-fold rate decrements in DNA cleavage and religation by H265A. We surmise that vaccinia topoisomerase catalyzes strand transfer to DNA and non-DNA nucleophiles via a common reaction pathway in which His-265 stabilizes the scissile phosphate in the transition state rather than acting as a general acid or base.     

Kinetic investigations of the reactions of cytochrome c oxidase with hydrogen peroxide

The reaction of H2O2 with reduced cytochrome c oxidase was investigated with rapid-scan/stopped-flow techniques. The results show that the oxidation rate of cytochrome a3 was dependent upon the peroxide concentration (k = 2 X 10(4) M-1 X s-1). Cytochrome a and CuA were oxidised with a maximal rate of approx. 20 s-1, indicating that the rate of internal electron transfer was much slower with H2O2 as the electron acceptor than with O2 (k greater than or equal to 700 s-1). Although other explanations are possible, this result strongly suggests that in the catalytic cycle with oxygen as a substrate the internal electron-transfer rate is enhanced by the formation of a peroxo-intermediate at the cytochrome a3-CuB site. It is shown that H2O2 took up two electrons per molecule. The reaction of H2O2 with oxidised cytochrome c oxidase was also studied. It is shown that pulsed oxidase readily reacted with H2O2 (k approximately 700 M-1 X s-1). Peroxide binding is followed by an H2O2-independent conformational change (k = 0.9 s-1). Resting oxidase partially bound H2O2 with a rate similar to that of pulsed oxidase; after H2O2 binding the resting enzyme was converted into the pulsed conformation in a peroxide-independent step (k = 0.2 s-1). Within 5 min, 55% of the resting enzyme reacted in a slower process. We conclude from the results that oxygenated cytochrome c oxidase probably is an enzyme-peroxide complex.     

The oxidation of cytochrome c oxidase by hydrogen peroxide

The reaction of H2O2 with mixed-valence and fully reduced cytochrome c oxidase was investigated by photolysis of fully reduced and mixed-valence carboxy-cytochrome c oxidase in the presence of H2O2 under anaerobic conditions. The results showed that H2O2 reacted rapidly (k = (2.5-3.1) X 10(4) M-1 X s-1) with both enzyme species. With the mixed-valence enzyme, the fully oxidised enzyme was reformed. On the time-scale of our experiments, no spectroscopically detectable intermediate was observed. This demonstrates that mixed-valence cytochrome c oxidase is able to use H2O2 as a two-electron acceptor, suggesting that cytochrome c oxidase may under suitable conditions act as a peroxidase. Upon reaction of H2O2 with the fully reduced enzyme, cytochrome a was oxidised before cytochrome a3. From this observation it was possible to estimate that the rate of electron transfer from cytochrome a to a3 is about 0.5-5 s-1.     

A novel intermediate in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase.

An unusual intermediate bound to the enzyme was detected in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase. This intermediate had absorbance maxima at 464 and 440 nm. Such spectra are characteristic of resonance stabilized intermediates detected in the interaction of substrates and quasi-substrates with pyridoxal phosphate enzymes. An intermediate of this kind has not been detected in the interaction of thiosemicarbazide with other pyridoxal phosphate enzymes. This intermediate was generated slowly (t 1/2 = 4 min) following the addition of thiosemicarbazide (200 microM) to sheep liver serine hydroxymethyltransferase (5 microM). It was bound to the enzyme as evidenced by circular dichroic bands at 464 and 440 nm and the inability to be removed upon Centricon filtration. The kinetics of interaction revealed that thiosemicarbazide was a slow binding reversible inhibitor in this phase with a k(on) of 11 M-1 s-1 and a k(off) of 5 x 10(-4) s-1. The intermediate was converted very slowly (k = 4 x 10(-5) s-1) to the final products, namely the apoenzyme and the thiosemicarbazone of pyridoxal phosphate. A minimal kinetic mechanism involving the initial conversion to the intermediate absorbing at longer wavelengths and the conversion of this intermediate to the final product, as well as, the formation of pyridoxal phosphate-thiosemicarbazone directly by an alternate pathway is proposed.     

The reaction of choline acetyltransferase with sulfhydryl reagents. Methoxycarbonyl-CoA disulfide as an active site-directed reagent.

The reaction of choline acetyltransferase with methoxycarbonyl alkyl disulfides leads to a progressive loss in enzyme activity as the size of the alkyl group increases from methyl to n-butyl. Reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or methoxycarbonyl coenzyme A (CoA) disulfide, leads to a total loss of enzyme activity. DTNB inactivation is biphasic (k1 = approximately 9 x 10(2) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1) with the slow phase being diminished by acetyl-CoA. Methoxycarbonyl-CoA disulfide inactivation is also biphasic (k1 = approximately 2.1 x 10(3) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1), with the rapid phase being diminished in the presence of acetyl-CoA. Inactivation by methoxycarbonyl methyl disulfide, ethyl disulfide, or hydroxyethyl disulfide, or by methyl methanethiosulfonate is not biphasic. Pretreatment of the enzyme with methyl methanethiosulfonate, which leads to a 25% loss in enzyme activity, abolishes the fast phase of DTNB inactivation, the slow phase of methoxycarbonyl-CoA disulfide inactivation, and any further inactivation by methoxycarbonyl ethyl disulfide. These results are interpreted to suggest that choline acetyltransferase contains two classes of reactive sulfhydryl groups, neither of which are required for enzyme activity.     

Laser flash photolysis study of intermolecular and intramolecular electron transfer in trimethylamine dehydrogenase

Laser flash photolysis has been used to investigate the kinetics of reduction of trimethylamine dehydrogenase by substoichiometric amounts of 5-deazariboflavin semiquinone, and the subsequent intramolecular electron transfer from the FMN cofactor to the Fe4S4 center. The initial reduction event followed second-order kinetics (k = 1.0 x 10(8) M-1 s-1 at pH 7.0 and 6.4 x 10(7) M-1 s-1 at pH 8.5) and resulted in the formation of the neutral FMN semiquinone and the reduced iron-sulfur cluster (in a ratio of approximately 1:3). Following this, a slower, protein concentration independent (and thus intramolecular) electron transfer was observed corresponding to FMN semiquinone oxidation and iron-sulfur cluster reduction (k = 62 s-1 at pH 7.0 and 30 s-1 at pH 8.5). The addition of the inhibitor tetramethylammonium chloride to the reaction mixture had no effect on these kinetic properties, suggesting that this compound exerts its effect on the reduced form of the enzyme. Treatment of the enzyme with phenylhydrazine, which introduces a phenyl group at the 4a-position of the FMN cofactor, decreased both the rate constant for reduction of the protein and the extent of FMN semiquinone production, while increasing the amount of iron-sulfur center reduction, consistent with the results obtained with the native enzyme. Experiments in which the kinetics of reduction of the enzyme were determined during various stages of partial reduction were also consistent with these results, and further indicated that the FMN semiquinone form of the enzyme is more reactive toward the deazariboflavin reductant than is the oxidized FMN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single electron reduction of 'slow' and 'fast' cytochrome-c oxidase.

Evidence is presented that single electron reduction is sufficient for rapid electron transfer (k greater than 20 s-1 at pH 8.0 in 0.43 M potassium EDTA) between haem a/CuA and the binuclear centre in 'fast' oxidase, whereas in 'slow' oxidase intramolecular electron transfer is slow even when both CuA and haem a are reduced (k congruent to 0.01 s-1). However, while a single electron can equilibrate rapidly between CuA, haem a and CuB in 'fast' oxidase, it seems that equilibration with haem a3 is relatively slow (k congruent to 2 s-1). Electron transfer between cytochrome c and CuA/haem a is similar for both types of enzyme (k congruent to 2.4 x 10(5) M-1.s-1).     

The effect of glycerol on the activity of beta-glucosidase from Botryodiplodia theobromae Pat.

1. In the activity of the high-Mr beta-glucosidase A (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. on o-nitrophenyl beta-D-glucopyranoside as substrate, both Vmax. and Km increased non-linearly with increasing concentration of glycerol, and the Vmax./Km(app.) ratio decreased non-linearly with increasing concentration of glycerol. 2. No increase in rate was observed with phenyl beta-D-glucopyranoside as substrate in the presence of up to 250 mM-glycerol, indicating that glucosylation is rate-limiting with this substrate. 3. With o-nitrophenyl beta-D-glucopyranoside, p-nitrophenyl beta-D-glucopyranoside and phenyl beta-D-glucopyranoside as substrates, kappa cat. values of 793.7 s-1, 62.8 s-1 and 5.4 s-1 respectively were calculated. 4. With o-nitrophenyl beta-D-glucopyranoside and phenyl beta-D-glucopyranoside as substrate, alpha-deuterium kinetic isotope effects of 1.9 +/- 0.03 and 1.01 +/- 0.01 respectively were found; in the presence of 200 mM-glycerol the values were 1.21 +/- 0.03 and 1.02 +/- 0.01 respectively. 5. In the presence of a large excess of o-nitrophenyl beta-D-glucopyranoside [( S] = 35.7 Km), the amount of o-nitrophenol and also of the transglucosylation product formed by beta-glucosidase action increased non-linearly, whereas that of glucose formed decreased non-linearly with increasing glycerol concentration. 6. All these results were found to fit the data calculated from rate equations derived on the basis of the proposed mechanism of enzyme action involving two ion-pair intermediates and a covalent alpha-D-glucosyl-enzyme in the reaction sequence [Umezurike (1987) Biochem. J. 241, 455-462].     

A kinetic study of cyclic adenosine 3':5'-monophosphate binding and mode of activation of protein kinase from Drosophila melanogaster embryos.

Cyclic AMP-dependent protein kinase and its regulatory subunit were isolated from Drosophila melanogaster embryos. The profiles of cyclic AMP binding by these proteins were significantly different. In order to explain such a difference and to find the mode of enzyme activation by cyclic AMP, a kinetic study of cyclic AMP binding was carried out. First, the association rate constant k1 and dissociation rate constant k-1 in the cyclic AMP-regulatory subunit interaction at 0 degrees C were estimated to be 2.3 X 10(6)M-1s-1 and 1.1 X 10(-3)s-1, respectively. Secondly, the three possible modes of enzyme activation by cyclic AMP were mathematically considered and could be described by a unique formula: r=APt + BQt (A + B=1) in which the parameters A, B, P, and Q are equivalent to rate constants in the sense that the rate constants are simply expressed by these parameters. Thirdly, the values of the parameters and subsequently the values of rate constants involved in the possible mechanisms were evaluated using a curve-fitting technique and compared with experimental observation. It was then found that the following mechanism was the only one which fitted the experimental observations. Namely, RC + L k3 equilibrium k-3 LRC k4 equilibrium k-4 RL + C where R, C, and L represent the regulatory and catalytic subunits and cyclic AMP as a ligand. Thus, our results indicate that in the presence of cyclic AMP the active enzyme (C) is released from a ternary intermediate which is the primary product of the cyclic AMP-holoenzyme interaction. The estimated values of the rate constants are: k3=3.5 X 10(6)M-1s-1;k-3=7.3 X 10(-1)s-1;and k4=3.8 X 10(-2)s. These estimates indicate that the reaction LRC leads to RL + C is relatively slow and limits the rate of the overall reaction. By comparing k-3 and k4, it is apparent that a large part of newly formed ternary intermediate reverts to the holoenzyme.