Mechanism of interaction of O-amino-D-serine with sheep liver serine hydroxymethyltransferase

The mechanism of interaction of O-amino-D-serine (OADS) with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in the enzyme activity, absorption spectra, circular dichroism (CD) spectra, and stopped-flow spectrophotometry. OADS was a reversible noncompetitive inhibitor (Ki = 1.8 microM) when serine was the varied substrate. The first step in the interaction of OADS with the enzyme was the disruption of enzyme-Schiff base, characterized by the rapid disappearance of absorbance at 425 nm (6.5 X 10(3) M-1 s-1) and CD intensity at 430 nm. Concomitantly, there was a rapid increase in absorbance and CD intensity at 390 nm. The spectral properties of this intermediate enabled its identification as pyridoxal 5'-phosphate (PLP). These changes were followed by a slow unimolecular step (2 X 10(-3) s-1) leading to the formation of PLP-OADS oxime, which was confirmed by its absorbance and fluorescence spectra and retention time on high-performance liquid chromatography. The PLP-OADS oxime was displaced from the enzyme by the addition of PLP as evidenced by the restoration of complete enzyme activity as well as by the spectral properties. The unique feature of the mechanism proposed for the interaction of OADS with sheep liver SHMT was the formation of PLP as an intermediate.     

Interactions of methoxyamine with pyridoxal-5'-phosphate-Schiff's base at the active site of sheep liver serine hydroxymethyltransferase.

The mechanism of interaction of methoxyamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in enzyme activity, visible absorption spectra, circular dichroism and fluorescence, and by evaluating the rate constant by stopped-flow spectrophotometry. Methoxyamine can be considered as the smallest substituted aminooxy derivative of hydroxylamine. It was a reversible noncompetitive inhibitor (Ki = 25 microM) of SHMT similar to O-amino-D-serine. Like in the interaction of O-amino-D-serine and aminooxyacetic acid, the first step in the reaction was very fast. This was evident by the rapid disappearance of the enzyme-Schiff base absorbance at 425 nm with a rate constant of 1.3 x 10(3) M-1 sec-1 and CD intensity at 430 nm. Concomitantly, there was an increase in absorbance at 388 nm (intermediate I). The next step in the reaction was the unimolecular conversion (1.1 x 10(-3) sec-1) of this intermediate to the final oxime absorbing at 325 nm. The identity of the oxime was established by its characteristic fluorescence emission at 460 nm when excited at 360 nm and by high performance liquid chromatography. These results highlight the specificity in interactions of aminooxy compounds with sheep liver serine hydroxymethyltransferase and that the carboxyl group of the inhibitors enhances the rate of the initial interaction with the enzyme.     

Binding of pyridoxal 5'-phosphate to the heme protein human cystathionine beta-synthase

Cystathionine beta-synthase (CBS), a pyridoxal 5'-phosphate (PLP) dependent enzyme, catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS was recently shown to be a heme protein. While the role of heme in CBS is unknown, catalysis by CBS can be explained solely by participation of PLP in the reaction mechanism. In this study, treatment of CBS with sodium borohydride selectively reduced the Schiff base but did not affect the heme. Purification and sequencing of the PLP-cross-linked peptide from a trypsin digest of the reduced enzyme revealed the evolutionarily conserved Lys119 to be the residue forming the Schiff base. Serine and hydroxylamine form an alpha-aminoacrylate and an oxime with PLP in CBS, respectively. The sulfhydryl-containing substrate, homocysteine, disturbs the heme environment but does not interact with PLP. In contrast to other PLP-dependent enzymes, CBS emits no PLP-related fluorescence when excited at 296 or 330 nm. PLP but not heme dissociates from the enzyme in the presence of hydroxylamine. The dissociation of PLP is a multistage process involving a short approximately 500 s lag phase, followed by a rapid inactivation and a slower PLP-oxime formation. PLP-free CBS exhibits a decrease of secondary structure as well as loss of CBS activity that can be only partially restored by PLP. This study constitutes the first comprehensive investigation of PLP interaction with a heme protein.     

The biological and biochemical properties of L-canaline, a naturally occurring structural analogue of L-ornithine.

Many leguminous plants synthesize L-canavanine and sequester this nitrogen-rich, non-protein amino acid in the seed (1,2). Arginase-mediated hydrolytic cleavage of L-canavanine, in a manner analogous to L-ornithine and urea formation from L-arginine, produces urea and L-canaline (3,4). The resulting canaline is distinctive in being the only naturally occurring amino acid which possesses the aminooxy group (Fig. 1). Canaline decomposes in several organic solvents employed for its analysis by partition and ion-exchange chromatography (5,6) but the important question of the overall stability of this substituted hydroxylamine has not been investigated.     

Chemical modification of 3 alpha,20 beta-hydroxysteroid dehydrogenase with diethyl pyrocarbonate. Evidence for an essential, highly reactive, lysyl residue

Diethyl pyrocarbonate inactivated the tetrameric 3 alpha,20 beta-hydroxysteroid dehydrogenase with second-order rate constants of 1.63 M-1 s-1 at pH 6 and 25 degrees C or 190 M-1 s-1 at pH 9.4 and 25 degrees C. The activity was slowly and partially restored by incubation with hydroxylamine (81% reactivation after 28 h with 0.1 M hydroxylamine, pH 9, 25 degrees C). NADH protected the enzyme against inactivation with a Kd (10 microM) very close to the Km (7 microM) for the coenzyme. The ultraviolet difference spectrum of inactivated vs. native enzyme indicated that a single histidyl residue per enzyme subunit was modified by diethyl pyrocarbonate, with a second-order rate constant of 1.8 M-1 s-1 at pH 6 and 25 degrees C. The histidyl residue, however, was not essential for activity because in the presence of NADH it was modified without enzyme inactivation and modification of inactivated enzyme was rapidly reversed by hydroxylamine without concomitant reactivation. Progesterone, in the presence of NAD+, protected the histidyl residue against modification, and this suggests that the residue is located in or near the steroid binding site of the enzyme. Diethyl pyrocarbonate also modified, with unusually high reaction rate, one lysyl residue per enzyme subunit, as demonstrated by dinitrophenylation experiments carried out on the treated enzyme. The correlation between inactivation and modification of lysyl residues at different pHs and the protection by NADH against both inactivation and modification of lysyl residues indicate that this residue is essential for activity and is located in or near the NADH binding site of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the heme and pyridoxal phosphate cofactors of human cystathionine beta-synthase reveals nonequivalent active sites

Cystathionine beta-synthase is an unusual enzyme that requires the cofactors heme and pyridoxal phosphate (PLP) to catalyze the condensation of homocysteine and serine to generate cystathionine. This transsulfuration reaction represents one of two major cellular routes for detoxification of homocysteine, which is a risk factor for atherosclerosis. While the beta-replacement reaction catalyzed by this enzyme suggests a role for the pyridoxal phosphate, the role of the heme is uncertain. In this study we have examined the effect of changing one of the ligands to the heme on the activity of the enzyme. Binding of carbon monooxide results in the displacement of a thiolate ligand to the ferrous heme, and is accompanied by complete loss of cystathionine beta-synthase activity. Furthermore, inhibition by CO is competitive with respect to homocysteine, providing the first indication that the homocysteine binding site is in the proximity of heme. Binding of both CO and cyanide to ferrous cystathionine beta-synthase occurs in two distinct isotherms and indicates that the hemes are nonequivalent. We have employed fluorescence spectroscopy to characterize the bound PLP and its interaction with serine. PLP bound to cystathionine beta-synthase is weakly fluorescent and exists as a mixture of the protonated and unprotonated tautomers. Reaction with hydroxylamine releases the oxime and greatly enhances the associated fluorescence. Binding of serine is accompanied by a shift to the unprotonated tautomer of the external aldimine as well as the appearance of a new fluorescent species at approximately 400 nm that could be due to the aminoacrylate or to a gemdiamine intermediate. These data provide the first characterization of the PLP bound to cystathionine beta-synthase. Treatment of cystathionine beta-synthase with hydroxylamine releases two PLPs after 1 day and results in complete loss of activity. Incubation for an additional 3-4 days results in the release of two more PLPs. These data lead us to revise the PLP stoichiometry to 4 per tetramer, and to the conclusion that the heme and PLP sites in cystathionine beta-synthase are nonequivalent.     

Delta 2- and delta 3-cephalosporins, penicillinate and 6-unsubstituted penems. Intrinsic reactivity and interaction with beta-lactamases and D-alanyl-D-alanine-cleaving serine peptidases.

The intrinsic reactivity of delta 2- and delta 3-deacetoxy-7-phenylacetamidocephalosporanates, penicillanate, unsubstituted, 2-methyl- and 2-phenyl-penems and other beta-lactam antibiotics has been expressed in terms of the second-order rate constant (M-1.s-1(OH-)) for the hydrolysis of the beta-lactam amide bond by OH- at 37 degrees C. The values thus obtained have been compared with the second-order rate constants (M-1.s-1(enzyme) for the opening of the same beta-lactam amide bond during interaction with the beta-lactamases of Streptomyces albus G and Actinomadura R39 and the D-alanyl-D-alanine-cleaving serine peptidases of Streptomyces R61 and Actinomadura R39. Depending on the cases, the accelerating effect due to enzyme action and expressed by the ratio M-1.s-1(enzyme)/M-1.s-1(OH) varies from less than 2 to more than 1 x 10(6). The primary parameter that governs enzyme action is the goodness of fit of the beta-lactam molecule to the enzyme cavity rather than its intrinsic reactivity. With the D-alanyl-D-alanine-cleaving serine peptidases, the three penems studied form intermediate complexes characterized by very short half lives of 14-100 s, values significantly lower than those exhibited by most beta-lactam compounds.     

The reaction mechanism of the novel vanadium-bromoperoxidase. A steady-state kinetic analysis

The reaction of vanadium-bromoperoxidase from the brown alga Ascophyllum nodosum with hydrogen peroxide, bromide, and 2-chlorodimedone has been subjected to an extensive steady-state kinetic analysis. Systematic variation of pH and the concentrations of these three components demonstrate that the reaction model includes four enzyme species: native bromoperoxidase, a bromoperoxidase-bromide inhibitory complex, a bromoperoxidase-hydrogen peroxide intermediate, and a bromoperoxidase-HOBr species. This latter intermediate did not display any direct interaction with the nucleophilic reagent as oxidized bromine species (Br-3, Br2, and/or HOBr) were the primary reaction products. The generation of oxidized bromine species was as fast as the bromination of 2-chlorodimedone. The enzyme did not show any specificity with regard to bromination of various organic compounds. Formation of the bromoperoxidase-bromide inhibitory complex was competitive with the reaction between hydrogen peroxide and enzyme. From the steady-state kinetic data lower limits for the second-order rate constants at various pH values were calculated for individual steps in the catalytic cycle. This pH study showed that native enzyme must be unprotonated prior to binding of hydrogen peroxide (second-order association rate constant of 2.5.10(6) M-1.s-1 at pH greater than 6). The pKa for the functional group controlling the binding of hydrogen peroxide was 5.7 and is ascribed to a histidine residue. The reaction rate between bromide and enzyme-hydrogen peroxide intermediate also depended on pH (second-order association rate constant of 1.7.10(5) M-1.s-1 at pH 4.0).     

High resolution crystal structures of human kynurenine aminotransferase‐I bound to PLP cofactor,and in complex with aminooxyacetate

In this study, we report two high‐resolution structures of the pyridoxal 5′ phosphate (PLP)‐dependent enzyme kynurenine aminotransferase‐I (KAT‐I). One is the native structure with the cofactor in the PLP form bound to Lys247 with the highest resolution yet available for KAT‐I at 1.28 Å resolution, and the other with the general PLP‐dependent aminotransferase inhibitor, aminooxyacetate (AOAA) covalently bound to the cofactor at 1.54 Å. Only small conformational differences are observed in the vicinity of the aldimine (oxime) linkage with which the PLP forms the Schiff base with Lys247 in the 1.28 Å resolution native structure, in comparison to other native PLP‐bound structures. We also report the inhibition of KAT‐1 by AOAA and aminooxy‐phenylpropionic acid (AOPP), with IC50s of 13.1 and 5.7 μM, respectively. The crystal structure of the enzyme in complex with the inhibitor AOAA revealed that the cofactor is the PLP form with the external aldimine linkage. The location of this oxime with the PLP, which forms in place of the native internal aldimine linkage of PLP of the native KAT‐I, is away from the position of the native internal aldimine, with the free Lys247 substantially retaining the orientation of the native structure. Tyr101, at the active site, was observed in two conformations in both structures.     

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.     

Prostaglandin H synthase and hydroperoxides: peroxidase reaction and inactivation kinetics

The reaction kinetics of the peroxidase activity of prostaglandin H synthase have been examined with 15-hydroperoxyeicosatetraenoic acid and hydrogen peroxide as substrates and tetramethylphenylenediamine as cosubstrate. The apparent Km and Vmax values for both hydroperoxides were found to increase linearly with the cosubstrate concentration. The overall reaction kinetics could be interpreted in terms of an initial reaction of the synthase with hydroperoxide to form an intermediate equivalent to horseradish peroxidase Compound I, followed by reduction of this intermediate by cosubstrate to regenerate the resting enzyme. The rate constants estimated for the generation of synthase Compound I were 7.1 X 10(7) M-1 s-1 with the lipid hydroperoxide and 9.1 X 10(4) M-1 s-1 with hydrogen peroxide. The rate constants estimated for the rate-determining step in the regeneration of resting enzyme by cosubstrate were 9.2 X 10(6) M-1 s-1 in the case of the reaction with lipid hydroperoxide and 3.5 X 10(6) M-1 s-1 in the case of reaction with hydrogen peroxide. The intrinsic affinities of the synthase peroxidase for substrate (Ks) were estimated to be on the order of 10(-8) M for lipid hydroperoxide and 10(-5) M for hydrogen peroxide. These affinities are quite similar to the reported affinities of the synthase for these hydroperoxides as activators of the cyclooxygenase. The peroxidase activity was found to be progressively inactivated during the peroxidase reaction. The rate of inactivation of the peroxidase was increased by increases in hydroperoxide level, and decreased by increases in peroxidase cosubstrate. The inactivation of the peroxidase appeared to occur by a hydroperoxide-dependent process, originating from synthase Compound I or Compound II.     

Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism

Stopped-flow techniques were used to investigate the kinetics of the formation of manganese peroxidase compound I (MnPI) and of the reactions of MnPI and manganese peroxidase compound II (MnPII) with p-cresol and MnII. All of the rate data were obtained from single turnover experiments under pseudo-first order conditions. In the presence of H2O2 the formation of MnPI is independent of pH over the range 3.12-8.29 with a second-order rate constant of (2.0 +/- 0.1) x 10(6) M-1 s-1. The activation energy for MnPI formation is 20 kJ mol-1. MnPI formation also occurs with organic peroxides such as peracetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid with second-order rate constants of 9.7 x 10(5), 9.5 x 10(4), and 5.9 x 10(4) M-1 s-1, respectively. The reactions of MnPI and MnPII with p-cresol strictly obeyed second-order kinetics. The second-order rate constant for the reaction of MnPII with p-cresol is extremely low, (9.5 +/- 0.5) M-1 s-1. Kinetic analysis of the reaction of MnII with MnPI and MnPII showed a binding interaction with the oxidized enzymes which led to saturation kinetics. The first-order dissociation rate constants for the reaction of MnII with MnPI and MnPII are (0.7 +/- 0.1) and (0.14 +/- 0.01) s-1, respectively, when the reaction is conducted in lactate buffer. Rate constants are considerably lower when the reactions are conducted in succinate buffer. Single turnover experiments confirmed that MnII serves as an obligatory substrate for MnPII and that both oxidized forms of the enzyme form productive complexes with MnII. Finally, these results suggest the alpha-hydroxy acids such as lactate facilitate the dissociation of MnIII from the enzyme.     

Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance

Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1,000,000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of an oxyferryl porphyrin pi-cation-radical intermediate in the reaction between hydrogen peroxide and a mutant yeast cytochrome c peroxidase. Evidence for tryptophan-191 involvement in the radical site of compound I

Peroxide oxidation of a mutant cytochrome c peroxidase, in which Trp-191 has been replaced by Phe through site-directed mutagenesis, produces an oxidized intermediate whose stable UV/visible absorption spectrum is very similar to that of compound I of the native yeast enzyme. This spectrum is characteristic of an oxyferryl, Fe(IV), heme. Stopped-flow studies reveal that the reaction between the mutant enzyme and hydrogen peroxide is biphasic with the transient formation of an intermediate whose absorption spectrum is quite distinct from that of either the native ferric enzyme or the final product. Rapid spectral scanning of the intermediate provides a spectrum characteristic of an oxyferryl porphyrin pi-cation-radical species. At pH 6, 100 mM ionic strength, and 25 degrees C, the rate constant for formation of the oxyferryl pi-cation radical has a lower limit of 6 X 10(7) M-1 s-1 and the rate of conversion of the transient intermediate to the final oxidized product is 51 +/- 4 s-1. Evidence is presented indicating that Trp-191 either is the site of the radical in CcP compound I or is intimately involved in formation of the radical.     

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)     

Aminooxyphosphonates as Slow Binding Inhibitors of Aspartate and Alanine Aminotransferases from Porcine Heart

Abstract

Aminooxymethylphosphonic (AOMP), 1–aminooxyethylphosphonic (1-AOEP) and 2-aminooxyethyl-phosphonic (2-AOEP) acids have been synthesised and were found to be potent slow binding inhibitors of aspartate- and alanine-aminotransferases with K_i ranging from nanomolar to micromolar values. The half-life of the inhibited complexes varied from 8 min (AspAT-2-AOEP) to 11 h (AspAT-AOMP). Kinetic analysis of the interaction of both enzymes with AOMP suggested the formation of an E-I complex in a single slow binding process. In the case of other compounds, attempt to discriminate between a single- or a double-step mechanism, consistent with an E-I intermediate followed by a slow E-I to E-I* isomerisation process could not be clearly resolved. Spectral studies of the complex formed between PLP-bound enzyme and the aminooxy compound resulted in a shift from 362 nm, the absorption maximum of the native enzyme, to 380 nm, characteristic of the oxime produced. The kinetic parameters for aminooxyphosphonates were compared to those for their carboxylic and aminophosphonic analogues.     

Pregnancy zone protein, a proteinase binding alpha-macroglobulin. Stopped-flow kinetic studies of its interaction with chymotrypsin

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein, strongly related to alpha 2-macroglobulin (alpha 2M). The proteinase binding reaction of PZP is investigated using chymotrypsin as a model enzyme. The time-course of the interaction is studied by measuring the change in intrinsic protein fluorescence of PZP-chymotrypsin reaction mixtures as a function of time after rapid mixing in a stopped-flow apparatus. Titrations show the changes of fluorescence at equilibrium to correspond with the formation of a chymotrypsin-PZP(tetramer) species. The kinetic results show the formation of the species to take place in an overall second-order process dependent on the concentrations of chymotrypsin and of PZP(dimers), k = 5 x 10(5) M-1 x s-1. Reactions of PZP-thiol groups do not give rise to fluorescence changes. The fluorescence changes most likely reflect the formation of an intermediate with intact thiol esters. Further analysis of the kinetic results suggests that the chymotrypsin-PZP(tetramer) intermediate is formed in two reaction steps: (1) initially native PZP(dimers) are cleaved at bait regions by enzyme molecules, and that is the rate determining reaction of the fluorescence changes; (2) association with another PZP(dimer) or PZP(dimer)-chymotrypsin complex in a very fast reaction that leads to the formation of 1:1 -chymotrypsin-PZP(tetramer) intermediate, probably with intact thiol esters. The interactions studied apparently are established early in the path of the reaction and the fluorescence changes probably reflect noncovalent enzyme-PZP contacts, which are not changed when covalent binding occurs. Further, fluorescence changes are seen only in reactions of PZP with enzymes, not with methylamine.     

Reactions of the Wurster's red radical cation with hemoglobin and glutathione during the cooxidation of N,N-dimethyl-p-phenylenediamine [correction of phenlenediamine] and oxyhemoglobin in human red cells.

N,N-Dimethyl-p-phenylenediamine (DMPD) reacted directly with oxyhemoglobin under formation of ferrihemoglobin and, presumably, the N,N-dimethyl-p-phenylenediamine radical cation (DMPP.+). The apparent second-order rate constant of this reaction was 1 M-1 s-1 (pH 7.4, 37 degrees C). The reaction rate was diminished by catalase (by 1/3) and by superoxide dismutase (by 1/5). The apparent second-order rate constant of ferrihemoglobin formation by DMPD.+ was 5 x 10(3) M-1 s-1. Since DMPD.+ is disproportionated by 50% at pH 7.4, the quinonediimine could not be excluded as the ultimate ferrihemoglobin forming oxidant. To prove this hypothesis, the disproportionation equilibrium was shifted to the radical side by addition of excess DMPD. Ferrihemoglobin formation was thereby increased, indication that the radical was the responsible oxidant. In contrast to ferrihemoglobin formation, reactions with glutathione occurred predominantly with the quinonediimine. The second-order rate constant of this reaction was 4 x 10(5) M-1 s-1 which approaches the value obtained with p-benzoquinone. In contrast to the corresponding reactions of the N,N,N',N'-tetramethyl-p-phenylenediamine radical cation, the disporportionation reaction of DMPD.+ was very fast, k = 2 x 10(6) M-1 s-1. Formation of glutathione disulfide was negligible and the main reaction products were two isomeric glutathione adducts, 2- and 3-(glutathione-S-yl)-N,N-dimethyl-p-phenylenediamine. In human erythrocytes, DMPD produced many equivalents of ferrihemoglobin, diminished glutathione and produced both thioethers. In contrast to ferrihemoglobin formation, DMPD and glutathione disappearance as well as thioether appearance occured only after a marked lag phase. The calculated steady state concentration of DMPD.+ was only 4 x 10(-6) the DMPD concentration, as long as ferrihemoglobin was low. At increasing ferrihemoglobin higher steady state concentrations of the radical are attained. In fact, preformed ferrihemoglobin in red cells significantly accelerated DMPD and glutathione disappearance. This effect was completely prevented in the presence of ferrihemoglobin-complexing cyanide. The presented experiments once more appoint blood as a metabolically competent organ for the biotransformation of aromatic amines.     

The reductive half-reaction of xanthine oxidase. Identification of spectral intermediates in the hydroxylation of 2-hydroxy-6-methylpurine.

The reaction of xanthine oxidase with 2-hydroxy-6-methylpurine (also called 2-oxo-6-methylpurine) has been studied under both anaerobic and aerobic conditions. Reaction of enzyme with substoichiometric concentrations of hydroxymethylpurine in aerobic 0.1 M 3-(cyclohexylamino)propanesulfonic acid, 0.1 N KCl, 0.3 mM EDTA, pH 10.0, exhibits two reaction intermediates detectable by UV-visible spectrophotometry. The rate constants for formation of the first intermediate, conversion of the first to the second, and the decay of the second to give oxidized enzyme are 18, 1.2, and 0.13 s-1, respectively. The difference spectra of these two intermediates relative to oxidized enzyme are characterized by absorbance maxima at 470 and 540 nm, respectively, with extinction changes (relative to oxidized enzyme) of approximately 410 M-1 cm-1. The 0.13 s-1 decay of the second intermediate agrees well with kcat of 0.11 s-1 determined under the same conditions. Based on a comparison of the kinetics of the reaction as monitored by UV-visible absorption and electron paramagnetic resonance spectrometry, it is concluded that these spectral intermediates arise from the molybdenum center of the enzyme in the MoIV and MoV valence states, respectively, the latter corresponding to the species exhibiting the "very rapid" MoV EPR signal known to be formed in the course of the reaction. This conclusion is supported by the results of experiments using cytochrome c reduction to follow the formation of superoxide production in the course of the aerobic reaction of xanthine oxidase with substoichiometric hydroxymethylpurine, which demonstrate unequivocally that the species exhibiting the very rapid EPR signal is formed by one-electron oxidation of a MoIV species rather than direct one-electron reduction of MoVI by substrate. No evidence is found for the formation of any of the MoV EPR signals designated "rapid" in the present studies, and it is concluded that this species is not a bona fide catalytic intermediate in the reductive half-reaction of xanthine oxidase.     

Stopped-flow kinetic study of the peroxidase reactions of mangano-microperoxidase-8

We have investigated the kinetics for the peroxidase-type reaction of mangano microperoxidase 8 (Mn(III)-MP8) by the time-resolved and single-wavelength stopped-flow technique. The formation of intermediate and its subsequent reaction with substrates were studied separately. Oxidation of Mn(III)-MP8 by H2O2 at pH 10.7 yields an intermediate (1) with a rate constant of 2.9 x10(4) M-1 s-1. The formation of 1 exhibits no deuterium solvent isotope effect, favoring the homolytic cleavage of the Mn(III)-MP8 bound hydroperoxide. The rate for the formation of 1 increases sharply as the pH increases and no other intermediate was detected in the entire pH range. Addition of substrate to 1 leads to the regeneration of Mn(III)-MP8. Monitoring the conversion of 1 to Mn(III)-MP8 allows the determination of the substrate reactivity. The substrate reactivity varies by more than two orders of magnitude ranging from 1.04 x 10(6) M-1 s-1 for ascorbic acid to 4.61 x 10(3) M-1s-1 for aniline. It is linearly correlated with the reduction potential for most of the substrates studied, with the easier oxidized species showing greater reactivity. The substrate reactivity drops rapidly as the pH increases. The substrate reactivity at pH 10.7 for the Mn(III)-MP8 system is smaller than that of the corresponding Fe(III)-MP8 system by 2- to 25-fold, depending on the substrate used.