scholarly journals Microbial oxidation of amines. Spectral and kinetic properties of the primary amine dehydrogenase of Pseudomonas AM 1

1971 ◽  
Vol 123 (5) ◽  
pp. 757-771 ◽  
Author(s):  
R. R. Eady ◽  
P. J. Large
Keyword(s):  
Fluorescence Emission ◽  
Electrophoretic Analysis ◽  
Enzyme Mechanism ◽  
Heat Denaturation ◽  
Kinetic Properties ◽  
Microbial Oxidation ◽  
Prosthetic Group ◽  
Iron And Zinc ◽  
Amine Dehydrogenase ◽  
Phenazine Methosulphate

1. An improved procedure is reported for purification of the amine dehydrogenase from methylamine-grown Pseudomonas AM1 which yielded a product homogeneous by sedimentation and disc-electrophoretic analysis, with molecular weight of 133000. 2. The purified enzyme had absorption maxima at 280 and 430nm. On aging, a third peak appeared at 325nm, and the 430nm peak decreased in intensity. This spectrum was independent of pH. 3. Addition of 2.5mm-semicarbazide, phenylhydrazine, hydrazine or hydroxylamine produced modified spectra with maxima respectively at 400, 440, 395 and 425nm. 4. Aerobic addition of methylamine resulted in a bleaching of the 430nm peak and the appearance of a new one at 325nm. This spectral change was retained after removal of the methylamine by dialysis. The original spectrum could be restored on addition of phenazine methosulphate. 5. Addition of borohydride partially inactivated the enzyme and produced spectral changes similar to those observed with methylamine. Pre-treatment with methylamine prevented the inactivation by borohydride. The degree of inactivation could be increased by alternate phenazine methosulphate and borohydride treatments. 6. The addition of methylamine or borohydride each caused shifts in the fluorescence emission maximum from 348 to 380nm. 7. Lineweaver–Burk plots of reciprocal activity against reciprocal concentration of either of the substrates n-butylamine or phenazine methosulphate were consistent with a mechanism that involves interconversion of two free forms of the enzyme by the two substrates. 8. The enzyme, although spectrally modified, was not inactivated by dialysis against diethyldithiocarbamate, and contained about 0.27 g-atom of copper/mol, with small traces of cobalt, iron and zinc. 9. Conventional methods of resolution did not release the prosthetic group. Heat denaturation after treatment of the enzyme with methylamine liberated a yellow chromophore which did not reactivate resolved aspartate aminotransferase, and whose spectral, electrophoretic and fluorescence properties did not agree with any recognizable pyridoxal derivatives. 10. Despite the inconclusive results with the isolated chromophore, the observations on the enzyme suggest that it may contain a pyridoxal derivative bound as a Schiff's base which is converted into the pyridoxamine form on aerobic treatment with methylamine and reconverted into the pyridoxal form with phenazine methosulphate. 11. The copper detected is probably not involved in the enzyme mechanism, since most copper-chelating agents are not inhibitory, and since the enzyme does not react with oxygen.

Spectral and kinetic properties of a cationic peroxidase secreted by cultured peanut cells

1985 ◽  
Vol 63 (10) ◽  
pp. 1086-1092 ◽  
Author(s):  
Anne-Marie Lambeir ◽  
H. Brian Dunford ◽  
Robert B. van Huystee ◽  
Jerzy Lobarzewski
Keyword(s):  
Dissociation Constant ◽  
Native Enzyme ◽  
Kinetic Properties ◽  
Compound I ◽  
Prosthetic Group ◽  
Equilibrium Studies ◽  
Alkaline Transition ◽  
Cyanide Binding ◽  
Ph Range ◽  
Compound Ii

It is demonstrated that the cationic peroxidase isolated from the growth medium of cultured peanut cells reacts via the same mechanism as other peroxidases, namely conversion of the native enzyme into compound I by reaction with hydrogen peroxide, followed by two reductions by one-electron donors to compound II and then back to the native enzyme. From the pyridine hemochromogen spectrum it is concluded that the prosthetic group of the native enzyme is ferriprotoporphyrin IX. Optical spectra are recorded for (i) the native (ferric) enzyme and its cyanide, azide, fluoride and alkaline forms, (ii) ferrous peroxidase and its cyanide and carbon monoxide complexes, and (iii) compounds I, II, and III. Equilibrium studies show that the ferric cyanide complex has a dissociation constant of 3.0 ± 0.5 μM over the pH range 3–8. The fluoride complex has a dissociation constant which varies from 1.6 μM at pH 4.0 to 28 μM at pH 4.8. Azide has a much lower affinity than fluoride. The alkaline transition occurs with an apparent pKa value of 9.2. Rate constants were recorded for cyanide binding, the alkaline transition, compound I formation, and for the reactions of compound II with a series of substrates. Similarities and differences to horseradish peroxidase are discussed.

scholarly journals Effects of ionic strength on the catalysis and stability of prolyl oligopeptidase

1995 ◽  
Vol 312 (1) ◽  
pp. 267-271 ◽  
Author(s):  
L Polgár
Keyword(s):  
Ionic Strength ◽  
Fluorescence Emission ◽  
Water Structure ◽  
Emission Spectra ◽  
Heat Denaturation ◽  
Prolyl Oligopeptidase ◽  
Fluorescence Emission Spectra ◽  
Water Shell ◽  
Serine Protease Family ◽  
High Concentrations

Prolyl oligopeptidase is the prototype of a new serine protease family, unrelated to trypsin and subtilisin. In contrast with these proteases, prolyl oligopeptidase is remarkably sensitive to ionic strength, being more active in the presence of high concentrations of salt. The enzyme has two catalytic forms, which interconvert with changing pH. To reveal the structural bases of these phenomena, the effects of 0.5 M NaCl on the stability of the enzyme were investigated by studying its denaturation as a function of pH, temperature, and urea concentration. The three independent methods have unequivocally demonstrated that denaturation of the enzyme is promoted in the presence of NaCl. Furthermore, destabilization of the low-pH form by urea is more significant than that of the high-pH form. Examination of the fluorescence emission spectra of various denatured forms indicates that the enzyme is not fully unfolded in 8 M urea, nor at acidic pH. The tryptophan residues in the acid-denatured state are mainly buried. The results are interpreted in terms of the decay of the protective water shell at the higher ionic strength. The higher enthalpy and entropy of activation for heat denaturation provide further evidence that a more ordered water structure stabilizes the protein in the absence of salt. The biphasic kinetics obtained with denaturation by heat and urea suggest that the enzyme has two domains of different stabilities.

scholarly journals Kinetic analysis of duck ε-crystallin, a lens structural protein with lactate dehydrogenase activity

1990 ◽  
Vol 267 (1) ◽  
pp. 51-58 ◽  
Author(s):  
S H Chiou ◽  
H J Lee ◽  
G G Chang
Keyword(s):  
Lactate Dehydrogenase ◽  
Kinetic Analysis ◽  
Substrate Inhibition ◽  
Product Inhibition ◽  
Enzyme Mechanism ◽  
Kinetic Properties ◽  
Structural Protein ◽  
Lactate Dehydrogenase Activity ◽  
Chicken Heart ◽  
Inhibition Studies

Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.

Gamma-Glutamyltransferase: kinetic properties and assay conditions when gamma-glutamyl-4-nitroanilide and its 3-carboxy derivative are used as donor substrates.

1977 ◽  
Vol 23 (1) ◽  
pp. 79-85 ◽  
Author(s):  
L M Shaw ◽  
J W London ◽  
D Fetterolf ◽  
D Garfinkel
Keyword(s):  
Enzyme Mechanism ◽  
Simultaneous Occurrence ◽  
Kinetic Properties ◽  
Gamma Glutamyltransferase ◽  
Gamma Glutamyl ◽  
Ping Pong ◽  
Clinical Interest ◽  
Kinetics Of ◽  
Substrate Concentrations ◽  
Assay Conditions

Abstract The kinetics of human serum gamma-glutamyltransferase (EC 2.3.2.2) were investigated, with use of glycylglycine as a gamma-glutamyl acceptor substrate and gamma-glutamyl-4-nitroanilide and its carboxy derivative, gamma-glutamyl-3-carboxy-4-nitroanilide, as donor substrates. The simultaneous occurrence of both gamma-glutamyltransfer and autotransfer was established by descending paper chromatography. Constant-ratio double-reciprocal plots confirm that the enzyme mechanism is nonsequential (ping-pong bi-bi). Inhibition by either donor was not found, and inhibition by glycylglycine was only observed at concentrations above those of clinical interest. Kinetic constants obtained by nonlinear regression analysis of initial velocity data were used to determine reagent substrate concentrations for the assay of this enzyme. An assay with use of 4 mmol of gamma-glutamyl-3-carboxy-4-nitroanilide and 100 mmol of glycylglycine per liter yielded equivalent activities to those by assay with use of 4 mmol of gamma-glutamyl-4-nitroanilide and 40 mmol of glycylglycine per liter. These concentrations of the carboxy donor and glycylglycine are also "cost optimal" and present no procedural problems when used.

scholarly journals Kinetic and chemical mechanisms for the effects of univalent cations on the spectral properties of aromatic amine dehydrogenase

1998 ◽  
Vol 329 (1) ◽  
pp. 175-182 ◽  
Author(s):  
Zhenyu ZHU ◽  
L. Victor DAVIDSON
Keyword(s):  
Absorption Spectrum ◽  
Reaction Mechanism ◽  
Aromatic Amine ◽  
Ph Dependence ◽  
Spectral Change ◽  
Univalent Cations ◽  
Prosthetic Group ◽  
Cation Concentration ◽  
Amine Dehydrogenase ◽  
Spectral Perturbation

Univalent cations and pH influence the UV-visible absorption spectrum of the tryptophan tryptophylquinone (TTQ) enzyme, aromatic amine dehydrogenase (AADH). Little spectral perturbation was observed when pH was varied in the absence of univalent cations. The addition of alkali metal univalent cations (K+, Na+, Li+, Rb+ or Cs+) to oxidized AADH caused significant changes in its absorption spectrum. The apparent Kd for each cation, determined from titrations of the spectral perturbation, decreased with increasing pH. Transient kinetic studies involving rapid mixing of AADH with cations and pH jump revealed that the rate of the cation-induced spectral changes initially decreased with increasing cation concentration to a minimum value, then increased with increasing cation concentration. A kinetic model was developed to fit these data, determine the true pH-independent Kd values for K+ and Na+, and explain the pH-dependence of the apparent Kd. A chemical reaction mechanism, based on the kinetic data, is presented in which the metallic univalent cation facilitates the chemical modification of the TTQ prosthetic group to form an hydroxide adduct which gives rise to the spectral change. Addition of NH4+/NH3 to AADH caused changes in the absorption spectrum which were very different form those caused by addition of the metallic univalent cations. The kinetics of the reaction induced by addition of NH4+/NH3 were also different, being simple saturation kinetics. Another reaction mechanism is proposed for the NH4+/NH3-induced spectral change that involves nucleophilic addition of the unprotonated NH3 to TTQ. The general relevance of these data and models to the physiological reactions of TTQ-dependent enzymes and to the roles of univalent cations in modulating enzyme activity are discussed.

scholarly journals Purification and properties of an amine dehydrogenase from Pseudomonas AM1 and its role in growth on methylamine

1968 ◽  
Vol 106 (1) ◽  
pp. 245-255 ◽  
Author(s):  
R R Eady ◽  
P J Large
Keyword(s):  
Ph Value ◽  
Ammonium Salts ◽  
Tertiary Amines ◽  
Brilliant Cresyl Blue ◽  
Whole Cells ◽  
Cresyl Blue ◽  
Purification And Properties ◽  
Km Value ◽  
Amine Dehydrogenase ◽  
Phenazine Methosulphate

1. Whole cells of Pseudomonas AM1 grown on methylamine oxidize methylamine, formaldehyde and formate. Crude extracts oxidize methylamine only if supplemented with phenazine methosulphate. 2. By using a spectrophotometric assay, the methylamine-oxidizing enzyme has been purified 20-fold in 31% yield. 3. The enzyme is a dehydrogenase, unable to utilize oxygen, NAD, NADP, flavines or menadione as electron acceptors, but able to utilize phenazine methosulphate, ferricyanide, cytochrome c or brilliant cresyl blue. 4. The enzyme is non-specific, readily oxidizing aliphatic monoamines and diamines, histamine and ethanol-amine. Secondary and tertiary amines, quaternary ammonium salts and aromatic amines are not oxidized. 5. The pH optima for methylamine, n-pentylamine and putrescine are respectively 7·6, 8·0 and 8·5. 6. The Km value for methylamine is 5·2μm and that for phenazine methosulphate 56μm. 7. The enzyme will withstand heating for 15min. at 80° without loss of activity, but is inactivated at higher temperatures. It is not inactivated by any pH value between 2·6 and 10·6. 8. The dehydrogenase is inhibited by semicarbazide (Ki 3·35μm), isoniazid (Ki 1·17μm), cuprizone (Ki 0·49μm), p-chloromercuribenzoate (Ki 0·45mm) and quinacrine (Ki 12·1mm). 9. The enzyme is absent from succinate-grown cells, and, during adaptation from succinate to methylamine, activity appears before growth on methylamine begins.

scholarly journals Replacement of enzyme-bound calcium with strontium alters the kinetic properties of methanol dehydrogenase

1994 ◽  
Vol 300 (1) ◽  
pp. 175-182 ◽  
Author(s):  
T K Harris ◽  
V L Davidson
Keyword(s):  
Paracoccus Denitrificans ◽  
Competitive Inhibitor ◽  
Methanol Dehydrogenase ◽  
Native Enzyme ◽  
Kinetic Properties ◽  
Host Bacterium ◽  
Prosthetic Group ◽  
Dependent Activity ◽  
Cyanide Inhibition ◽  
Endogenous Activity

Methanol dehydrogenase (MEDH) possesses tightly bound Ca2+ in addition to its pyrroloquinoline quinone (PQQ) prosthetic group. Ca2+ was replaced with Sr2+ by growing the host bacterium, Paracoccus denitrificans, in media in which Ca2+ was replaced with Sr2+. MEDH, which was purified from these cells (Sr-MEDH), exhibited an increased absorption coefficient for the PQQ chromophore, and displayed certain kinetic properties which were different from those of native MEDH. Native MEDH exhibits an endogenous activity which is not stimulated by substrate and which is inhibited by cyanide. Sr-MEDH exhibited lower endogenous activity which was stimulated by substrate, and was much less sensitive to inhibition by cyanide. The Vmax. for the methanol-dependent activity of Sr-MEDH was 3-fold greater than that of the native enzyme, and the Ks for methanol was altered. Cyanide also acts as an obligatory activator and competitive inhibitor of methanol-dependent activity in native MEDH from P. denitrificans [Harris and Davidson (1993) Biochemistry 32, 4362-4368]. Sr-MEDH exhibited a similar K1 for cyanide inhibition of methanol-dependent activity, but the KA for cyanide activation of this activity was 17-fold greater than that for the native enzyme. The activation energy of Sr-MEDH was 13.4 kJ (3.2 kcal)/mol lower than that of the native enzyme. These data confirm and significantly extend the conclusions from genetic [Richardson and Anthony (1992) Biochem. J. 287, 709-715] and crystallographic [White, Boyd, Mathews, Xia, Dai, Zhang and Davidson (1993) Biochemistry 32, 12955-12958] studies that suggest an apparently unique role for Ca2+ in MEDH compared with other Ca(2+)-dependent proteins and enzymes.

scholarly journals Purification and kinetic analysis of a baculovirus ecdysteroid UDP-glucosyltransferase

1998 ◽  
Vol 330 (3) ◽  
pp. 1265-1270 ◽  
Author(s):  
P. Owain EVANS ◽  
David R. O'REILLY
Keyword(s):  
Kinetic Analysis ◽  
Biochemical Characterization ◽  
Active Form ◽  
Gel Filtration ◽  
Enzyme Mechanism ◽  
The Other ◽  
Kinetic Properties ◽  
Autographa Californica Nucleopolyhedrovirus ◽  
Moulting Hormones

The baculovirus ecdysteroid UDP-glucosyltransferase (EGT) disrupts the hormonal balance of the insect host by catalysing the conjugation of ecdysteroids, the moulting hormones, with the sugar moiety from UDP-glucose or UDP-galactose. In this study, Autographa californica nucleopolyhedrovirus EGT has been overproduced and purified, and its kinetic properties determined. The enzyme was purified 1100-fold to near-homogeneity using only two major steps, ion-exchange and gel-filtration chromatography. EGT activity was eluted from the gel-filtration column as a single peak corresponding to a 260±50 kDa protein, suggesting that the enzyme is an oligomer of three to five subunits, as the subunit molecular mass is approximately 56 kDa. Kinetic analysis showed that EGT has broadly similar specificities for UDP-galactose and UDP-glucose (kcat/Km = 1790.8 and 902.1 respectively) when ecdysone is used as the other substrate. On the other hand, it shows marked differences in specificity for the various ecdysteroids tested. Ecdysone seems to be the optimal substrate (kcat/Km = 7101.1), whereas 3-dehydroecdysone, an ecdysone precursor in Lepidoptera, is seven times less favourable (kcat/Km = 1085.7). Notably, 20-hydroxyecdysone, the active form of the hormone, is conjugated very poorly (kcat/Km = 31.6). Analysis of the data revealed that the enzyme mechanism involves the formation of an ecdysteroid-UDP-sugar-enzyme ternary complex. This work represents the most detailed biochemical characterization of an EGT to date.

Functional Characterisation of Dictyostelium Myosin II with Conserved Tryptophanyl Residue 501 Mutated to Tyrosine

1999 ◽  
Vol 380 (7-8) ◽  
pp. 1017-1023 ◽  
Author(s):  
R. Batra ◽  
D. J. Manstein
Keyword(s):  
Myosin Ii ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Spectroscopic Studies ◽  
Chain Gene ◽  
Kinetic Properties ◽  
Skeletal Muscle Myosin ◽  
Multicellular Development ◽  
Functional Characterisation ◽  
Tryptophanyl Residue

AbstractWe created aDictyostelium discoideummyosin II mutant in which the highly conserved residue Trp-501 was replaced by a tyrosine residue. The mutant myosin alone, when expressed in aDictyosteliumstrain lacking the functional myosin II heavy chain gene, supported cytokinesis and multicellular development, processes which require a functional myosin inDictyostelium. Additionally, we expressed the W501Y mutant in the soluble myosin head fragment M761-2R (W501Y-2R) to characterise the kinetic properties of the mutant myosin motor domain. The affinity of the mutant myosin for actin was approximately 6-fold decreased, but other kinetic properties of the protein were changed less than 2-fold by the W501Y mutation. Based on spectroscopic studies and structural considerations, Trp-501, corresponding to Trp-510 in chicken fast skeletal muscle myosin, has been proposed to be the primary ATP-sensitive tryptophanyl residue. Our results confirm these conclusions. While the wild-type construct displayed a 10% fluorescence increase, addition of ATP to W501Y-2R was not followed by an increase in tryptophan fluorescence emission.

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