scholarly journals Mechanism of d-fructose isomerization by Arthrobacterd-xylose isomerase

1992 ◽  
Vol 283 (1) ◽  
pp. 223-233 ◽  
Author(s):  
M Rangarajan ◽  
B S Hartley
Keyword(s):  
Ring Opening ◽  
Ph Dependence ◽  
Xylose Isomerase ◽  
Competitive Inhibitor ◽  
Apparent Dissociation Constant ◽  
Ph Values ◽  
Rate Limiting Step ◽  
Competitive Inhibitors ◽  
Rate Limiting ◽  
Very High

The mechanism of D-fructose isomerization by Arthrobacter D-xylose isomerase suggested from X-ray-crystallographic studies was tested by detailed kinetic analysis of the enzyme with various metal ions at different pH values and temperatures. At D-fructose concentrations used in commercial processes Mg2+ is the best activator with an apparent dissociation constant of 63 microM; Co2+ and Mn2+ bind more strongly (apparent Kd 20 microM and 10 microM respectively) but give less activity (45% and 8% respectively). Ca2+ is a strict competitive inhibitor versus Mg2+ (Ki 3 microM) or Co2+ (Ki 105 microM). The kinetics show a compulsory order of binding; Co2+ binds first to Site 2 and then to Site 1; then D-fructose binds at Site 1. At normal concentrations Mg2+ binds at Site 1, then D-fructose and then Mg2+ at Site 2. At very high Mg2+ concentrations (greater than 10 mM) the order is Mg2+ at Site 1, Mg2+ at Site 2, then D-fructose. The turnover rate (kcat.) is controlled by ionization of a residue with apparent pKa at 30 degrees C of 6.0 +/- 0.07 (Mg2+) or 5.3 +/- 0.08 (Co2+) and delta H = 23.5 kJ/mol. This appears to be His-219, which is co-ordinated to M[2]; protonation destroys isomerization by displacing M[2]; Co2+ binds more strongly at Site 2 than Mg2+, so competes more strongly against H+. The inhibition constant (Ki) for the two competitive inhibitors 5-thio-alpha-D-glucopyranose and D-sorbitol is invariant with pH, but Km(app.) in the Mg[1]-enzyme is controlled by ionization of a group with pKa 6.8 +/- 0.07 and delta H = 27 kJ/mol, which appears to be His-53. This shows that Km(app.) is a complex constant that includes the rate of the ring-opening step catalysed by His-53, which explains the pH-dependence. In the Mg[1]Mg[2]-enzyme or Co[1]Co[2]-enzyme, the pKa is lower (6.2 +/- 0.1 or 5.6 +/- 0.08) because of the extra adjacent cation. Hence the results fit the previously proposed pathway, but show that the mechanisms differ for Mg2+ and Co2+ and that the rate-limiting step is isomerization and not ring-opening as previously postulated.

scholarly journals Kinetics of the diamine oxidase reaction

1973 ◽  
Vol 131 (3) ◽  
pp. 459-469 ◽  
Author(s):  
William G. Bardsley ◽  
M. James C. Crabbe ◽  
Julian S. Shindler
Keyword(s):  
Diamine Oxidase ◽  
Experimental Conditions ◽  
Ph Values ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Deuterium Substitution ◽  
Effects Of Temperature ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Oxidase Reaction

1. The oxidation of p-dimethylaminomethylbenzylamine was followed spectrophotometrically by measuring the change in E250 caused by the p-dimethylaminomethylbenzaldehyde produced under a wide variety of experimental conditions. 2. The effect of variations in concentrations of both substrates (amine and oxygen) and all products (aminoaldehyde, hydrogen peroxide and ammonia) on this reaction was studied and the results used to develop a formal mechanism. 3. The nature of the rate-limiting step was elucidated by studying the effects of alterations in ionic strength, dielectric constant and deuterium substitution on the velocity of the forward reaction. 4. Thermodynamic activation energy parameters were obtained at several pH values from the effects of temperature on the reaction.

scholarly journals Using pH dependence to understand mechanisms in electrochemical CO reduction

2021 ◽  
Author(s):  
Georg Kastlunger ◽  
Lei Wang ◽  
Nitish Govindarajan ◽  
Hendrik H. Heenen ◽  
Stefan Ringe ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Modern Technology ◽  
Catalyst Design ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Wide Range ◽  
Alkaline Conditions ◽  
Novel Catalyst ◽  
Co Reduction ◽  
Rate Limiting

Electrochemical conversion of CO(2) into hydrocarbons and oxygenates is envisioned as a promising path towards closing the carbon cycle in modern technology. To this day, however, the reaction mechanisms towards the plethora of products are disputed, complicating the search for novel catalyst materials. In order to conclusively identify the rate-limiting steps in CO reduction on Cu, we analyzed the mechanisms on the basis of constant potential DFT kinetics and experiments at a wide range of pH values (3 - 13). We find that *CO dimerization is energetically favoured as the rate limiting step towards multi-carbon products. This finding is consistent with our experiments, where the reaction rate is nearly unchanged on an SHE potential scale, even under acidic conditions. For methane, both theory and experiments indicate a change in the rate-limiting step with electrolyte pH from the first protonation step in acidic/neutral conditions to a later one in alkaline conditions. We also show, through a detailed analysis of the microkinetics, that a surface combination of *CO and *H is inconsistent with the measured current densities and Tafel slopes. Finally, we discuss the implications of our understanding for future mechanistic studies and catalyst design.

scholarly journals Studies of the esterase activity of cytosolic aldehyde dehydrogenase with resorufin acetate as substrate

1997 ◽  
Vol 322 (3) ◽  
pp. 701-708 ◽  
Author(s):  
Trevor M. KITSON ◽  
Kathryn E. KITSON
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Point Of View ◽  
Ph Profile ◽  
Rate Limiting Step ◽  
The Common ◽  
Aldehyde Oxidation ◽  
Rate Limiting ◽  
Hydrolysis Of ◽  
Very High ◽  
Putative Catalytic Residue

Resorufin acetate is a very good substrate for sheep liver cytosolic aldehyde dehydrogenase, both from the point of view of practical spectrophotometry and in terms of information provided about the nature of the catalysis shown by this enzyme. p-Nitrophenyl (PNP) acetate competes against resorufin acetate for the enzyme's active site (although relatively weakly as the latter substrate has the lower Michaelis constant), but acetaldehyde (in the presence of NAD+) inhibits the hydrolysis of resorufin acetate only at very high aldehyde concentration. In the absence of cofactor, the rate-limiting step in the hydrolysis of resorufin acetate and of PNP acetate is hydrolysis of the common acetyl-enzyme, as shown by the observation of bursts of chromophoric product and very similar values of kcat. In the presence of NAD+ or NADH, however, the deacylation step with resorufin acetate is greatly accelerated until acylation seems to become rate-limiting, because no burst is seen under these conditions. Millimolar concentrations of Mg2+ activate the hydrolyis of resorufin acetate both in the presence and absence of cofactors. With both Mg2+ and cofactor the kcat for hydrolysis of resorufin acetate is 30–35 s-1; this is three orders of magnitude higher than the kcat for aldehyde oxidation in the presence of Mg2+, showing that the enzyme's potential catalytic efficency is very much hampered by the slowness with which NADH dissociates from its binding site. The pH profile for the hydrolysis of resorufin acetate in the presence of NAD+ or NADH fits well to a theoretical ionization curve of pKa approx. 8.2; it is suggested that this might belong to the enzyme's putative catalytic residue (Cys-302).

Diagnostic Enzymology of a-L-Iduronidase with Special Reference to a Sulphated Disaccharide Derived from Heparin

1982 ◽  
Vol 62 (2) ◽  
pp. 193-201 ◽  
Author(s):  
J. J. Hopwood ◽  
Vivienne Muller
Keyword(s):  
Enzyme Activity ◽  
Competitive Inhibitor ◽  
Ph Profile ◽  
Ph Values ◽  
Substrate Structure ◽  
Cultured Skin ◽  
Bivalent Cations ◽  
Competitive Inhibitors ◽  
Specific Ion Effect ◽  
Hydrolysis Of

1. Iduronosyl anhydro[1-3H]mannitol 6-sulphate (IMs), iduronosyl anhydro[1-3H]mannitol, phenyl iduronide (PhI) and 4-methylumbelliferyl iduronide have been compared as substrates for the diagnostic estimation of α-l-iduronidase activity present in human leucocyte and cultured skin fibroblast homogenates. The pH profile of leucocyte and fibroblast iduronidase activity was dependent on substrate structure and concentration, the ionic strength and the nature of the buffer ion used in the assay mixture. 2. NaCl, KBr and Na2SO4 were shown to be parabolic competitive inhibitors of IMs activity, the K1 with fibroblast homogenates being 34, 13.4 and 0.22 mmol/l respectively. NaCl and KBr were shown to have a primary salt effect on the interaction between enzyme and substrate but Na2SO4 appeared to have a specific ion effect at a cationic binding site. 3. NaCl inhibited the hydrolysis of IMs at all pH values studied, whereas NaCl concentrations of 0.2 mol/l inhibited the hydrolysis of PhI at pH values below 3.8 but activated the enzyme at higher incubation pH values. 4. Cu2+ was shown to be a potent non-competitive inhibitor of IMs enzyme activity with an apparent Kl, of approximately 0.02 mmol/l. The enzyme activity was inhibited by Fe2+ (Kl 4 mmol/l), Hg2+ and Ag+, but has not significantly been affected by other univalent or bivalent cations. 5. The presence of solvent and salt effects on apparent Km but not the Vmax. suggest that the binding of IMs to the enzyme involved charge neutralization, and it is inferred that two cationic binding sites are present at the active site. It is postulated that one site specifically binds to the iduronic acid carboxyl group, the other to the 6-sulphate of the anhydromannitol moiety.

Kinetics of reaction of 1,2-diaminobenzene with phenylglyoxal

1990 ◽  
Vol 55 (5) ◽  
pp. 1216-1222 ◽  
Author(s):  
Jiří Klicnar ◽  
Jaromír Mindl ◽  
Vojeslav Štěrba
Keyword(s):  
Reaction Kinetics ◽  
Cyclization Reaction ◽  
Amino Group ◽  
Ph Values ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Kinetics Of Reaction ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Aldehydic Group

The cyclization reaction kinetics of phenylglyoxal monohydrate with 1,2-diaminobenzene have been studied in formate, acetate, and phosphate buffers. At high pH values and low buffer concentrations the rate-limiting step consists in the protonation of the intermediate formed by addition of the first amino group to aldehydic group of phenylglyoxal. With increasing concentrations of formate and acetate buffers the rate-limiting step shifts to the formation of the intermediate. In phosphate buffers the catalysis by the basic buffer component makes itself felt, too. At higher concentrations of 1,2-diaminobenzene, the dehydration of phenylglyoxal monohydrate gradually becomes the rate-limiting step.

Removal of toxic elements from aqueous solution using bentonite modified with l-histidine

2014 ◽  
Vol 70 (12) ◽  
pp. 2022-2030 ◽  
Author(s):  
E. N. Bakatula ◽  
E. M. Cukrowska ◽  
I. M. Weiersbye ◽  
L. Mihaly-Cozmuta ◽  
H. Tutu
Keyword(s):  
Aqueous Solution ◽  
Freundlich Isotherm ◽  
Batch Mode ◽  
Operating Variables ◽  
Ph Values ◽  
Surface Areas ◽  
Rate Limiting Step ◽  
Thermodynamic Constants ◽  
Pseudo Second Order ◽  
Rate Limiting

This study proposes the use of bentonite modified with l-histidine for the removal of Cu, Co, Cr, Fe, Hg, Ni, U and Zn from aqueous solutions such as those impacted by acidic drainage. The surface areas of natural bentonite and bentonite–histidine were 73.8 and 61.2 m2 g−1, respectively. Elemental analysis showed an increase in the amount of carbon (0.258%) and nitrogen (0.066%) for the bentonite–histidine. At a fixed solid/solution ratio, the operating variables affecting the adsorption of metal ions from aqueous solution such as pH, initial concentration, contact time and temperature were studied in batch mode. The Freundlich isotherm model yielded a better fit than the Langmuir for the adsorption of Cu, Co, Ni and Zn, implying adsorption on a heterogeneous surface. Adsorption kinetics followed a pseudo-second-order model, suggesting chemisorption as the rate-limiting step. The apparent activation energy was greater than 40 kJ mol−1 for Cu, Zn, Ni, Co and U, which is characteristic of a chemically controlled reaction. Thermodynamic constants ΔG and ΔH showed that the adsorption of metals was endothermic and spontaneous. Adsorption of heavy metals onto bentonite–histidine was efficient at low pH values, meaning that the adsorbent could be useful for remediating acid mine water.

scholarly journals Using pH dependence to understand mechanisms in electrochemical CO reduction

2021 ◽  
Author(s):  
Georg Kastlunger ◽  
Lei Wang ◽  
Nitish Govindarajan ◽  
Hendrik H. Heenen ◽  
Stefan Ringe ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Modern Technology ◽  
Catalyst Design ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Wide Range ◽  
Alkaline Conditions ◽  
Novel Catalyst ◽  
Co Reduction ◽  
Rate Limiting

Electrochemical conversion of CO(2) into hydrocarbons and oxygenates is envisioned as a promising path towards closing the carbon cycle in modern technology. To this day, however, the reaction mechanisms towards the plethora of products are disputed, complicating the search for novel catalyst materials. In order to conclusively identify the rate-limiting steps in CO reduction on Cu, we analyzed the mechanisms on the basis of constant potential DFT calculations and experiments at a wide range of pH values (3 - 13). We find that *CO dimerization is energetically favoured as the rate limiting step towards multi-carbon products. This finding is consistent with experiments, where the reaction rate is nearly unchanged on an SHE potential scale, even under acidic conditions. For methane, both theory and experiments indicate a change in the rate-limiting step with electrolyte pH from the first protonation step in acidic/neutral conditions to a later one in alkaline conditions. We also show, through a detailed analysis of the microkinetics, that a surface combination of *CO and *H is inconsistent with the measured current densities and Tafel slopes. Finally, we discuss the implications of our understanding for future mechanistic studies and catalyst design.

Formation of the Meisenheimer spiro adduct of N-(2,4,6-trinitrophenyl)alanine methylamide and its rearrangement to 2-amino-N-methyl-N-(2,4,6-trinitrophenyl)propanamide

1986 ◽  
Vol 51 (9) ◽  
pp. 1972-1985 ◽  
Author(s):  
Vladimír Macháček ◽  
Makky M. M. Hassanien ◽  
Vojeslav Štěrba ◽  
Antonín Lyčka
Keyword(s):  
Oxygen Atom ◽  
Nitro Group ◽  
Hydrogen Chloride ◽  
Ph Values ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Neutral Compound ◽  
Rate Limiting

N-(2,4,6-trinitrophenyl)alanine methylamide (I) undergoes base-catalyzed cyclization in methanol to give the spiro adduct II. In aniline-anilinium chloride buffers, the spiro adduct is protonated at the oxygen atom of 2-nitro group to give the neutral compound III. In 4-bromoaniline buffers or by action of methanolic hydrogen chloride, the compound III is opened to E and Z isomers of 2-amino-N-methyl-N-(2,4,6-trinitrophenyl)propanamide hydrochloride (IV). The rate-limiting step of cyclization of compound Z-IV to compound III consists in the isomerization Z-IV → E-IV. At higher pH values (acetate buffers), the rate-limiting step is gradually changed to the isomerization of 2-amino-N-methyl-N-(2,4,6-trinitrophenyl)propanamide (Z-V → E-V).

BIOSYNTHESE VON STEROIDGLUCURONIDEN BEI VERSCHIEDENEN STOFFWECHSELZUSTÄNDEN

1965 ◽  
Vol 50 (1) ◽  
pp. 25-34 ◽  
Author(s):  
Von Herbert Schriefers ◽  
Brigitte Keck ◽  
Margot Otto
Keyword(s):  
Glycogen Content ◽  
Chemical Nature ◽  
Glucuronic Acid ◽  
Reduction Rate ◽  
Reaction Sequence ◽  
Daily Production ◽  
Rate Limiting Step ◽  
Rate Limiting ◽  
Hydrolysis Of ◽  
Very High

ABSTRACT A method is described with which the synthesis of steroid glucuronides in the rat liver can be measured directly. Though, as shown in perfusion experiments with testosterone as substrate, the capacity of the liver to synthesize glucuronides is very high (9.5 μg androsterone-glucuronide equivalents/min · g corresponding to a daily production of about 100 mg), its efficiency depends on an undisturbed carbohydrate metabolism: fasting reduces the glucuronide synthesis to one fifth of the control values and alloxan-diabetes causes a decrease of thirty per cent. Intraperitoneal injection of glucose into fasting animals increases the glucuronide synthesis within two hours up to values near the normal. The decline in the rate of glucuronide production cannot be accounted for by the concomitant decrease in testosterone reduction rate. In fasting animals the lowering of the glucuronide synthesis is more pronounced than the ring A reduction rate. On the basis of these facts it is suggested that in the reaction sequence, which proceeds via the formation of dihydro- and tetrahydrocompounds to steroid glucuronides, the transfer of glucuronic acid is the rate limiting step. Obviously the fall in glucuronide synthesis is due to a decrease of liver glucose-6-phosphate and glycogen content, both of which may lead to a diminished supply of UDP-glucuronic acid. Among the aglucones liberated following enzymatic hydrolysis of the chromatographically separated glucuronide fraction, testosterone amounts to only 5 ± 2.04% of the yield expected according to the glucuronic acid estimation. Most of the aglucone fraction consists of products from testosterone hydrogenation. A report dealing with their quantitative distribution and clarifying their chemical nature is in preparation.

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