scholarly journals Pig heart lactate dehydrogenase. Binding of pyruvate and the interconversion of pyruvate-containing ternary complexes

1975 ◽  
Vol 151 (3) ◽  
pp. 715-727 ◽  
Author(s):  
M J Boland ◽  
H Gutfreund
Keyword(s):  
Lactate Dehydrogenase ◽  
Dissociation Constant ◽  
Turnover Rate ◽  
Nadh Oxidation ◽  
Lactate Oxidation ◽  
First Order ◽  
Single Turnover ◽  
Ph Values ◽  
Km Value ◽  
Rate Limiting

1. Lactate oxidation catalysed by pig heart lactate dehydrogenase was studied in the presence of inhibitory concentrations of pyruvate. Experimental results show the presence of an intermediate which occurs immediately after the hydride transfer step, but before the dissociation of pyruvate and the H+ produced by the reaction. The rate constant for pyruvate dissociation and the dissociation constant for pyruvate from the ternary complex differ from those obtained in pyruvate reduction experiments. 2. In single-turnover pyruvate reduction by pig heart lactate dehydrogenase at pH8.0 pyruvate can bind to the enzyme before a H+ is taken up, and the subsequent uptake of a H+ is governed by a step that is also rate-limiting for single-turnover and steady-state NADH oxidation. 3. Observation of various intermediates in the single-turnover pyruvate reduction experiments has made it possible to determine separately the dissociation constant and Km value for pyruvate at pH8.0, and also the catalytic turnover rate and Km for pyruvate under first-order conditions at different pH values. 4. Further studies on single-turnover pyruvate reduction carried out in 2H2O, or in water at low temperature, show another step which, under these conditions, is slower than that controlling H+ uptake and rate-limiting for NADH oxidation. A scheme is presented which explains these results.

scholarly journals Transient-kinetic studies of pig muscle lactate dehydrogenase

1971 ◽  
Vol 121 (2) ◽  
pp. 235-240 ◽  
Author(s):  
R. A. Stinson ◽  
H. Gutfreund
Keyword(s):  
Steady State ◽  
Lactate Dehydrogenase ◽  
Hydride Transfer ◽  
Kinetic Studies ◽  
Muscle Lactate ◽  
First Order ◽  
Ph Values ◽  
Liver Alcohol Dehydrogenase ◽  
Pig Muscle ◽  
Rate Limiting

1. The very fast pre-steady-state formation of NADH catalysed by pig M4 lactate dehydrogenase was equivalent to the enzyme-site concentration at pH values greater than 8.0 and to one-half the site concentration at pH6.8. 2. The rate of dissociation of NADH from the enzyme at pH8.0 (450s−1) in the absence of other substrates is faster than the steady-state oxidation of lactate (80s−1). The latter process is therefore controlled by a step before NADH dissociation but subsequent to the hydride transfer. 3. The oxidation of enzyme–NADH by excess of pyruvate was studied as a first-order process at pH9.0. There was no effect of NADD on this reaction and it was concluded that the ternary complex undergoes a rate-limiting change before the hydride-transfer step. 4. Some conclusions about the reactions catalysed by the M4 isoenzyme were drawn from a comparison of these results with those obtained with the H4 isoenzyme and liver alcohol dehydrogenase.

Pulsradiolyse des Schwefelwasserstoffs in wäßriger Lösung

1967 ◽  
Vol 22 (3) ◽  
pp. 273-282 ◽  
Author(s):  
W. Karmann ◽  
G. Meissner ◽  
A. Henglein
Keyword(s):  
Dissociation Constant ◽  
Rate Constant ◽  
Hydrated Electron ◽  
Alkaline Solutions ◽  
Oh Radical ◽  
Acid Solutions ◽  
Fast Reaction ◽  
First Order ◽  
Broad Maximum ◽  
Ph Values

The reactions of the OH radical with H2S and SH⊖ are very fast (k = 1.1·1010 and 5.4·109 mole-1 liter sec-1, respectively). The hydrated electron reacts with H2S (1.1·1010) but does not react with SH⊖. The rate constant of the reaction CNS+SH⊖ CNS⊖ +SH was found to be equal to 9.8·108. Species absorbing at 3800 Å (e ∼ 104 mole-1 liter cm-1) are formed in these reactions of H2S and SH⊖ The intensity of this absorption is very low in strongly acid solutions, increases with pH, goes through a broad maximum at about pH=7, reaches a lower plateau between pH = 8.5 -10.5 and decreases in strongly alkaline solutions. At pH values below 7, the intensity is dependent on the concentrations of both SH⊖ and free H2S, i. e. at constant SH⊖ concentration a stronger absorption occurs if more free H2S is present. In the plateau below pH = 8.5 -10.5, the intensity is practically independent of the SH⊖ concentration above 4·10-5 mole/l. The decay of the 3800 A absorption after the pulse is first order with τ1/2 = 5 μsec independent of the OH⊖ and SH⊖ concentrations in alkaline solutions.Below pH = 7, the absorption disappears by second order, the rate constant being larger at lower pH values. SH or S⊖ absorb weakly in the near UV and do not contribute to the 3800 Å absorption. This absorption is attributed to the ions H2S2⊖ or H2S2⊖ in alkali and to H2S2⊖or higher complexes such as H3S4⊖ in acid. The first order decay of 5 μsec is attributed to the spontaneous decomposition of S2H2⊖ into S⊖ +SH⊖. Two S⊖ ions form S22⊖ (2 k=2·109 mole-1 liter sec-1). The complex H2S2⊖ is formed in the fast reaction SH + SH⊖ ↔ S2H2⊖ (dissociation constant about 4·10-5 mole/l). The formation of an intermediate complex SHOH⊖ in the reaction of OH with SH⊖ is discussed. This intermediate reacts with either SH⊖ to form S2H2⊖ or with OH⊖ (or other proton acceptors such as ΗΡΟ42Θ) to form SΘ+ H2O+OHΘ.

scholarly journals pH-controlled hydrogen-bonding (Short Communication)

1974 ◽  
Vol 143 (3) ◽  
pp. 775-777 ◽  
Author(s):  
John L. Wood
Keyword(s):  
Hydrogen Bonding ◽  
Dissociation Constant ◽  
Small Proportion ◽  
Association Constant ◽  
Short Communication ◽  
Dibasic Acid ◽  
Ph Dependence ◽  
Ph Values ◽  
Conjugate Acid ◽  
Total Base

The pH-dependence of the degree of hydrogen-bonding between a base and its conjugate acid is considered. When only a small proportion of the total base is complexed, the amount complexed is proportional to (1+coshp)−1 where p=2.303 (pKa–pH), pKa being the dissociation constant of the conjugate acid. This represents sharp pH-dependence. As the proportion complexed increases, the curve broadens, eventually becoming flat-topped, with more than half the base complexed over the range of pH values pKa±logKC, approximately. (K is the complex association constant and C is the formal base concentration, including all forms.) There are similarities to the extent of mono-protonation of a dibasic acid.

scholarly journals Kinetics of thiamin cleavage by sulphite

1969 ◽  
Vol 113 (4) ◽  
pp. 611-615 ◽  
Author(s):  
J. Leichter ◽  
M. A. Joslyn
Keyword(s):  
Aqueous Solutions ◽  
Rate Constant ◽  
Sulphur Dioxide ◽  
First Order ◽  
Ph Values ◽  
Rate Of Reaction ◽  
Kinetics Of

Results are presented on the rate of thiamin cleavage by sulphite in aqueous solutions as affected by temperature (20–70°), pH(2·5–7·0), and variation of the concentration of either thiamin (1–20μm) or sulphite (10–5000μm as sulphur dioxide). Plots of the logarithm of percentage of residual thiamin against time were found to be linear and cleavage thus was first-order with respect to thiamin. At pH5 the rate was also found to be proportional to the sulphite concentration. In the pH region 2·5–7·0 at 25° the rate constant was 50m−1hr.−1 at pH5·5–6·0, and decreased at higher or lower pH values. The rate of reaction increased between 20° and 70°, indicating a heat of activation of 13·6kcal./mole.

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

2021 ◽  
Author(s):  
Biswash Thakuri ◽  
Bruce O'Rourke ◽  
Amanda Graves ◽  
Matthew Liptak
Keyword(s):  
Dynamic Equilibrium ◽  
Ionization Mass ◽  
Esi Ms ◽  
Enzymatic Mechanism ◽  
Single Turnover ◽  
Rate Limiting Step ◽  
Planar Substrate ◽  
Out Of Plane ◽  
Substrate Conformation ◽  
Rate Limiting

The non-canoncial heme oxygenase MhuD from <i>Mycobacterium tuberculosis</i> binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation resulting in a refined enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring population of the ruffled heme conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a refined enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.<br>

A note on some factors influencing muscle pH values in commercial pig carcasses

1978 ◽  
Vol 26 (2) ◽  
pp. 229-232 ◽  
Author(s):  
W. C. Smith ◽  
A. Wilson
Keyword(s):  
Least Squares ◽  
Practical Significance ◽  
Longissimus Dorsi ◽  
Post Mortem ◽  
First Order ◽  
Commercial Grade ◽  
Ph Values ◽  
Factors Influencing ◽  
Muscle Ph ◽  
Pig Carcasses

ABSTRACTMeasurements of longissimus dorsi pH at 45 min post mortem (pH1) were taken in 27 727 pig carcasses at four bacon factories. The overall least squares mean pH1 was 6·26 and 15·5% of carcasses had pHi values of 5·9 or less. Sex and commercial grade of carcass had little influence on muscle pHi or the incidence of values below pH 6·0. Factory differences were recorded in the distribution of muscle and within each there were marked producer effects in mean and the incidence of values below pH 6·0. Carcasses of Large Whites had fewer muscle pH1 values below 6·0 (12·4%) than those of British Landrace (18·7%), this being mainly due to a large breed effect at one factory. Although breed and testing background of sires of the carcasses examined and the first order interactions with factory had significant effects on muscle pHi and the incidence of values below pH 6·0, they were of little practical significance.

scholarly journals An Antipersister Strategy for Treatment of Chronic Pseudomonas aeruginosa Infections

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Martina Koeva ◽  
Alina D. Gutu ◽  
Wesley Hebert ◽  
Jeffrey D. Wager ◽  
Lael M. Yonker ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lactate Dehydrogenase ◽  
Stationary Phase ◽  
Persister Cells ◽  
Content Type ◽  
Ph Values ◽  
Potentiation Effect ◽  
Metabolite Concentrations ◽  
Ldh Assay

ABSTRACTBacterial persisters are a quasidormant subpopulation of cells that are tolerant to antibiotic treatment. The combination of the aminoglycoside tobramycin with fumarate as an antibacterial potentiator utilizes an antipersister strategy that is aimed at reducing recurrentPseudomonas aeruginosainfections by enhancing the killing ofP. aeruginosapersisters. Stationary-phase cultures ofP. aeruginosawere used to generate persister cells. A range of tobramycin concentrations was tested with a range of metabolite concentrations to determine the potentiation effect of the metabolite under a variety of conditions, including a range of pH values and in the presence of azithromycin or cystic fibrosis (CF) patient sputum. In addition, 96-well dish biofilm and colony biofilm assays were performed, and the cytotoxicity of the tobramycin-fumarate combination was determined utilizing a lactate dehydrogenase (LDH) assay. Enhanced killing of up to 6 orders of magnitude ofP. aeruginosapersisters over a range of CF isolates, including mucoid and nonmucoid strains, was observed for the tobramycin-fumarate combination compared to killing with tobramycin alone. Furthermore, significant fumarate-mediated potentiation was seen in the presence of azithromycin or CF patient sputum. Fumarate also reduced the cytotoxicity of tobramycin-treatedP. aeruginosato human epithelial airway cells. Finally, in mucoid and nonmucoid CF isolates, complete eradication ofP. aeruginosabiofilm was observed in the colony biofilm assay due to fumarate potentiation. These data suggest that a combination of tobramycin with fumarate as an antibacterial potentiator may be an attractive therapeutic for eliminating recurrentP. aeruginosainfections in CF patients through the eradication of bacterial persisters.

scholarly journals Kinetics and mechanism of action of muscle pyruvate kinase

1978 ◽  
Vol 169 (1) ◽  
pp. 39-54 ◽  
Author(s):  
Leighton G. Dann ◽  
Hubert G. Britton
Keyword(s):  
Steady State ◽  
Dissociation Constant ◽  
Pyruvate Kinase ◽  
Alternative Pathway ◽  
Slow Step ◽  
Velocity Data ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Muscle Pyruvate Kinase ◽  
Rate Limiting

1. The mechanism of rabbit muscle pyruvate kinase was investigated by measurements of fluxes, isotope trapping, steady-state velocity and binding of the substrates. All measurements were made at pH8.5 in Tris/HCl buffer and at 5mm-free Mg2+. 2. Methods of preparing [32P]phosphoenolpyruvate from [32P]Pi in high yield and determining [32P]-phosphoenolpyruvate and [8-14C]ADP are described. 3. The ratio Flux of ATP to ADP/Flux of ATP to phosphoenolpyruvate (measured at equilibrium) increased hyperbolically with ADP concentration from unity to about 2.1 at 2mm-ADP, but was unaffected by phosphoenolpyruvate concentration. Since the ratio is greater than unity, one pathway for the addition of substrates must involve phosphoenolpyruvate adding first to the enzyme in a rate-limiting step. However, the substrates must also add in the alternative order, because of the non-linear increase in the ratio with ADP concentration and because the rate of increase is very much less than that predicted from the steady-state velocity data for an ordered addition. The lack of influence of phosphoenolpyruvate on the ratio is consistent with the rapid addition of ADP in the alternative pathway. At low ADP concentrations the alternative pathway contributes less than 33% to the total reaction. 4. Isotope trapping was observed with [32P]phosphoenolpyruvate, confirming that when phosphoenolpyruvate adds first to the enzyme it is in a rate-limiting step. The release of phosphoenolpyruvate from the ternary complex must also be a slow step. Trapping was not observed with [8-14C]ADP, hence the addition of ADP to the free enzyme must be rapid unless its dissociation constant is very large (>20mm). 5. Binding studies showed that 4mol of [32P]phosphoenolpyruvate binds to 1mol of the enzyme, probably unligated to Mg2+, with a dissociation constant appropriate to the mechanism indicated above. Binding of [8-14C]ADP could not be detected, and hence the binding of ADP occurs by a low-affinity step. The latter is also demanded by the steady-state velocity data. 6. The ratio Flux of phosphoenolpyruvate to ATP/Flux of phosphoenolpyruvate to pyruvate (determined from the incorporation of label into phosphoenolpyruvate from [3-14C]-pyruvate or [γ-32P]ATP during the forward reaction) did not differ significantly from unity. Steady-state velocity data predicted grossly different flux ratios for ordered dissociations of the products, and the results indicate that the dissociation must be rapid and random. The data also exclude a Ping-Pong mechanism. 7. Permissible rate constants for the above mechanism are calculated. The results indicate a high degree of cooperativity in binding, whatever the order of addition of substrate.

scholarly journals A stopped-flow kinetic study of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath)

1989 ◽  
Vol 259 (1) ◽  
pp. 167-172 ◽  
Author(s):  
J Green ◽  
H Dalton
Keyword(s):  
Electron Transfer ◽  
Protein C ◽  
Turnover Rate ◽  
Protein A ◽  
Methylococcus Capsulatus ◽  
Bond Breakage ◽  
Conversion Of Methane ◽  
Protein Components ◽  
A Charge ◽  
Rate Limiting

1. The roles of the three protein components of soluble methane mono-oxygenase were investigated by the use of rapid-reaction techniques. The transfer of electrons through the enzyme complex from NADH to methane/O2 was also investigated. 2. Electron transfer from protein C, the reductase component, to protein A, the hydroxylase component, was demonstrated. Protein C was shown to undergo a three-electron--one-electron catalytic cycle. The interaction of protein C with NADH was investigated. Reduction of protein C was shown to be rapid, and a charge-transfer interaction between reduced FAD and NAD+ was observed; this intermediate was also found in static titration experiments. Thus the binding of NADH, the reduction of protein C and the intramolecular transfer of electrons through protein C were shown to be much more rapid than the turnover rate of methane mono-oxygenase. 3. The rate of transfer of electrons from protein C to protein A was shown to be lower than the reduction of protein C but higher than the turnover rate of methane mono-oxygenase. Association of the proteins was not rate-limiting. The amount of protein A present in the system had a small effect on the rate of reduction of protein C, indicating some co-operativity between the two proteins. 4. Protein B was shown to prevent electron transfer between protein C and protein A in the absence of methane. On addition of saturating concentrations of methane electron transfer was restored. With saturating concentrations of methane and O2 the observed rate constant for the conversion of methane into methanol was 0.26 s-1 at 18 degrees C. 5. By the use of [2H4]methane it was demonstrated that C-H-bond breakage is likely to be the rate-limiting step in the conversion of methane into methanol.

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.

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