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The kinetics of the reversible inhibition of heart lactate dehydrogenase through formation of the enzyme-oxidized nicotinamide-adenine dinucleotide-pyruvate compound

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Abstract

The inhibition of lactate dehydrogenase at high pyruvate concentration was studied in three ways. First, a rapid decrease in the rate of the enzyme reaction was observed; secondly, the rate of formation of a pyruvate-NAD(+) compound was followed by the change in E(325); thirdly, the rate of quenching of the protein fluorescence was measured. The data obtained at pH6.0 at different temperatures and ionic strengths as functions of pyruvate, NAD(+) and enzyme concentrations show that the extent of inhibition can be correlated with the reversible formation of a compound between pyruvate and enzyme-bound NAD(+). It is suggested that the detailed kinetic analysis of the formation of this abortive ternary compound will give pertinent information about properties of the enzyme-NAD(+) compound involved in the normal catalytic process.

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... Rather longstanding and perhaps forgotten metabolic studies have suggested that high concentrations of pyruvate can induce substrate inhibition of LDH extracted from various cells, including cardiac cells, erythrocytes and muscle cells (32,33). To extend these previous observations to tumor cellextracted enzymes (32)(33)(34), we first validated a classic coupled-enzyme kinetic assay using commerciallyavailable bovine heart LDH, purified human recombinant LDHA and recombinant LDHB in response to a broad range of pyruvate concentrations. ...
... Rather longstanding and perhaps forgotten metabolic studies have suggested that high concentrations of pyruvate can induce substrate inhibition of LDH extracted from various cells, including cardiac cells, erythrocytes and muscle cells (32,33). To extend these previous observations to tumor cellextracted enzymes (32)(33)(34), we first validated a classic coupled-enzyme kinetic assay using commerciallyavailable bovine heart LDH, purified human recombinant LDHA and recombinant LDHB in response to a broad range of pyruvate concentrations. Because initial velocities were evaluated as a change (decrease) in NADH absorbance, which was coupled with pyruvate to lactate conversion, it was important to affirm the specificity of initial velocity signals. ...
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Cellular pyruvate is an essential metabolite at the crossroads of glycolysis and oxidative phosphorylation, capable of supporting fermentative glycolysis by reduction to lactate mediated by lactate dehydrogenase (LDH) among other functions. Several inherited diseases of mitochondrial metabolism impact extracellular (plasma) pyruvate concentrations, and [1-¹³C]pyruvate infusion is used in isotope-labeled metabolic tracing studies, including hyperpolarized magnetic resonance spectroscopic imaging (MRSI). However, how these extracellular pyruvate sources impact intracellular metabolism is not clear. Herein, we examined the effects of excess exogenous pyruvate on intracellular LDH activity, extracellular acidification rates (ECAR) as a measure of lactate production, and hyperpolarized [1-¹³C]pyruvate-to-[1-¹³C]lactate conversion rates across a panel of tumor and normal cells. Combined LDH activity and LDHB/LDHA expression analysis intimated various hetero-tetrameric isoforms comprised of LDHA and LDHB in tumor cells, not only canonical LDHA. Millimolar concentrations of exogenous pyruvate induced substrate inhibition of LDH activity in both enzymatic assays ex vivo and in live cells, abrogated glycolytic ECAR, and inhibited hyperpolarized [1-¹³C]pyruvate-to-[1-¹³C]lactate conversion rates in cellulo. Importantly, the extent of exogenous pyruvate-induced inhibition of LDH and glycolytic ECAR in live cells was highly dependent on pyruvate influx, functionally mediated by monocarboxylate transporter-1 (MCT1) localized to the plasma membrane. These data provided evidence that highly concentrated bolus injections of pyruvate in vivo may transiently inhibit LDH activity in a tissue type- and MCT1-dependent manner. Maintaining plasma pyruvate at submillimolar concentrations could potentially minimize transient metabolic perturbations, improve pyruvate therapy, and enhance quantification of metabolic studies, including hyperpolarized [1-¹³C]pyruvate MRSI and stable isotope tracer experiments.
... It has been demonstrated that incubation of LDH in the presence of pyruvate and NAD + results in the formation of a strong inhibitor (NAD-Pyr) in the active center of the enzyme. In NAD-Pyr the β-carbon atom of pyruvate is covalently linked to the C-4 position of the nicotinamide ring678. The NAD-and 3-substituted NAD (APAD) pyruvate adducts also spontaneously form during the incubation of pyruvate and the dinucleotides at pH 11 [9,10]. ...
... The absorption of the inhibitor in the complex at 327 nm is equal to 0.125 corresponding to the extinction coefficient of NADH-GA of 6.5±0.5 mM −1 cm −1 . This value is similar to that of NADH and its derivatives in the near-UV spectral region [7,8,30].Figure 4A shows the results of treating cultured adult mouse cardiac myocytes with varying concentrations of NADH-GA ranging from 10 nM to 1 μM during 16 hr of reoxygenation following 3 hours of hypoxia. Control cells were incubated under normoxic conditions for the duration of the experiment and are assigned a survival of 100%. ...
Article
Alkaline incubation of NADH results in the formation of a very potent inhibitor of lactate dehydrogenase. High resolution mass spectroscopy along with NMR characterization clearly showed that the inhibitor is derived from attachment of a glycolic acid moiety to the 4-position of the dihydronicotinamide ring of NADH. The very potent inhibitor is competitive with respect to NADH. The inhibitor added in submicromolar concentrations to cardiomyocytes protects them from damage caused by hypoxia/reoxygenation stress. In isolated mouse hearts, addition of the inhibitor results in a substantial reduction of myocardial infarct size caused by global ischemia/reperfusion injury.
... Interestingly, the activity of LDHB has been reported to be inhibited by excess pyruvate, the so-called substrate-inhibition effect [28][29][30][31] . This inhibition could be largely removed if the serine 162 is replaced by leucine 32,33 , which prompted us to explore whether serine 162 phosphorylation modulates the substrateinhibition effect. ...
... Remarkably, it was reported that replacement of LDHA/B S162 by leucine efficiently relieved the substrate-inhibition by pyruvate 32,33 , indicating S162 is indeed critical for the substrateinhibition of LDHB. It was suggested that the change from serine to leucine reduces the affinity between LDHB and NADH, and disrupts the formation of the LDHB-pyruvate-NAD + adduct 32,33 , a covalent adduct causing the substrate-inhibition effect [28][29][30]35 . In our study, molecular modeling showed that the nicotinamide ring of NADH is pushed away from S162 of LDHB after the serine is phosphorylated (Supplementary Fig. 4a, b). ...
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Overexpressed Aurora-A kinase promotes tumor growth through various pathways, but whether Aurora-A is also involved in metabolic reprogramming-mediated cancer progression remains unknown. Here, we report that Aurora-A directly interacts with and phosphorylates lactate dehydrogenase B (LDHB), a subunit of the tetrameric enzyme LDH that catalyzes the interconversion between pyruvate and lactate. Aurora-A-mediated phosphorylation of LDHB serine 162 significantly increases its activity in reducing pyruvate to lactate, which efficiently promotes NAD+ regeneration, glycolytic flux, lactate production and bio-synthesis with glycolytic intermediates. Mechanistically, LDHB serine 162 phosphorylation relieves its substrate inhibition effect by pyruvate, resulting in remarkable elevation in the conversions of pyruvate and NADH to lactate and NAD+. Blocking S162 phosphorylation by expression of a LDHB-S162A mutant inhibited glycolysis and tumor growth in cancer cells and xenograft models. This study uncovers a function of Aurora-A in glycolytic modulation and a mechanism through which LDHB directly contributes to the Warburg effect.
... LDH is a well-characterised enzyme which reversibly converts pyruvate and reduced nicotinamide adenine dinucleotide (NADH) to lactate and NAD + . The reaction mechanism for LDH ( Fig. 5) involves the ordered binding of the coenzymes (NADH or NAD + ) to LDH followed by the subsequent binding of its corresponding substrate (pyruvate or lactate, respectively) [35]. The transfer of an electron between the coenzyme and the substrate then reversibly occurs in the ternary complex as part of the catalytic process. ...
... The kinetic parameters associated with them are shown in the shaded region. The remaining reactions adapted from[35]. ...
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Quasi steady-state enzyme kinetic models are increasingly used in systems modelling. The Michaelis Menten model is popular due to its reduced parameter dimensionality, but its low-enzyme and irreversibility assumption may not always be valid in the in vivo context. While the total quasi-steady state assumption (tQSSA) model eliminates the reactant stationary assumptions, its mathematical complexity is increased. Here, we propose the differential quasi-steady state approximation (dQSSA) kinetic model, which expresses the differential equations as a linear algebraic equation. It eliminates the reactant stationary assumptions of the Michaelis Menten model without increasing model dimensionality. The dQSSA was found to be easily adaptable for reversible enzyme kinetic systems with complex topologies and to predict behaviour consistent with mass action kinetics in silico. Additionally, the dQSSA was able to predict coenzyme inhibition in the reversible lactate dehydrogenase enzyme, which the Michaelis Menten model failed to do. While the dQSSA does not account for the physical and thermodynamic interactions of all intermediate enzyme-substrate complex states, it is proposed to be suitable for modelling complex enzyme mediated biochemical systems. This is due to its simpler application, reduced parameter dimensionality and improved accuracy.
... Janin and Iwatsubo (14) correlated the rates of several spectroscopic changes with the rate of inhibition of homoserine dehydrogenase by threonine as first measured by Barber and Bright (13). Gutfreund et al. (19) correlated the rate of inhibition of lactate dehydrogenase by pyruvate with the rate of formation of the enzyme. NAD+ pyruvate complex. ...
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Stopped flow spectrophotometry was used to investigate the kinetics of the transition of the phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD oxidoreductase, EC 1.1.1.95) reaction from the active to the inhibited rate upon the addition of the physiological inhibitor serine. The transition was characterized by a single first order rate constant (kobs,i) which was independent of enzyme concentration. At pH 8.5, kobs,i increased in a hyperbolic manner with serine concentration from 2 to 8 s-1. The increase in kobs,i occurred at serine concentrations where the steady state inhibition was virtually complete. These results indicate that serine inhibition is an allosteric process involving a conformational change in the enzyme. A model is presented in which serine at low concentrations binds exclusively to the inhibited state of the enzyme and shifts the equilibrium toward that state; at high serine concentrations, serine binds to the active state, facilitating its conversion to the inhibited state. An alternative model, which we favor, proposes two classes of inhibitor binding sites. The kinetics of the fluorescence quenching of enzyme-bound NADH by serine (Sugimoto, E., and Pizer, L.I. (1968) J. Biol. Chem. 243, 2090-2098), measured by stopped flow fluorimetry, was also characterized by a single first order rate constant (kobs,f.q.) which was independent of enzyme concentration. At pH 8.5, kobs,f.q. ranged from 0.4 s-1 at low serine concentrations to 1.1 s-1 at high serine concentrations. These results indicate that the fluorescence quenching induced by serine is a manifestation of a structural change in the enzyme. Enzyme and excess NADH were mixed with substrate and serine in the stopped flow instrument, and enzyme-bound NADH fluorescence was monitored by exciting through the protein at 285 nm. A rapid fluorescence quenching process, which occurred within the mixing time, was followed by a slower fluorescence enhancement process which terminated in a steady state level corresponding to the quenched fluorescence of the enzyme NADH serine complex. The rapid quenching was the result of substrate binding (Dubrow, R., and Pizer, L.I. (1977) J. Biol. Chem. 252, 1539-1551). The fluorescence enhancement was characterized by a single first order rate constant whose value for a given serine concentration corresponded with Kobs,j. This data shows that the quenched state of the enzyme-NADH-complex is the state which is directly responsible for the inhibition of enzyme activity. During catalysis the quenched state is achieved from a different initial conformation, and consequently at a different rate, than in the absence of substrate. kobs,j and kobs,f.q. were also measured using glycine, another inhibitor. The ultraviolet difference spectrum between enzyme and enzyme plus serine was determined and proposed to be the result of the same structural change which is responsible for the fluorescence quenching by serine.
... For dehydrogenases, several molecular mechanisms of substrate inhibition have been proposed, including the formation of a covalent adduct between the oxidized forms of substrate and cofactor, allosteric inhibition (which occurs away from the active site, e.g., in D-3-phosphoglycerol dehydrogenase from Mycobacterium tuberculosis), and the formation of a nonproductive enzyme complex with cofactor and/or substrate (6)(7)(8)(9)(10)(11)(12)(13). The latter mechanism can be associated with the dehydrogenase residues located both in the substrate and in cofactor binding sites. ...
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Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from Staphylococcus aureus is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from Escherichia coli, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semi-conserved Gly234 (to Ser, Thr or Ala) located in the BetB NAD(+) binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and non-productive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the non-productive binding of betaine aldehyde.
... In our hands, FK866 also stimulated a mild and dose-independent decrease in NADH levels. This differential effect of FK866 on NAD + and NADH levels in cardiomyocytes could be explained by the pyruvate-lactate conversion mediated by lactate dehydrogenase [38,39] and by the activity of ubiquinone oxidoreductase and other dehydrogenases [40]. However, this hypothesis should be tested in future studies. ...
... In our hands, FK866 also stimulated a mild and dose-independent decrease in NADH levels. This differential effect of FK866 on NAD + and NADH levels in cardiomyocytes could be explained by the pyruvate–lactate conversion mediated by lactate dehydrogenase [38,39] and by the activity of ubiquinone oxidoreductase and other dehydrogenases [40]. However, this hypothesis should be tested in future studies. ...
... In our hands, FK866 also stimulated a mild and dose-independent decrease in NADH levels. This differential effect of FK866 on NAD + and NADH levels in cardiomyocytes could be explained by the pyruvate–lactate conversion mediated by lactate dehydrogenase [38,39] and by the activity of ubiquinone oxidoreductase and other dehydrogenases [40]. However, this hypothesis should be tested in future studies. ...
... In addition, we observed that the NAD + /NADH ratio was reduced following OGDH suppression in the sensitive PIK3CA MUT cells, indicative of an altered redox state that paralleled sensitivity to OGDH (Fig. 5E). NADH produced in the cytosol must be regenerated to NAD + for glycolysis to proceed, which can be achieved through the activity of lactate dehydrogenase (22)(23)(24), glycerol-3-phosphate shuttle (25), or the malateaspartate shuttle. The malate-aspartate shuttle activity regenerates NAD + in the cytosol, which brings the reducing equivalents into the mitochondria (26,27) (Fig. 5F). ...
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Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate-aspartate shuttle, which is important for cytoplasmic NAD(+) regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate-aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD(+)/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene.
... Asymmetric biocatalysis has become increasingly popular in the synthesis of chiral pharmaceuticals, and dehydrogenases have received much attention for their use in industrial bioreduction of various chemicals (Pan et al., 2014;Xu et al., 2015aXu et al., , 2015b. However, substrate inhibition is widespread in dehydrogenases, such as lactate dehydrogenase (Eszes et al., 1996;Gutfreund et al., 1968;Hewitt et al., 1999;Shoemark et al., 2007), drosophila alcohol dehydrogenase (DADH) (Benach et al., 1999;Winberg and Mckinley-Mckee, 1988), phenylethanol dehydrogenase (PED) (Hoffken et al., 2006) and salutaridine reductase (SalR) (Geissler et al., 2007). Thus, it is imperative to solve the issue between substrate inhibition of enzymes and the high substrate loading required for economically feasible biotransformations (Hollmann et al., 2011;Huisman et al., 2010). ...
Article
Substrate inhibition of enzymes is one of the main obstacles encountered frequently in industrial biocatalysis. Haloketone reductase SsCR was seriously inhibited by substrate 2,2',4'-trichloroacetophenone. In this study, two essential loops were found that have a relationship with substrate binding by conducting X-ray crystal structure analysis. Three key residues were selected from the tips of the loops and substituted with amino acids with lower hydrophobicity to weaken the hydrophobic interactions that bridge the two loops, resulting in a remarkable reduction of substrate inhibition. Among these variants, L211H showed a significant attenuation of substrate inhibition, with a Ki of 16 mM, which was 16 times that of the native enzyme. The kinetic parameter kcat/Km of L211H was 3.1 × 103 s-1 mM-1, showing the comparable catalytic efficiency to that of the wild-type enzyme (WT). At the substrate loading of 100 mM, the space time yield of variant L211H in asymmetric reduction of the haloketone was 3-fold higher than that of the WT.
... The LDHd isplays substrate inhibition at high pyruvate concentrations. [22] NADH binds first to LDH, followed by pyruvate to form the catalytically active complex.A fter reduction, the LDH-NAD + -lactatec omplex is obtained, leading to the release of lactate upon turnover.A th igh concentrations of pyruvate, the inactive LDH-NAD + -pyruvate binary complex is formed leading to LDH inhibition. The substrate inhibition is slightly affected by the aggregate, which displays ad ecreased K i compared with the free enzyme. ...
Article
Herein, we report on enzyme aggregates assembled around covalently cross-linked streptavidin tetramers. The streptavidin oligomeric matrix (SavMatrix) is produced using the SpyTag - SpyCatch technology and binds tightly to fusion proteins bearing a streptavidin-binding peptide (SBP). Fusing the SBPs to different enzymes leads to precipitation of the streptavidin-enzyme aggregates upon mixing the complementary components. This straightforward strategy can be applied to crude cell-free extract, allowing the one-step assembly and purification of catalytically active aggregates. Enzyme cascade assemblies can be produced upon adding different SBP-fused enzymes to the SavMatrix. The reaction rate for lactate dehydrogenase (LDH) is improved tenfold (compared to the soluble enzyme) upon precipitation with the SavMatrix from crude cell-free extracts. Additionally, the kinetic parameters are improved. A cascade combining a transaminase with LDH for the synthesis of enantiopure amines from prochiral ketones displays nearly threefold rate enhancement for the synthesis of (R)-α-methylbenzylamine compared to the free enzymes in solution.
... For example, in the heart of fishes and many other animals, the isoform LDH-B predominates, which is inhibited by the accumulation of its substrate, the pyruvate. The LDH-B isoform would protect such sensitive tissues against the aggressive effects of lactate accumulation under hypoxia (Gutfreund et al., 1968;Almeida-Val & Val, 1993). The reduction in LDH activity, under hypoxia, could be reached by allosteric effectors (Brown & Christian, 1974), by shifting between dimer and tetramer forms (Yamamoto & Storey, 1988), or by peptides that affect the interaction between LDH subunits (Döbeli & Schoenenberger, 1983). ...
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Background In the wild, matrinchã ( Brycon amazonicus ) and tambaqui ( Colossoma macropomum ) rely strongly on their swimming capacity to perform feeding, migration and reproductive activities. Sustained swimming speed in fishes is performed almost exclusively by aerobic red muscles. The white muscle has high contraction power, but fatigue quickly, being used mainly in sprints and bursts, with a maximum duration of few seconds. The Ucrit test, an incremental velocity procedure, is mainly a measure of the aerobic capacity of a fish, but with a high participation of anaerobic metabolism close to the velocity of fatigue. Our previous study has indicated a high swimming performance of matrinchã (Ucrit) after hypoxia exposure, despite increased levels of lactate in plasma. In contrast, tambaqui with high lactate levels in plasma presented very low swimming performance. Therefore, we aimed to study the resistance of matrinchã and tambaqui to the increased lactate levels in muscle over an incremental velocity test (Ucrit). As a secondary aim, we analyzed the differences in anaerobic metabolism in response to environmental hypoxia, which could also support the better swimming performance of matrinchã, compared to tambaqui. Methods We measured, over incremented velocities in both species, the metabolic rate (the oxygen consumption by the fish; MO 2 ), and the concentrations of lactate and nitrites and nitrates (NOx) in muscles. NOx was measured as an indicator of nitric oxide and its possible role in improving cardiorespiratory capacity in these fishes, which could postpone the use of anaerobic metabolism and lactate production during the swimming test. Also, we submitted fishes until fatigue and hypoxia (0.5 mg L ⁻¹ ) and measured, in addition to the previous parameters, lactate dehydrogenase activity (LDH; the enzyme responsible for lactate production), since that swimming performance could also be explained by the anaerobic capacity of producing ATP. Results Matrinchã exhibited a better swimming performance and higher oxygen consumption rates. Lactate levels were higher in matrinchã only at the moment of fatigue. Under hypoxia, LDH activity increased in the white muscle only in tambaqui, but averages were always higher in matrinchã. Discussion and conclusions The results suggest that matrinchã is more resistant than tambaqui regarding lactate accumulation in muscle at the Ucrit test, but it is not clear how much it contributes to postpone fatigue. The higher metabolic rate possibly allows the accumulated lactate to be used as aerobic fuel by the matrinchã, improving swimming performance. More studies are needed regarding matrinchã’s ability to oxidize lactate, the effects of exercise on muscle acidification, and the hydrodynamics of these species, to clarify why matrinchã is a better swimmer than tambaqui.
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The renaturation process of different lactate dehydrogenase isozymes (llactate:NAD+ oxidoreductase, EC 1.1.1.27) from their unfolded subunits was investigated using a number of techniques, (a) kinetics of activity regain, (b) the kinetics of fluorescence change of the protein tryptophans, (c) kinetics of regain of the fluorescence properties of a covalently attached fluorescence probe (fluorescein) and (d) the kinetics of assembly, by following the intermediate oligomeric species appearing in the assembly pathway from monomers to tetramers. The results indicate that the unfolded polypeptide is converted to the active oligomeric species by the following scheme: Step I and step II are first-order where step II is rate limiting. The ligands NAD+ and NADH accelerate step II, thus converting step I to the rate-limiting process. The fact that partially folded lactate dehydrogenase subunits are capable of coenzyme binding may indicate the possible role of these ligands in the assembly of lactate dehydrogenase in vivo. Steps III and IV were found to be fast. The intermediate formation of an enzyme dimer which then dimerizes to the tetrameric species is found to be the major assembly pathway. Only a small portion of the lactate dehydrogenase tetramer is formed through the intermediate formation of a trimer intermediate.
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l-Lactate dehydrogenase (l-LDH) of Lactobacillus casei (LCLDH) is a typical bacterial allosteric l-LDH that requires fructose 1,6-bisphosphate (FBP) for its enzyme activity. A mutant LCLDH was designed to introduce an inter-subunit salt bridge network at the Q-axis subunit interface, mimicking Lactobacillus pentosus non-allosteric l-LDH (LPLDH). The mutant LCLDH exhibited high catalytic activity with hyperbolic pyruvate saturation curves independently of FBP, and virtually the equivalent K(m) and V(m) values at pH 5.0 to those of the fully activated wild-type enzyme with FBP, although the K(m) value was slightly improved with FBP or Mn(2+) at pH 7.0. The mutant enzyme exhibited a markedly higher apparent denaturating temperature (T(1/2)) than the wild-type enzyme in the presence of FBP, but showed an even lower T(1/2) without FBP, where it exhibited higher activation enthalpy of inactivation (ΔH(‡)). This result is consistent with the fact that the active state is more unstable than the inactive state in allosteric equilibrium of LCLDH. The LPLDH-like network appears to be conserved in many bacterial non-allosteric l-LDHs and dimeric l-malate dehydrogenases, and thus to be a key for the functional divergence of bacterial l-LDHs during evolution.
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In order to evaluate the effectiveness of l-lactate dehydrogenase (LDH) from rabbit muscle as a regenerative catalyst of the biologically important cofactor nicotinamide adenine dinucleotide (NAD), the kinetics over broad concentrations were studied to develop a suitable kinetic rate expression. Despite robust literature describing the intricate complexations, the mammalian rabbit muscle LDH lacks a quantitative kinetic rate expression accounting for simultaneous inhibition parameters, specifically at high pyruvate concentrations. Product inhibition by l-lactate was observed to reduce activity at concentrations greater than 25 mM, while expected substrate inhibition by pyruvate was significant above 4.3 mM concentration. The combined effect of ternary and binary complexes of pyruvate and the coenzymes led to experimental rates as little as a third of expected activity. The convenience of the statistical software package JMP allowed for effective determination of experimental kinetic constants and simplification to a suitable rate expression: $$ v = \frac{{{V_{max}}\left( {AB} \right)}}{{{K_{ia}}{K_b} + {K_b}A + {K_a}B + AB + \frac{P}{{{K_{I - Lac}}}} + \frac{{{B^2}A}}{{{K_{I - Pyr}}}} + \frac{{{B^2}Q}}{{{K_{I - Pyr - NAD}}}}}} $$where the last three terms represent the inhibition complex terms for lactate, pyruvate, and pyruvate–NAD, respectively. The corresponding values of K I–Lac, K I–Pyr, and K I–Pyr–NAD for rabbit muscle LDH are 487.33 mM−1 and 29.91 mM and 97.47 mM at 22 °C and pH 7.8.
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Preassay-incubation of the highly purified human erythrocyte adenine phosphoribosyltransferase (EC 2.4.2.7) (AMP pyrophosphorylase) with one of its substrates, 5-phosphoribosyl 1-pyrophosphate (PRibPP), changes the apparent V max value of the enzyme reaction. The extent of inhibition by preassay-incubation with an inhibitor, fructose 1,6-diphosphate (FDP), or a destabilizer, hypoxanthine (Hx), is found not to be proportional to the amount of the inhibitor present. The maximum inhibition achieved by preassay-incubation was about 40%. The PRibPP, FDP, and Hx induced changes in AMP pyrophosphorylase do not require the presence of divalent ions. The inhibtion of AMP pyrophosphorylase produced by preincubation with Hx was prevented when PRibPP was added to the preassay-incubation system. However, the preassay-incubation effect of FDP was only partially diminished under the same conditions. Contrary to the PRibPP-bound AMP pyrophosphorylase, the adenine-bound enzyme was found to be more heat labile than the unbound enzyme. Similar thermal instability was also observed with FDP- and Hx-bound enzyme. Our experimental results indicate that a conformational change of AMP pyrophosphorylase induced by the binding of metabolites is a slow process as compared to the overall catalytic reaction. This hysteretic characteristic of AMP pyrophosphorylase may be one of the regulatory mechanisms in purine intermediary metabolism.
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A kinetic study of the rate of pyruvate reduction by goldfish LDH-M4 (the homotetrameric form of lactate dehydrogenase which predominates in skeletal muscle) provided an analysis of the effects of pH and temperature on v (reaction velocity) and estimates of how temperature might affect catalysis in vivo, where the physiological pH regulation imposes a temperature coefficient of −0.015 to −0.020 pH unit/ °C. Consistent with published data for other LDHs, (i) V (maximum reaction velocity) was pH insensitive over a physiological pH range, (ii) the Km for pyruvate, KP, was sensitive to both pH and temperature, and (iii) the Km for NADH and the dissociation constant for NADH were both sensitive to temperature, but not to pH. V approximately doubled with each 10 °C (Ea = 11.7 kcal/mol). The effects of pH and temperature on KP were consistent with two enthalpic contributions, an ionization enthalpy (ΔHi∘) of 7.2 kcal/mol (probably a histidine imidazole), and an enthalpy (ΔHSO) of 5.8 kcal/mol for the combination of pyruvate with the nonionized (pH ⪡ pK′) LDH-NADH complex. When the pH was varied according to the physiological temperature coefficient, v was more sensitive to temperature than for conditions of constant pH, the usual design of kinetic experiments. This effect was due to the decreased temperature sensitivity of KP caused by partial concellation of the ΔHi∘ effect by the pH regulation: . At constant pH, on the other hand, KP increased strongly with temperature and had the effect of offsetting (especially at higher pH values) the large increases in V. It was suggested that the magnitudes of ΔHi∘ and ΔHSO might have been important in the evolution of LDHs and other enzymes of cold-blooded animals.
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The mechanism of the inhibitory effect of high concentrations of pyruvate on human erythrocyte lactate dehydrogenase has been studied by the use of a new parameter, delta, defined as the difference between the reciprocals of initial reaction rates obtained from experimental measurements and hypothetical linear Lineweaver-Burk plots. This parameter served as a method for differentiating between the competitive and umcompetitive substrate inhibition. Results of this study indicate that pyruvate is a competitive substrate inhibitor. It is suggested that the inhibitory effect of pyruvate is due to its competition with NADH for binding to the free enzyme and formation of an inactive enzyme-pyruvate binary complex. The competitive nature of pyruvate inhibition is further supported by the results of a kinetic study with NADH as the variable substrate. The dissociation constnat of the inactive enzyme-pyruvate binary complex was determined to be 101 micrometer. The physiological significance of the inhibitory effect could be to preserve a level of NADH concentration necessary for other vital enzymic reactions of living cells despite the presence of a high concentration of pyruvate.
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Chicken liver lactate dehydrogenase L-lactate : NAD+ oxidoreductase, EC1.1.1.27) reversibly catalyses the conversion of hydroxypyruvate to L-glycerate. The variation of the initial reaction rate with the substrate or coenzyme (NADH) concentration together with the inhibition caused by the reaction products and excess substrates, reveal that the kinetic mechanism of the reaction, with hydroxypyruvate as substrate, is of the rapid-equilibrium, ordered-ternary-complex type; NADH is the first substrate in the reaction sequence. Rate equations have been developed for the hydroxypyruvate.E.NADH system without inhibitors, with excess substrates, and with reaction products. Comparison of the rate equations obtained with those calculated theoretically from an ordered-ternary-complex mechanism reveals the existence of E.NAD.NADH,E.NAD-hydroxypyruvate and E.hydroxypyruvate complexes.
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In the presence of NAD, pyruvate inhibits various isozymes of lactate dehydrogenase via ( 1 ) the rapidly reversible formation of a dead-end, (abortive) ternary complex, E· NAD·pyruvate, and (2) the slowly reversible formation of a binary enzyme-inhibitor complex in which the inhibitor is the adduct of pyruvate and NAD, NAD-Pyr. Thus, pyruvate-induced inhibition patterns obtained from initial velocity studies of the normal enzymic reaction, NADH + pyruvate → NAD + lactate, are caused solely by the dead-end ternary complex (E·NAD·pyruvate). Because of weak binding, it is unlikely that this complex is important in vivo in mammalian systems. The curvature of the product-time plots obtained with saturating substrate concentrations and product concentrations far from equilibrium is produced by the slow formation of the binary E·NAD-Pyr complex (t1/2 = 5 min at 15°C for dogfish A4 enzyme). In the limiting steady state, at pH 7, and at saturating NADH concentration formation of the adduct complex reduces the fraction of active enzyme only by about 50%. But as the system approaches equilibrium a much greater reduction in active enzyme can be observed. In addition, the decomposition of the adduct complex is slow (t1/2 = 1 to 5 min under pseudophysiological conditions). However, significant amounts of the adduct complex cannot be detected in extracts of rat heart that were rapidly prepared under conditions where the adduct complex is relatively stable. Hence, the adduct complex, as well, is unlikely to be important, physiologically, in mammalian systems.
Article
These studies determine the levels of malate dehydrogenase isoenzymes in cardiac muscle by a steady state kinetic method which depends on the differential inhibition of these isoenzyme forms by high concentrations of oxaloacetate. This inhibition is similar to that exhibited by lactate dehydrogenase in the presence of high concentrations of pyruvate. The results obtained by this method are comparable in resolution to those obtained by CM-Sephadex fractionation and by differential centrifugation for the analyses of mitochondrial malate dehydrogenase and cytoplasmic malate dehydrogenase in tissues. The use of standard curves of percent inhibition of malate dehydrogenase activity plotted against the ratio of mitochondrial MDH activity to the total of mMDH and cMDH activities [ malate dehydrogenase ratio] (percent m-type) is introduced for studies of comparative mitochondrial function in heart muscle of different species or in different tissues of the same species.
Article
The pyruvate-to-lactate assay for determining lactate dehydrogenase (EC 1.1.1.27) can now yield linearity equal to or better than that obtained by the lactate-to-pyruvate assay. In addition, there are significant advantages to the pyruvate-to-lactate reaction: (a) a greater change in absorbance per unit time, which allows more accurate spectrophotometric readout; (b) lower reactant concentrations are required, which substantially reduces the cost per assay; (c) solid reagents are used to prepare the assay solution; and (d) reagent solutions are more stable. However, impurities present in commercial NADH preparations may substantially affect measured lactated dehydrogenase activities; therefore, a Standard Reference Material for NADH is being developed for issuance by the National Bureau of Standards.
Article
1. The rate of adduct formation between NAD+ and enol-pyruvate at the active site of lactate dehydrogenase is determined by the rate of enolization of pyruvate in solution. 2. The proportion of enol-pyruvate solutions is less than 0.01%. 3. The overall dissociation constant of adduct formation is less than 5 X 10(-8) M for pig heart lactate dehydrogenase at pH 7.0. 4. The unusual kinetics for adduct formation previously observed in the case of rabbit muscle lactate dehydrogenase [Griffin & Criddle (1970) Biochemistry 9, 1195--1205] may be attributed to the concentration of enol-pyruvate in solution being considerably less than the concentration of enzyme.
Article
From the analysis of the configuration of structural elements in protein molecules it has been possible to formulate some general principles describing the parameters and forces important in defining polypeptide conformation and interaction. Attempts to apply these principles to the prediction of the three-dimensional structure led to semi-empirical methods which succeeded in verifying local conformations in given structures of proteins. The calculation of the gross three-dimensional structure is not feasible at the present stage of potential theory and quantum-chemical approximations. Recent developments of physical and chemical techniques have established complete knowledge of the amino acid sequences of a great number of proteins and the implications of the primary structure to the higher levels of protein conformation, folding and subunit association. The catalytic function of the subunits in oligomeric enzymes is still an unsolved problem. From the point of view of evolution, association is considered to cause regulation of enzymatic activity rather than activity itself. Contrary to this the usual procedures of subunit dissociation in general lead to inactive monomers. Carrying out a systematic study of the particle weight of some glycolytic enzymes at concentrations comparable to those in the enzymatic test (c ≦ 1 μg/ml) the correlation of quarternary structure and catalytic activity turns out to be different for different enzymes. In the case of the apoenzyme of lactic dehydrogenase, high dilution is accompanied by deactivation and partial dissociation to the dimer; in the presence of the coenzyme or the substrate the active tetramer is stabilized even at concentrations below the nM range. Similar results are obtained for the apoenzyme of glyceraldehyde-3-phosphate dehydrogenase in which case dissociation leads to dimers and monomers at concentrations of the enzymatic test; the holoenzyme again is shown to be stable towards dissociation as well as deactivation and proteolysis. Aldolase is split reversibly by urea while dilution does not cause detectable dissociation. The aforementioned results of equilibrium studies are confirmed by kinetic measurements of the reactivation, refolding and reassociation of the enzymes after preceding splitting of the native quaternary structure. Under conditionsin vivo (high enzyme concentrations, high levels of other proteins, coenzymes and substrates) the subunit structure of the enzymes is expected to be stable. Therefore, the potential equilibrium between active oligomers and inactive subunits cannot play an important role in the regulation of the enzymes under concern.
1.1. NAD+, NADH, lactate, pyruvate and the substrate analogues oxamate and oxalate all protected pig lactate dehydrogenase (EC 1.1.1.27) isoenzymes M4 and H4 against inactivation at 52–64° in Tris-Chl buffer (pH 7.5). The time-course of inactivation could be described as a single first-order reaction, both in the presence and absence of substrates. Protection by substrates was dependent on substrated concentration.2.2. For kinetic treatment of the data, protection was defined as the difference between the first-order rate constants of inactivation measured in the absence and in the presence of substrate (Δk), respectively. On the assumption that protection was due to the equilibrium formation of enzyme-substrate complexes, simple equations were derived for calculation of the dissociation constants involved. A convenient procedure was found for distinguishing between the random order and compulsory order formation of a ternary complex.3.3. Protection, defined as Δk, was a hyperbolic function of the concentration of NAD+ and NADH indicating the expected proportionality between protection and coenzyme binding. The dissociation constants calculated from these hyperbolic functions were of the order of 1·10−5-1·10−4 M which, in turn, conformed with the idea that protection was due to binding of coenzyme to the specific binding sites. Lactate, pyruvate, oxamate and oxalate, when added to lactate dehydrogenase in the absence of coenzyme and at concentrations lower than about 1·10−2 M, did not protect lactate dehydrogenase against heat inactivation. The protection observed at higher concentrations was most marked with oxalate and it was definitely a nonhyperbolic function of oxalate concentration.4.4. Lactate, pyruvate, oxamate and oxalate, when added to lactate dehydrogenase in the presence of coenzyme, had a marked protective effect at concentrations as low as 1·10−4 M. Detailed investigations were carried out with the lactate dehydrogenase-H4 + NAD+ + pyruvate ternary system. Assuming that protection was due to the equilibrium formation of a ternary enzyme-substrate complex, an attempt was made to determine the reaction sequence and to calculate the dissociation constants involved. By applying the above kinetic equations, we foudn that the ternary complex was formed in a compulsory order, with pyruvate being bound only to the lactate dehydrogenase-NAD+ complex and not to the free enzyme. A dissociation constant of 0.75·10−3–0.96·10−3M was calculated for pyruvate in the ternary complex, whilst the dissociation constant obtained for NAD+ was 1.5·10−3M.
Article
The catalytic properties of the purified horseshoe crab and seaworm d-lactate dehydrogenases were determined and compared with those of several l-lactate dehydrogenases. Apparent Km's and degrees of substrate inhibition have been determined for both enzymes for pyruvate, d-lactate, NAD+ and NADH. They are similar to those found for l-lactate dehydrogenases. The Limulus “muscle”-type lactate dehydrogenase is notably different from the “heart”-type lactate dehydrogenase of this organism in a number of properties.
Article
This chapter discusses the lactate dehydrogenase. Lactic acid is the end product of anaerobic glycolysis in muscle tissue has been known for all of this century. Cell-free extracts able to catalyze the oxidation of lactate to pyruvate. The five different permutations of two different polypeptide chains readily explained the electrophoretic patterns. The distribution of these two polypeptide chains was dependent on whether the extract originated in aerobic tissue, such as heart or in anaerobic tissue as in skeletal muscle. The NAD+ binding structure found in L-lactate dehydrogenase (LDH) occurs frequently in other dehydrogenases and other proteins. In LDH the problem of catalysis is presented in stark simplicity. The complications of metal ions, linked substrate phosphorylation, or of ammonia uptake are absent. LDH is the only simpler dehydrogenase where both structure and sequence are known at present. The concept of multiple molecular forms of LDH has stimulated many investigations into the nature, function, and control of isozymes. There are only two major structural genes and there is a complex variety of other LDH genes, which can be expressed in some tissues at certain stages of development.
Article
Acetylcholinesterase is a very rapid enzyme, essential in the process of nerve impulse transmission at cholinergic synapses. It is the target of all currently approved anti-Alzheimer drugs and further progress in the modulation of its activity requires structural as well as dynamical information. Here we report three experimental approaches that allowed to gain structural insight into the dynamics of acetylcholinesterase. First, the structure of its complex with a putative secondgeneration anti-Alzheimer drug is presented. Next, a steady-state approach for determining atomic-resolution structures of enzyme/substrate complexes is detailed, which permitted to structurally follow the traffic of substrates within the active site of the acetylcholinesterase. Lastly, a new kinetic-crystallography strategy is described, in which UV-laser induced cleavage of a photolabile precursor of the enzymatic product is combined with temperaturecontrolled X-ray crystallography, in order to enable the molecular motions necessary for the expulsion of photolysis products to occur. Taken together, the results described herein permit a detailed description of the traffic of substrates and products within acetylcholinesterase, and provide insights into its conformational energy landscape.
Article
The cell is an extremely complex environment, notably highly crowded, segmented and confining. Overall, there is overwhelming and ever-growing evidence that, to understand how biochemical reactions proceed in vivo, one cannot separate the biochemical actors from their environment. Effects such as excluded volume, obstructed diffusion, weak non-specific interactions and fluctuations all team up to steer biochemical reactions often very far from what observed in ideal conditions. In this paper we use Ficoll PM70 and PEG 6000 to build artificial crowded milieux of controlled composition and density in order to assess how such environments influence the bio-catalytic activity of lactate dehydrogenase (LDH). Our measurements show that the normalized apparent affinity and maximum velocity decrease in the same fashion, a behavior reminiscent of uncompetitive inhibition, with PEG resulting in the largest reduction. In line with previous studies on other enzymes of the same family, and in agreement with the known role of a surface loop involved in enzyme isomerization and regulation of access to the active site, we suggest that the crowding matrix interferes with the conformational ensemble of the enzyme. This likely results in both impaired enzyme-complex isomerization and thwarted product release. Molecular dynamics simulations confirm that excluded-volume effects lead to an entropic force that effectively tend to push the loop closed, thereby effectively shifting the conformational ensemble of the enzyme in favor of a more stable complex isoform. Overall, our study substantiates the idea that most biochemical kinetics cannot be fully explained without including the subtle action of the environment where they take place naturally, in particular accounting for important factors such as excluded-volume effects, and also weak non-specific interactions when present, confinement and fluctuations.
Chapter
The enzyme lactate dehydrogenase (LDH) catalyses the conversion of lactate to pyruvate in the presence of the coenzyme nicotinamide adenine dinucleotide (NAD). It is composed of four subunits each of molecular weight 35,000 (Appella and Markert, 1961; Cahn et al, 1962). The subunits are single polypeptide chains of either H (heart) or M (muscle) variety (Wieland and Pfleiderer, 1957; Markert and Møller, 1959). Hybridization of these chains gives rise to five possible isoenzymes per species. This paper concerns itself with the properties of the M4 isoenzyme of dogfish (Squalus acanthius). Rossmann et al (1967) have previously reported that the apo-enzyme crystallizes in space group F422 with a = 146.9, c = 155.2A, and one polypeptide chain per asymmetric unit. Adams et al (1969) showed that the molecular center coincided with the intersection of a defined set of mutually perpendicular two-fold axes in the crystal lattice. They also reported a low resolution (5.0A) structure in which the boundaries of the molecule and subunits could be traced. In addition some properties of the binary coenzyme complex were discussed.
Chapter
For a number of years my associates and I at Duke and at Kentucky have been interested in the mechanism of action of lactate dehydrogenase. To provide the background for our present results it seems appropriate briefly to outline results obtained previously.
Chapter
I would like to thank the Organizers of this Symposium for inviting me to present some ideas on the concept of hysteresis. At first, it seemed to me that such a concept might be somewhat removed from the regulation of metabolic control by the enzymatically catalyzed interconversion of enzyme forms. However, I later realized that under some conditions one can apply the predictions of the hysteresis concept to the kind of interconvertible enzyme systems which are the topic of this Symposium. I appreciate the efforts of the Organizers for their round- about way of bringing this fact to my attention.
Chapter
Considerable progress in the understanding of cardiomyopathies of unknown etiology had been expected by endomyocardial biopsy. The absence of any specific morphological features, however, makes a diagnosis of congestive cardiomyopathy (COCM) unsatisfactory and is of such limited value from the diagnostic standpoint [1, 2, 3], although there is generally a good correlation between the severity of the condition clinically and the extent of the morphological changes [4, 5, 6]. Thus by ultrastructural examination of the biopsy material an assessment of prognosis was attempted [7, 8, 9].
Chapter
The NAD+ dependent tetrameric enzyme lactate dehydrogenase (EC 1.1.1.27), of molecular weight 140,000 Daltons, catalyzes the interconversion of L (+) lactate and pyruvate. Five isoenzymes result from the two subunit types abundant in tissues. The H subunit type is predominant in heart tissue and the M type in skeletal muscle. The M4 isoenzyme (LDH-5) from dogfish (squalus acanthius) has been most studied in this laboratory, while investigations are now proceeding on the H4 (LDH-1) and M4 (LDH-5) isoenzyme of pig.
Chapter
Nicotinamide-nucleotide-linked dehydrogenases provide much of the original stimulus for the necessary extension of kinetic theory already developed for one-substrate and hydrolytic enzymes. This was partly because of the convenience and precision with which rates can be measured by means of the light absorption or fluorescence emission of the reduced coenzymes and because of the changes of these properties, which accompany the binding of reduced coenzymes to many dehydrogenases. Knowledge of the static structure of an enzyme puts the same advantageous position as the chemist studying the mechanism of a reaction between simpler compounds of known structure. The nicotinamide nucleotides are coenzymes, and not prosthetic groups, and can be considered as substrates from the kinetic point of view. They also form stable and reversible compounds with dehydrogenases. It is reasonable to suppose that the enzyme-coenzyme compounds are intermediates in the overall catalytic reaction. A compulsory-order mechanism with the coenzyme combining first with the enzyme and dissociating last, has been established for a few dehydrogenases.
Article
In this study, a dry assay of L-lactate via the enzymatic chromatographic test (ECT) was developed. An L-lactate dehydrogenase plus an NADH regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The (0.5 μL, 2(-6) U/μL) diaphorase was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5μL, 12mM), L-lactate dehydrogenase (1μL, 0.25 U/μL), and NAD(+) (2μL, 1.5×10(-5) M) were added into the mobile phase (100μL) composed of 0.1% (w/w) Tween 20 in 10 mM phosphate buffer (pH 9.0), and the process was left to run for ten minutes. This detection had a linear range of 0.039-5 mM with a detection limit of 0.047 mM. This quantitative analysis process for L-lactate was easy to operate with good stability and was proper for the point-of-care testing applications. Copyright © 2014. Published by Elsevier Inc.
Article
The requirements for enzymic cofactor recycling have been investigated in a system employing alcohol and lactate dehydrogenases. The interactions of various combinations of free dehydrogenases or dehydrogenases immobilized either to the same or separate supports, with free NAD, a soluble highmolecular weight derivative of NAD or an insoluble derivative of NAD have been examined.
Article
Lactate dehydrogenase from Blastocladiella emersonii was purified. The enzyme has a molecular weight of about 70,000 and is specific for d-lactate. Normal Michaelis-Menten kinetics were found for NADH with a Km of 0.03 mm. The enzyme exhibited negative cooperativity for pyruvate. The cooperativity increased with decreasing NADH concentration. S0.5 for pyruvate was 2.5 mm. NAD inhibited the enzyme in a competitive manner with regard to NADH. All other combinations of product inhibition were noncompetitive. The results indicated that lactate and pyruvate can affect the enzyme activity in several ways. It is suggested that the reaction occurs according to an ordered sequential mechanism with NADH as leading substrate and NAD as the last product released. Under the conditions used, 50% inhibition of the enzyme was obtained with 50 to 200 mm salts. Salt inhibition depends on the size of the anions, the ionic strength being of less importance.
Article
A novel enzyme sensor based on CdSe quantum dots (QDs)/polycaprolactone (PCL) composite porous fibers has been prepared via a simple electrospinning method. We choose PCL, a biocompatible polymer, as the host polymer for the electrospun fibers and prepare CdSe QDs as sensing probes. Experiments demonstrate that the CdSe QDs are miscible and chemically inert to PCL in the electrospinning solvent mixture containing N,N-dimethylformamide and chloroform. Therefore, the CdSe QDs are uniformly distributed within the PCL fibers, leading to good preservation of their original fluorescence properties. In addition, secondary porous structures of the fibers are produced by introducing a porogen to accelerate inner-fiber analyte diffusion and increase the sensitivity of this sensor. These prepared fluorescent fibers can be reversibly quenched by nicotinamide adenine dinucleotide (NAD) due to the electron transfer (ET) process between NAD and the CdSe QDs. Based on the films formed by these fibers and an enzyme-catalyzed reaction for converting NAD to its reduced form NADH, this sensor has successfully realized the fluorescence turn-on detection of an NAD-dependent enzyme, lactate dehydrogenase (LDH). The detection time is only 10 min per sample, and linear calibration plots of the activity of LDH are obtained from 200–2400 U L−1. These QD composite porous fibers can be developed as a facile, rapid, sensitive and stable NAD-dependent enzyme detection platform and may find more applications in novel optical devices.
Article
It has been found that in the determination of lactate dehydrogenase activity (substrate: lactate, coenzyme: NAD+) as proposed by Amador et al.1 the linearity between Δ A and time hardly holds true. This appeared to be the result of an inhibition, a phenomenon which increases with time.The best method for calculating the enzyme activity with this forward reaction was by means of a polynomial curve-fitting procedure. As NADH and pyruvate, when added to the reaction mixture, showed an inhibitory effect of the same order as found, it is probable that the inhibition is the result of these reaction products.
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The H4 isozyme of bovine lactate dehydrogenase was guanidinated with O-methyl isourea and its properties were compared with those of the native enzyme. Kinetic evidence suggests that coenzyme binding is not affected by guanidination but that the rate of dissociation of substrates is diminished. Fluorimetric titrations with NADH confirm the kinetic results and show that both oxamate and oxalate are bound more firmly by the complex between guanidinated enzyme and NADH than by the native enzyme·NADH complex. The changes in binding of inhibitors appear to result from the modification of several amino groups rather than from derivatization of a single essential group. It is proposed that strong interactions between carboxylate and guanidinium ions stabilize the ternary enzyme·coenzyme·inhibitor complex. Improved methods are presented for calculation of dissociation constants of enzyme·NADH·inhibitor complexes from fluorescence titration curves.
Article
Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)+-dependent succinate semialdehyde dehydrogenase YneI from S. yphimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD+ and NADP+ as cofactors, although affinity to NAD+ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD+ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD+ olecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site-directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD+. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. Proteins 2012. © 2012 Wiley Periodicals, Inc.
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Hysteretic enzymes are defined as those enzymes which respond slowly (in terms of some kinetic characteristic) to a rapid change in ligand, either substrate or modifier, concentration. Such slow changes, defined in terms of their rate relative to the over-all catalytic reaction, result in a lag in the response of the enzyme to changes in the ligand level. Several mechanisms, including ligand-induced isomerization of the enzyme, displacement of tightly bound ligands by other ligands, or polymerization and depolymerization, are discussed and it can be shown that the description of the time-dependent change in enzyme activity is similar for many different cases. Examination of the literature reveals that a large number of enzymes may fall into the category termed hysteretic, that such enzymes are frequently those which are important in metabolic regulation, that the time of conversion from one kinetic form to another may vary between seconds and minutes, and that there are experimental examples of all the mechanisms which are discussed theoretically. The possible relation between those enzymes which are hysteretic and regulation of complex metabolic processes is discussed in terms of the fact that the slow response of the hysteretic enzyme to changes in ligand level will lead to a time-dependent buffering of some metabolites and that this may be important with respect to pathways which utilize common intermediates or in which there are multiple branch points. It is suggested that the question of hysteresis in enzyme systems as defined here be systematically investigated in regulatory enzymes and that this concept may be of value in discussing the regulation of complex processes in vivo.
Article
Lactate dehydrogenase isoenzymes can be distinguished kinetically by the fact that isoenzyme H is strongly inhibited a few seconds after the reaction is started if high concentrations of pyruvate are present, in contrast to the M isoenzyme. A new instrument that exploits this fact can measure both the total activity and the proportion of H isoenzyme in serum or plasma in 8 to 10 s. The instrument consists of a simplified stopped-flow apparatus in which the plasma is assayed for lactate dehydrogenase activity, and an electronic device that measures the rate of the reaction at two pre-set time intervals. The first rate is taken between 0.2 and 0.4 s after the reaction is started, a time at which both isoenzymes are fully active, and at which the rate obtained thus reflects total lactate dehydrogenase activity in the plasma sample. The second rate is measured 4 to 6 s after the start of the reaction, at which time the H isoenzyme has become inhibited and the observed rate compared to the initial rate is therefore proportional to the percentage of H isoenzyme activity in the serum. These two rates are electronically displayed on two three-digit voltmeters, the first display being the total activity, the second a number proportional to the inhibited slope. The percentage of M isoenzyme can then be calculated from the initial and final rate. A total of five to six repeat assays may be done within a minute on 1 ml of plasma or serum. This instrument may be of significant value in following the progress of myocardial infarctions and other diseases.
Article
Previous interpretations of the mechanism of trypsin- and chymotrypsin-catalysed reactions in terms of two intermediates, the Michaelis complex and an acyl-enzyme, were based on steady-state studies and on the observation of individual steps under sub-optimum conditions. In the present paper new methods for the rapid analysis of chemical events and for the spectrophotometric detection of individual steps are applied to these two enzymes. These methods can be used to study reactions with specific amino acid ester substrates. It can be shown that there are at least three distinct steps between the Michaelis complex and the release of ethanol; the latter is likely to correspond to acyl-enzyme formation. The relative rates of these three steps are measured by rapid-flow techniques from observations of the displacement of chromophoric inhibitors and reactions with specific substrates containing chromophores, as well as from ethanol analyses during a single turnover of the enzyme reactions. It is concluded that the reactions of trypsin and chymotrypsin with their specific substrates involve the formation of a specially reactive conformation of the enzyme-substrate complex and that the rate constants involved in this rearrangement are at least as important for the overall reaction as those of the subsequent formation and decomposition of the acyl-enzyme.