Kinetic analysis of duck epsilon-crystallin, a lens structural protein with lactate dehydrogenase activity.
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ABSTRACT:
SUBMITTER: Chiou SH
PROVIDER: S-EPMC1131242 | biostudies-other | 1990 Apr
REPOSITORIES: biostudies-other
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Project description:During adaptive metabolic evolution a native glycerol dehydrogenase (GDH) acquired a d-lactate dehydrogenase (LDH) activity. Two active-site amino acid changes were detected in the altered protein. Biochemical studies along with comparative structure analysis using an X-ray crystallographic structure model of the protein with the two different amino acids allowed prediction of pyruvate binding into the active site. We propose that the F245S alteration increased the capacity of the glycerol binding site and facilitated hydrogen bonding between the S245 γ-O and the C1 carboxylate of pyruvate. To our knowledge, this is the first GDH to gain LDH activity due to an active site amino acid change, a desired result of in vivo enzyme evolution.
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Project description:Delta-Crystallin, the most abundant crystallin in the avian species, was isolated and purified from duck lenses. It was shown to possess endogenous argininosuccinate lyase activity catalysing the reversible cleavage of argininosuccinate to give fumarate and arginine with an equilibrium constant of 1.8 +/- 0.23 mM. In contrast, chicken lens delta-crystallin showed only 0.4-0.8% of the enzyme activity of duck delta-crystallin under identical assay conditions. Biochemical comparison of delta-crystallins from these two species revealed distinct differences in their structural and kinetic properties. An activity-staining method was developed for the easy detection of endogenous enzyme activity of delta-crystallin from crude lens extracts of different avian species. Two-dimensional gel electrophoresis of lens homogenates indicated that, in the chicken lens, delta-crystallin is composed mainly of a subunit with a pI of 5.9 and a subunit mass of 50 kDa, whereas that of duck lens possesses various 50 kDa subunits in a pI range of 5.9-6.8. Activity staining corroborated the fact that all charge isoenzymes of duck delta-crystallin possess enzyme activity whereas that of chicken delta-crystallin is devoid of activity. For duck delta-crystallin, variation of the enzyme activity with argininosuccinate concentration in the forward reaction followed saturation kinetics with an apparent Michaelis constant for the substrate of 17 +/- 5 microM. In the reverse reaction, initial-velocity studies showed intersecting patterns. Inhibitions of the forward reaction by products (fumarate and arginine) were both non-competitive with respect to argininosuccinate. Citrulline, an analogue of arginine, inhibited the enzyme activity in both directions and was competitive with respect to arginine but non-competitive with respect to fumarate or argininosuccinate. Succinate, which inhibited the bovine argininosuccinate lyase, did not affect the delta-crystallin enzyme activity in a concentration range between 1 and 300 mM. These results suggest a random Uni Bi kinetic mechanism for the argininosuccinate lyase activity of duck delta-crystallin with the formation of various abortive delta-crystallin-argininosuccinate-arginine, delta-crystallin-argininosuccinate-fumarate and delta-crystallin-argininosuccinate-citrulline ternary complexes.
| S-EPMC1131077 | biostudies-other
Project description:The endogenous argininosuccinate lyase activity of duck delta2-crystallin was specifically inactivated by the histidine-specific reagent, diethyl pyrocarbonate. The protein was protected by l-citrulline or l-arginine from the diethyl pyrocarbonate inactivation. To characterize further the chemical mechanism of the delta2-crystallin-catalysed reaction, deuterium-labelled argininosuccinate was enzymically synthesized from fumarate and l-arginine with delta2-crystallin in 2H2O. The argininosuccinate synthesized contained about 19% of the anhydride form; however, the deuterium was clearly demonstrated to be incorporated enantioselectively. Only the pro-HR atom at C-9 of the succinate moiety was labelled in the [2H]argininosuccinate-9-d synthesized, which indicates an anti-elimination mechanism for the endogenous argininosuccinate lyase activity of delta2-crystallin. The enzymic activity of duck lens delta2-crystallin in the pH range 5.5-8.5 was investigated using both protium- and deuterium-labelled argininosuccinate as the substrate. From the logkcat versus pH plot, two molecular pKa values of 6.18+/-0.02 and 8.75+/-0.03 were detected in the delta2-crystallin-argininosuccinate binary complex. The former must be dehydronated and the latter hydronated to achieve an optimum reaction rate. The logkcat/Km versus pH plot suggested two molecular pKa values of 5.96+/-0.09 and 8.29+/-0.10 for the free delta2-crystallin to be involved in the substrate binding. Small kinetic isotope effects of 1.17+/-0.02 and 1.05+/-0.09 were found for kcat and kcat/Km respectively. Combining results from labelling and kinetic analysis indicates that the endogenous argininosuccinate lyase activity of duck delta2-crystallin is compatible with a stepwise E1cB mechanism, the rate-limiting step probably at the C-N bond-cleavage step.
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Project description:Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR ("Critical sequence") contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.
| S-EPMC9963673 | biostudies-literature
Project description:Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR (“Critical sequence”) contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.
2023-01-31 | PXD035668 | Pride
Project description:Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40?°C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50?°C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 ?moles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50?°C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates.
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Project description:Glycolytic enzymes play a crucial role in the anaerobic glycolysis of apicomplexan parasites for energy generation. Consequently, they are considered as potential targets for new drug development. Previous studies revealed that lactate dehydrogenase (LDH), a glycolytic enzyme, is a potential drug target in different parasites, such as Plasmodium, Toxoplasma, Cryptosporidium, and Piroplasma. Herein, in order to investigate the structural basis of LDH in Babesia spp., we determined the crystal structure of apo Babesia orientalis (Bo) LDH at 2.67-Å resolution in the space group P1. A five-peptide insertion appears in the active pocket loop of BoLDH to create a larger catalytic pocket, like other protozoa (except for Babesia microti LDH) and unlike its mammalian counterparts, and the absence of this extra insertion inactivates BoLDH. Without ligands, the apo BoLDH takes R-state (relaxed) with the active-site loop open. This feature is obviously different from that of allosteric LDHs in T-state (tense) with the active-site loop open. Compared with allosteric LDHs, the extra salt bridges and hydrogen bonds make the subunit interfaces of BoLDH more stable, and that results in the absence of T-state. Interestingly, BoLDH differs significantly from BmLDH, as it exhibits the ability to adapt quickly to the synthetic co-factor APAD+. In addition, the enzymatic activity of BoLDH was inhibited non-competitively by polyphenolic gossypol with a K i value of 4.25 μM, indicating that BoLDH is sensitive to the inhibition of gossypol and possibly to its new derivative compounds. The current work provides the structural basis of BoLDH for the first time and suggests further investigation on the LDH structure of other Babesia spp. That knowledge would indeed facilitate the screening and designing of new LDH inhibitors to control the intracellular proliferation of Babesia spp.
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Project description:Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling.
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Project description:Although normally dormant, hair follicle stem cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier 1 (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allows them to remain dormant and yet quickly respond to appropriate proliferative stimuli.
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Project description:Testis-specific lactate dehydrogenase (LDH-C₄) is one of the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C₄ in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000-5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C₄ in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A₄ (LDH-A₄), Lactate Dehydrogenase B₄ (LDH-B₄), and LDH-C₄ were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km) of LDH-C₄ for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C₄ for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A₄ (Ki = 26.900 mmol/L), LDH-B₄ (Ki = 23.800 mmol/L), and LDH-C₄ (Ki = 65.500 mmol/L). These data suggest that inhibition of lactate by LDH-A₄ and LDH-B₄ were stronger than LDH-C₄. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C₄.
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