Dogfish M4 lactate dehydrogenase: reversible inactivation by pyridoxal 5'-phosphate and complete protection in complexes that mimic the active ternary complex.
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ABSTRACT:
SUBMITTER: Chen SS
PROVIDER: S-EPMC1172378 | biostudies-other | 1975 Nov
REPOSITORIES: biostudies-other
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Project description:1. Pig M4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.5 and 25 degrees C loses activity gradually. The maximum inactivation was 66%, and this did not increase with concentrations of pyridoxal 5'-phosphate above 1 mM. 2. Inactivation may be reversed by dialysis or made permanent by reducing the enzyme with NaBH4. 3. Spectral evidence indicates modification of lysine residues, and 6-N-pyridoxyl-lysine is present in the hydrolsate of inactivated, reduced enzyme. 4. A second cycle of treatment with pyridoxal 5'-phosphate and NaBH4 further decreases activity. After three cycles only 9% of the original activity remains. 5. Apparent Km values for lactate and NAD+ are unaltered in the partially inactivated enzyme. 6. These results suggest that the covalently modified enzyme is inactive; failure to achieve complete inactivation in a single treatment is due to the reversibility of Schiff-base formation and to the consequent presence of active non-covalently bonded enzyme-modifier complex in the equilibrium mixture. 7. Although several lysine residues per subunit are modified, only one appears to be essential for activity: pyruvate and NAD+ together (both 5mM) completely protect against inactivation, and there is a one-to-one relationship between enzyme protection and decreased lysine modification. 8. NAD+ or NADH alone gives only partial protection. Substrates give virtually none. 9. Pig H4 lactate dehydrogenase is also inactivated by pyridoxal 5'-phosphate. 10. The possible role of the essential lysine residue is discussed.
| S-EPMC1165597 | biostudies-other
Project description:1. The steady residual activity of ox liver glutamate dehydrogenase at equilibrium with the reversible inactivator pyridoxal 5'-phosphate was measured in the presence and absence of various protecting agents. 2. NAD(+) (up to 15mm) and its 3-acetylpyridine analogue (up to 5mm) both failed to protect, in contrast with NADH. 3. Partial protection was given by glutarate and by succinate. Adipate and pentanoate were much less effective. 4. Correspondingly, whereas succinate and glutarate were both shown to be strong inhibitors of the catalytic reaction, competitive with glutamate, adipate was only weakly competitive, and the still weaker inhibition by pentanoate was non-competitive. 5. When the enzyme was saturated with glutarate, NAD(+) became a good, although still partial, protecting agent. In the absence of protection, 1.8mm-pyridoxal 5'-phosphate decreased enzyme activity to 9%, in the presence of 150mm-glutarate to 29%, and with glutarate and 1mm-NAD(+) only to 73%. 6. 2-Oxoglutarate also promoted protection by NAD(+), but neither pentanoate nor succinate did so. The finding with succinate is remarkable in view of findings 3 and 4 above. 7. It seems possible that substrates or analogues possessing the glutarate structure promote a conformational change that alters the mode of NAD(+) binding. This may explain why glutamate is a much better substrate than norvaline or aspartate and why negative interactions in coenzyme binding occur only in the formation of ternary complexes with glutamate or its analogues.
| S-EPMC1168425 | biostudies-other
Project description:Human muscarinic receptor M4 belongs to the class A subfamily of the G-protein-coupled receptors (GPCRs). M4 has emerged as an attractive drug target for the treatment of Alzheimer's disease and schizophrenia. Recent results showed that M4-mediated cholinergic transmission is related to motor symptoms in Parkinson's disease. Selective ligand design for the five muscarinic acetylcholine receptor (mAchR) subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets. In order to obtain M4-selective antagonists, a new approach was tried to lock M4 into an inactive form by rationally designing an N4497.49R mutation, which mimics the allosteric sodium binding in the conserved sodium site usually found in class A GPCRs. In addition, the crystal structure of the mutation-induced inactive M4 was determined. By comparative analysis with other mAchR structures, followed by functional assays, the N4497.49R mutation was shown to stabilize M4 into an inactive state. Virtual screening of a focused ligand library using the crystal structure showed that the inactive M4 prefers antagonists much more than agonists. This study provides a powerful mutation strategy to stabilize GPCRs in inactive states and facilitate their structure determination.
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Project description:1. Mouse C4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.7 and 25 degrees C loses activity gradually; 1mM-pyridoxal 5'-phosphate causes 83% inactivation, and higher concentrations of the reagent cause no further loss of activity. 2. The final extent of inactivation is very pH-dependent, greater inactivation occurring at the high pH values. 3. Inactivation may be fully reversed by addition of cysteine, or made permanent by reducing the enzyme with NaBH4. 4. The absorption spectrum of inactivated reduced enzyme indicates modification of lysine residues. Inactivation by 80% corresponds to modification of at least 1.8 mol of lysine/mol of enzyme subunit. 5. There is no loss of free thiol groups after inactivation with pyridoxal 5'-phosphate and reduction of the enzyme. 6. NAD+ or NADH gives complete protection against inactivation. protection studies with coenzyme fragments indicate that the AMP moiety is largely responsible for the protective effect. Lactate (10 mM) gives no protection in the absence of added nucleotides, but greatly enhances the protection given by ADP-ribose (1 mM). Thus ADP-ribose is able to trigger the binding of lactate. 7. Pyridoxal 5'-phosphate also acts as a non-covalent inhibitor of mouse C4 lactate dehydrogenase. The inhibition is non-competitive with respect to both NAD+ and lactate. 8. Km values for the enzyme at pH 8.0 and 25 degrees C, with the non-varied substrate saturating, are 0.3 mM-lactate and 5 microM-NAD+. 9. These results are discussed and compared with pyridoxal 5'-phosphate modification of other lactate dehydrogenase isoenzymes and related dehydrogenases.
| S-EPMC1162226 | biostudies-other
Project description:We have determined the complete nucleotide sequence of the mitochondrial DNA (mtDNA) of the dogfish, Scyliorhinus canicula. The 16,697-bp-long mtDNA possesses a gene organization identical to that of the Osteichthyes, but different from that of the sea lamprey Petromyzon marinus. The main features of the mtDNA of osteichthyans were thus established in the common ancestor to chondrichthyans and osteichthyans. The phylogenetic analysis confirms that the Chondrichthyes are the sister group of the Osteichthyes.
| S-EPMC1460332 | biostudies-other
Project description:The burgeoning field of biomaterials for immunotherapy has aided in the understanding of foundational mechanisms of cancer immunology. In particular, implantable biomaterials can be engineered to investigate specific aspects of the tumor microenvironment either singularly or in combination. Of note, the metabolite - lactate, a byproduct of anaerobic glycolysis, is known to reprogram immune cells, resulting in increased tumor survival. An adequate model that can recapitulate intratumoral lactate concentrations does not exist. In this study, we demonstrate that a simple biomaterial platform could be developed as an instructive tool to decipher the effects of lactate in vivo. Briefly, we demonstrate that a peptide hydrogel loaded with granulocyte-macrophage colony stimulating factor and poly-(lactic-co-glycolic acid)/(lactic acid) microparticles can generate the localized lactate concentrations (∼2-22 mM) and cellular makeup of the tumor microenvironment, following subcutaneous implantation in mice. Furthermore, infiltrating immune cells adopt phenotypes similar to those seen in other in vitro and in vivo cancer models, including immunosupressive dendritic cells. This hydrogel system is a framework to interrogate immune cell modulation in cancer-like environments using safe and degradable biomaterials. Moreover, this system can be multifaceted, as incorporation of other cancer tumor environmental factors or chemotherapeutic drugs is facile and could be insightful in developing or improving immunotherapies.
| S-EPMC7606634 | biostudies-literature
Project description:Pyridoxal-5'-phosphate (PLP), in addition to its known metabolic functions, inactivates many DNA-dependent enzymes through conjugation to their critical amino groups. We have investigated the ability of PLP to inhibit bifunctional DNA repair glycosylases, which possess a catalytic amine. Of six enzymes tested, only endonuclease VIII-like protein 2 (NEIL2) was significantly inhibited by PLP. The inhibition was due to Schiff base formation between PLP and the enzyme. PLP-conjugated NEIL2 completely lost its ability to bind damaged DNA. Liquid chromatography/nanoelectrospray ionization tandem mass spectrometry of the products of proteolysis of pyridoxylated NEIL2 identified Lys50 as the site of modification. Thus, the beta2/beta3 loop where Lys50 is located in NEIL2 is important for DNA binding, presumably lies next to a phosphate-binding site, and may represent a target for regulation of the enzyme activity.
| S-EPMC2857644 | biostudies-literature
Project description:1. Pig heart mitochondrial malate dehydrogenase incubated with pyridoxal 5'-phosphate at pH 8.0 and 25 degrees C gradually loses activity. Such inactivation can be largely reversed by dialysis or by addition of L-lysine or L-cysteine, and can be made permanent by NaBH4 reduction. 2. Modification of malate dehydrogenase with pyridoxal 5'-phosphate at 35 degrees C involves two phases, an initial inactivation which is reversible and a slower irreversible second stage. 3. The initial reaction between pyridoxal 5'-phosphate and malate dehydrogenase appears to involve reversible formation of a Schiff base with the epsilon-amino group of a lysine residue. 4. Inactivation of malate dehydrogenase by pyridoxal 5'-phosphate at 10 degrees C involves only the reversible reaction. 5. At 10 degrees C repeated cycles of treatment with pyridoxal 5'-phosphate and NaBH4 reduction lead to a stepwise decline in residual activity. 6. Apparent Km values for malate and NAD+ are unaltered in the partially inactivated enzyme. 7. NAD+ and NADH give only partial protection against pyridoxal 5'-phosphate inactivation. Substrates give no effect.
| S-EPMC1172360 | biostudies-other
Project description:Under oxidative stress conditions, mitochondria are the major site for cellular production of reactive oxygen species (ROS) such as superoxide anion and H2O2 that can attack numerous mitochondrial proteins including dihydrolipoamide dehydrogenase (DLDH). While DLDH is known to be vulnerable to oxidative inactivation, the mechanisms have not been clearly elucidated. The present study was therefore designed to investigate the mechanisms of DLDH oxidative inactivation by mitochondrial reactive oxygen species (ROS). Mitochondria, isolated from rat brain, were incubated with mitochondrial respiratory substrates such as pyruvate/malate or succinate in the presence of electron transport chain inhibitors such as rotenone or antimycin A. This is followed by enzyme activity assay and gel-based proteomic analysis. The present study also examined whether ROS-induced DLDH oxidative inactivation could be reversed by reducing reagents such as DTT, cysteine, and glutathione. Results show that DLDH could only be inactivated by complex III- but not complex I-derived ROS; and the accompanying loss of activity due to the inactivation could be restored by cysteine and glutathione, indicating that DLDH oxidative inactivation by complex III-derived ROS was a reversible process. Further studies using catalase indicate that it was H2O2 instead of superoxide anion that was responsible for DLDH inactivation. Moreover, using sulfenic acid-specific labeling techniques in conjunction with two-dimensional Western blot analysis, we show that protein sulfenic acid formation (also known as sulfenation) was associated with the loss of DLDH enzymatic activity observed under our experimental conditions. Additionally, such oxidative modification was shown to be associated with preventing DLDH from further inactivation by the thiol-reactive reagent N-ethylmaleimide. Taken together, the present study provides insights into the mechanisms of DLDH oxidative inactivation by mitochondrial H2O2.
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Project description:Hydroxyacid dehydrogenases are responsible for the conversion of 2-keto acids to 2-hydroxyacids and have a wide range of biotechnological applications. In this study, a D-lactate dehydrogenase (D-LDH) from a Sporolactobacillus inulinus strain was experimentally verified to have both the D-LDH and glutamate dehydrogenase (GDH) activities (reversible deamination). The catalytic mechanism was demonstrated by identification of key residues from the crystal structure analysis and site-directed mutagenesis. The Arg234 and Gly79 residues of this enzyme play a significant role in both D-LDH and GDH activities. His295 and Phe298 in DLDH744 were identified to be key residues for lactate dehydrogenase (LDH) activity only whereas Tyr101 is a unique residue that is critical for GDH activity. Characterization of the biochemical properties contributes to understanding of the catalytic mechanism of this novel D-lactate dehydrogenase enzyme.
| S-EPMC4580590 | biostudies-literature