The reaction of N-acetylneuraminate lyase with chloropyruvate.
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ABSTRACT:
SUBMITTER: Barnett JE
PROVIDER: S-EPMC1168410 | biostudies-other | 1974 Nov
REPOSITORIES: biostudies-other
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Project description:Selenocysteine lyase (SCL) catalyzes the pyridoxal 5'-phosphate-dependent removal of selenium from l-selenocysteine to yield l-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward l-selenocysteine and no activity to its cognate l-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with l-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with l-cysteine. Cys-375 on the flexible loop directed l-selenocysteine, but not l-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system.
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Project description:1. A method of preparation and purification of citrate oxaloacetate-lyase (EC 4.1.3.6) from Aerobacter aerogenes is described. 2. The equilibrium of this reaction has been determined at pH 8.4 and 25 degrees . It has been shown that K, i.e. [citrate(3-)]/[oxaloacetate(keto) (2-)][acetate (-)], is 3.08+/-0.72, but that K(app.), i.e. [total citrate]/[total oxaloacetate][total acetate], is markedly affected by the initial concentrations of the reactants and magnesium. 3. The free-energy change during the cleavage of citrate has been calculated and compared with data from other sources. 4. The free energy of hydrolysis of acetyl-CoA has been evaluated from the present data. 5. A detailed knowledge of the interactions of the reactants with metal ions has been shown to be important in the calculation of the equilibrium constant and related thermodynamic functions.
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Project description:The enzymatic reactions leading to the deamination of β-lysine, lysine, or 2-aminoadipic acid are of great interest for the metabolic conversion of lysine to adipic acid. Enzymes able to carry out these reactions are not known, however ammonia lyases (EC 4.3.1.-) perform deamination on a wide range of substrates. We have studied 3-methylaspartate ammonia lyase (MAL, EC 4.3.1.2) as a potential candidate for protein engineering to enable deamination towards β-lysine, that we have shown to be a competitive inhibitor of MAL. We have characterized MAL activity, binding and inhibition properties on six different compounds that would allow to define the molecular determinants necessary for MAL to deaminate our substrate of interest. Docking calculations showed that β-lysine as well as the other compounds investigated could fit spatially into MAL catalytic pocket, although they probably are weak or very transient binders and we identified molecular determinants involved in the binding of the substrate. The hydrophobic interactions formed by the methyl group of 3-methylaspartic acid, together with the presence of the amino group on carbon 2, play an essential role in the appropriate binding of the substrate. The results showed that β-lysine is able to fit and bind in MAL catalytic pocket and can be potentially converted from inhibitor to substrate of MAL upon enzyme engineering. The characterization of the binding and inhibition properties of the substrates tested here provide the foundation for future and more extensive studies on engineering MAL that could lead to a MAL variant able to catalyse this challenging deamination reaction.
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Project description:The urea cycle consists of six consecutive enzymatic reactions that convert waste nitrogen into urea. Deficiencies of any of these enzymes of the cycle result in urea cycle disorders (UCDs), a group of inborn errors of hepatic metabolism that often result in life-threatening hyperammonemia. Argininosuccinate lyase (ASL) catalyzes the fourth reaction in this cycle, resulting in the breakdown of argininosuccinic acid to arginine and fumarate. ASL deficiency (ASLD) is the second most common UCD, with a prevalence of ~1 in 70,000 live births. ASLD can manifest as either a severe neonatal-onset form with hyperammonemia within the first few days after birth or as a late-onset form with episodic hyperammonemia and/or long-term complications that include liver dysfunction, neurocognitive deficits, and hypertension. These long-term complications can occur in the absence of hyperammonemic episodes, implying that ASL has functions outside of its role in ureagenesis and the tissue-specific lack of ASL may be responsible for these manifestations. The biochemical diagnosis of ASLD is typically established with elevation of plasma citrulline together with elevated argininosuccinic acid in the plasma or urine. Molecular genetic testing of ASL and assay of ASL enzyme activity are helpful when the biochemical findings are equivocal. However, there is no correlation between the genotype or enzyme activity and clinical outcome. Treatment of acute metabolic decompensations with hyperammonemia involves discontinuing oral protein intake, supplementing oral intake with intravenous lipids and/or glucose, and use of intravenous arginine and nitrogen-scavenging therapy. Dietary restriction of protein and dietary supplementation with arginine are the mainstays in long-term management. Orthotopic liver transplantation (OLT) is best considered only in patients with recurrent hyperammonemia or metabolic decompensations resistant to conventional medical therapy.
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