Mechanism of regulation of the malic enzyme from Fusarium

1974 ◽  
Vol 20 (4) ◽  
pp. 443-454 ◽  
Author(s):  
M. W. Zink
Keyword(s):  
Malic Acid ◽  
Malic Enzyme ◽  
Kinetic Studies ◽  
Oxidative Decarboxylation ◽  
Mercaptosuccinic Acid ◽  
Substrate Activation ◽  
Phosphate Inhibition ◽  
Phosphoglyceric Acid ◽  
Variable Substrate ◽  
Text Component

The NADP-specific malic enzyme which catalyzes the oxidative decarboxylation of malic acid has been partially purified from extracts of Fusarium oxysporum and some of its kinetic parameters studied. Reciprocal velocity plots with malate as variable substrate are nonlinear except at high NADP concentration, while with NADP as variable substrate the reciprocal plots are linear. Substrate activation by malic acid describes a biphasic character in reciprocal plots. Kinetic studies indicate that there are two binding sites for malic acid. At saturating levels of NADP, two Km components for malic acid, designated as [Formula: see text] and [Formula: see text] are evident. The separation of the two components may have been accomplished; [Formula: see text] component by mercaptosuccinic acid and [Formula: see text] component by either low pH or by phosphate. Inhibition of the purified malic enzyme is nontotal with fructose 1,6-diphosphate, 6-phosphogluconic acid, and 3-phos-phoglyceric acid. The kinetic relationship between fructose 1,6-diphosphate or 6-phospho-gluconate and malic acid is competitive with respect to the [Formula: see text] component and noncompetitive with [Formula: see text] component. Inhibition by 3-phosphoglyceric acid and oxaloacetate with respect to both components is noncompetitive and competitive, respectively. The significance of negative cooperativity and the effect of the above-mentioned inhibitors is discussed.

REGULATION OF THE "MALIC" ENZYME IN NEUROSPORA CRASSA

1967 ◽  
Vol 13 (9) ◽  
pp. 1211-1221 ◽  
Author(s):  
M. W. Zink
Keyword(s):  
Cell Growth ◽  
Neurospora Crassa ◽  
Growth Phase ◽  
Pyruvic Acid ◽  
Malic Acid ◽  
Malic Enzyme ◽  
Enzyme Preparation ◽  
Growth Cycle ◽  
Oxidative Decarboxylation ◽  
Enzyme Repression

"Malic" enzyme has been isolated from Neurospora crassa which can bring about the reversible carboxylation of pyruvic acid. The enzyme is specific to L-malate and NADP and is activated by Mn++ and Mg++. The partially purified enzyme does not decarboxylate oxaloacetate but is competitively inhibited by it. This enzyme is synthesized only during the early stages of the growth cycle and is repressed by acetate. In addition, the oxidative decarboxylation of malic acid is competitively inhibited by aspartic acid; the degree of inhibition depends upon the cell growth phase from which the enzyme is extracted. "Malic" enzyme isolated from a 12-h culture is not significantly inhibited by aspartate. However, this inhibition increases to 90% if an enzyme preparation from a 24-h culture is used. The significance of enzyme repression by acetate and the inhibition of the activity by aspartate are discussed.

scholarly journals Unidirectional inhibition and activation of ‘malic’ enzyme of Solanum tuberosum by meso-tartrate

1975 ◽  
Vol 149 (2) ◽  
pp. 349-355 ◽  
Author(s):  
K H Do Nascimento ◽  
D D Davies ◽  
K D Patil
Keyword(s):  
Solanum Tuberosum ◽  
Organic Acids ◽  
Malic Acid ◽  
Active Site ◽  
Malic Enzyme ◽  
Control Site ◽  
Oxidative Decarboxylation ◽  
Reductive Carboxylation ◽  
Similar Explanation ◽  
Stimulation Of

A kinetic study of ‘malic’ enzyme (EC 1.1.1.40) from potato suggests that the mechanism is Ordered Bi Ter with NADP+ binding before malate, and NADPH binding before pyruvate and HCO3-. The analysis is complicated by the non-linearity that occurs in some of the plots. meso-Tartrate is shown to inhibit the oxidative decarboxylation of malate but to activate the reductive carboxylation of pyruvate. To explain these unidirectional effects it is suggested that the control site of ‘malic’ enzyme binds organic acids (including meso-tartrate) which activate the enzyme. meso-Tartrate, however, competes with malate for the active site and thus inhibits the oxidative decarboxylation of malate. Because meso-tartrate does not compete effectively with pyruvate for enzyme-NADPH, its binding at the control site leads to a stimulation of the carboxylation of pyruvate. A similar explanation is advanced for the observation that malic acid stimulates its own synthesis.

Alternative Substrates for Malic Enzyme:  Oxidative Decarboxylation ofl-Aspartate†

Biochemistry ◽  
2002 ◽  
Vol 41 (40) ◽  
pp. 12200-12203 ◽  
Author(s):  
Dali Liu ◽  
Chi-Ching Hwang ◽  
Paul F. Cook
Keyword(s):  
Malic Enzyme ◽  
Oxidative Decarboxylation ◽  
Alternative Substrates

scholarly journals Identification and Characterization ofMAE1, the Saccharomyces cerevisiae Structural Gene Encoding Mitochondrial Malic Enzyme

1998 ◽  
Vol 180 (11) ◽  
pp. 2875-2882 ◽  
Author(s):  
Eckhard Boles ◽  
Patricia de Jong-Gubbels ◽  
Jack T. Pronk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activity ◽  
Pyruvate Kinase ◽  
Malic Enzyme ◽  
Fold Increase ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Oxidative Decarboxylation ◽  
Mrna Levels ◽  
Malic Enzyme Activity

ABSTRACT Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression ofYKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures,MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.

Adenylate Kinase from Maize Leaves: True Substrates, Inhibition by P1 , P5-di(adenosine-5′) pentaphosphate and Kinetic Mechanism

1990 ◽  
Vol 45 (6) ◽  
pp. 607-613 ◽  
Author(s):  
Leszek A. Kleczkowski ◽  
Douglas D. Randall ◽  
Warren L. Zahler
Keyword(s):  
Adenylate Kinase ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Reverse Reaction ◽  
Plant Tissues ◽  
Forward Reaction ◽  
Higher Plant ◽  
Maize Leaves ◽  
Variable Substrate ◽  
Free Magnesium

Abstract Purified maize leaf adenylate kinase (AK) was shown to use one molecule each of free ADP and Mg-ADP as well as free AM P and Mg-ATP as substrates in the forward and reverse reaction, respectively. This was deduced from substrate kinetic studies which were carried out under conditions of strictly defined concentrations of free and Mg-complexed adenylate species and under controlled free magnesium levels. Apparent Km values of the substrates of AK were 3 and 6 μM for ADP and Mg-ADP, respectively (forward reaction), and 69 and 25 μM for free AMP and Mg-ATP, respectively (reverse reaction). The enzyme was competitively inhibited by P1,P5-di(adenosine-5′)pentaphosphate (Ap5A), a bisubstrate analog of AK reaction, with apparent Ki values in the range of 11 -80 nM , depending on variable substrate. Substrate kinetic studies and inhibition patterns with Ap5A suggested a sequential random kinetic mechanism in both directions of the reaction. These properties of a higher plant AK are similar or analogous to those previously established for the enzyme from yeast and non-plant tissues.

The photosynthetic carbon metabolism of Zea mays and Gomphrena globosa: the location of the CO2 fixation and the carboxyl transfer reactions

1970 ◽  
Vol 48 (4) ◽  
pp. 777-786 ◽  
Author(s):  
J. A. Berry ◽  
W. J. S. Downton ◽  
E. B. Tregunna
Keyword(s):  
Zea Mays ◽  
Bundle Sheath ◽  
Oxidative Decarboxylation ◽  
Gomphrena Globosa ◽  
Phosphorylated Compounds ◽  
Bundle Sheath Cells ◽  
Phosphoglyceric Acid ◽  
Photosynthetic Carbon Metabolism ◽  
Gradient Separation ◽  
Density Gradient Separation

Zea mays and Gomphrena globosa form labeled aspartate and malate (C4-acids) via β-carboxylation of P-enolpyruvate during photosynthesis. Studies of the redistribution of 14C in pulse- and chase-type feedings of 14CO2 indicate that most labeled phosphorylated compounds are formed from the C4-acids. A mechanism involving CO2 as a transitory intermediate is advanced to explain the carboxyl transfer from the C4-acids to 3-phosphoglyceric acid (3-PGA). In this model, CO2 is generated through the oxidative decarboxylation of malic acid by "malic" enzyme, and is refixed by RuDP carboxylase to form 3-PGA. The pattern of labeling of photosynthetic products, the extractable enzyme activities, and the gas exchange properties of these plants appear to be consistent with this proposed sequence of reactions. The location of 14C-labeled compounds was determined by radioautography, and by nonaqueous density gradient separation. Differential grinding was used to study the location of some photosynthetic enzymes. These indicate that CO2 fixation by β-carboxylation occurs in the leaf mesophyll. The carboxyl transfer and the reactions leading to the photosynthesis of starch appear to be confined predominantly to the bundle sheath cells. Rapid transport of C4-acids from the site of CO2 fixation in the mesophyll to the bundle sheath may occur by plasmodesmata.

Determination of the rate-limiting steps for malic enzyme by the use of isotope effects and other kinetic studies

Biochemistry ◽  
1977 ◽  
Vol 16 (4) ◽  
pp. 571-576 ◽  
Author(s):  
Michael I. Schimerlik ◽  
C. E. Grimshaw ◽  
W. W. Cleland
Keyword(s):  
Malic Enzyme ◽  
Isotope Effects ◽  
Kinetic Studies ◽  
Rate Limiting

THE CARBOXYLASES OF LEAVES AND THEIR ROLE IN PHOTOSYNTHESIS

1952 ◽  
Vol 30 (4) ◽  
pp. 395-409 ◽  
Author(s):  
K. A. Clendenning ◽  
E. R. Waygood ◽  
P. Weinberger
Keyword(s):  
Sugar Beet ◽  
Hydrogen Cyanide ◽  
Malic Enzyme ◽  
Hill Reaction ◽  
Leaf Extracts ◽  
Root Extracts ◽  
Phosphoglyceric Acid ◽  
Reaction Capacity ◽  
The Hill

"Malic" enzyme isolated from the cytoplasm of parsley and sugar beet leaves was linked with illuminated spinach chloroplast fragments to effect photosynthesis in vitro. The model photosynthesis system containing excess "malic" enzyme was not inhibited by 5 × 10−4 M hydrogen cyanide. The "malic" enzyme system was inhibited by cyanide, however, at very low enzyme concentrations. The richest source of "malic" enzyme found in this study was the mature parsley leaf. Expressed on the same basis, the enzymatic capacities of parsley leaf "malic" enzyme and the Hill reaction capacity of isolated spinach chloroplasts are of similar magnitude. Higher "malic" enzyme and oxalacetic carboxylase activities were found in purified extracts of parsley leaves than in the corresponding root extracts. Oxalacetic, oxalsuccinic, α-ketoglutaric, and pyruvic carboxylases were not inhibited by 10−3 M hydrogen cyanide. The α-ketoglutaric and pyruvic carboxylases were much less abundant in leaves than in other plant organs; formic dehydrogenase was not detected in leaves although it is abundant in seeds. Glutamic carboxylase was found in the cytoplasm of wheat and sugar beet leaves, and with the aid of C14O2 was shown to be only weakly reversible. No evidence was obtained for the presence in leaf extracts of an enzyme, or mixture of enzymes, capable of decarboxylating phosphoglyceric acid in vitro.

scholarly journals Post-transcriptional regulation of redox homeostasis by the small RNA SHOxi in haloarchaea

2020 ◽  
Author(s):  
Diego Rivera Gelsinger ◽  
Rahul Reddy ◽  
Kathleen Whittington ◽  
Sara Debic ◽  
Jocelyne DiRuggiero
Keyword(s):  
Oxidative Stress ◽  
Malic Enzyme ◽  
Tricarboxylic Acid Cycle ◽  
Redox Homeostasis ◽  
Fine Tuning ◽  
Oxidative Decarboxylation ◽  
Seed Region ◽  
Stem Loop ◽  
Loop Region ◽  
Rna Interaction

ABSTRACTHaloarchaea are highly resistant to oxidative stress, however, a comprehensive understanding of the processes regulating this remarkable response is lacking. Oxidative stress-responsive small non-coding RNAs (sRNAs) have been reported in the model archaeon, Haloferax volcanii, but targets and mechanisms have not been elucidated. Using a combination of high throughput and reverse molecular genetic approaches, we elucidated the functional role of the most up-regulated intergenic sRNA during oxidative stress in H. volcanii, named Small RNA in Haloferax Oxidative Stress (SHOxi). SHOxi was predicted to form a stable secondary structure with a conserved stem-loop region as the potential binding site for trans-targets. NAD-dependent malic enzyme mRNA, identified as a putative target of SHOxi, interacted directly with a putative “seed” region within the predicted stem loop of SHOxi. Malic enzyme is an enzyme of the tricarboxylic acid cycle that catalyzes the oxidative decarboxylation of malate into pyruvate using NAD+ as a cofactor. The destabilization of malic enzyme mRNA, and the decrease in the NAD+/NADH ratio, resulting from the direct RNA-RNA interaction between SHOxi and its trans-target was essential for the survival of H. volcanii to oxidative stress. These findings indicate that SHOxi likely regulates redox homeostasis during oxidative stress by the post-transcriptional destabilization of malic enzyme mRNA. SHOxi-mediated regulation provides evidence that the fine-tuning of metabolic cofactors could be a core strategy to mitigate damage from oxidative stress and confer resistance. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in Archaea.

Intracellular Conversion of Malate and Localization of Enzymes Involved in the Metabolism of Malate in Photoautotrophic Cell Cultures of Chenopodium rubrum

1992 ◽  
Vol 47 (7-8) ◽  
pp. 545-552 ◽  
Author(s):  
Shin-ichi Amino
Keyword(s):  
Subcellular Localization ◽  
Cell Suspension ◽  
Malate Dehydrogenase ◽  
Cell Cultures ◽  
Malic Acid ◽  
Enzyme Activities ◽  
Malic Enzyme ◽  
Mitochondrial Fraction ◽  
Enzymatic Activities ◽  
Chenopodium Rubrum

Cells from photoautotrophic cultures of Chenopodium rubrum were fractionated for the isolation of purified chloroplasts and mitochondria. The subcellular localization of the enzymatic activities involved in the metabolism of malic acid was investigated. Highly purified chloroplasts were obtained from the protoplasts, whereas peroxisomes were still present in the mitochondrial fraction. NAD - and NADP-dependent malate dehydrogenase and malic enzyme activities were found in the mitochondrial and chloroplast fractions, respectively. Exogenously supplied [14C]labelled malate was metabolized by the photoautotrophic cell suspension, obviously to the greater part in mitochondria.

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