Optimum activity of the phosphofructokinase from Ascaris suum requires more than one metal ion.

Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose 6-phosphate (F6P) to give fructose 1,6-bisphosphate (FBP) using MgATP as the phosphoryl donor. As the concentration of Mg(2+) increases above the concentration needed to generate the MgATP chelate complex, a 15-fold increase in the initial rate was observed at low MgATP. The effect of Mg(2+) is limited to V/K(MgATP), and initial rate studies indicate an equilibrium-ordered addition of Mg(2+) before MgATP.

Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum phosphofructokinase.

The nucleotide sequence of a full-length cDNA encoding phosphofructokinase (PFK) enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2,653 bases comprises a single open reading frame of 2,452 bases and a noncoding region of 201 bases after the stop codon. The mature protein contains 812 amino acids and has a molecular mass of 90,900 Da.

A catalytic triad is responsible for acid-base chemistry in the Ascaris suum NAD-malic enzyme.

The pH dependence of kinetic parameters of several active site mutants of the Ascaris suum NAD-malic enzyme was investigated to determine the role of amino acid residues likely involved in catalysis on the basis of three-dimensional structures of malic enzyme. Lysine 199 is positioned to act as the general base that accepts a proton from the 2-hydroxyl of malate during the hydride transfer step. The pH dependence of V/K(malate) for the K199R mutant enzyme reveals a pK of 5.

Cloning, expression, and purification of fumarase from the parasitic nematode Ascaris suum.

The cDNA encoding fumarase, an enzyme catalyzing reversible hydration of fumarate to L-malate, from the parasitic roundworm Ascaris suum, has been cloned, sequenced, over-expressed in Escherichia coli, and purified. The single open reading frame translates into a protein of 50,502Da containing 467 amino acids. It shows 82, 77, and 58% identity with Caenorhabditis elegans, human, and E.

Ascaris suum NAD-malic enzyme is activated by L-malate and fumarate binding to separate allosteric sites.

The kinetic mechanism of activation of the mitochondrial NAD-malic enzyme from the parasitic roundworm Ascaris suum has been studied using a steady-state kinetic approach. The following conclusions are suggested. First, malate and fumarate increase the activity of the enzyme in both reaction directions as a result of binding to separate allosteric sites, i.

Crystallographic studies on Ascaris suum NAD-malic enzyme bound to reduced cofactor and identification of an effector site.

The crystal structure of the mitochondrial NAD-malic enzyme from Ascaris suum, in a quaternary complex with NADH, tartronate, and magnesium has been determined to 2.0-A resolution. The structure closely resembles the previously determined structure of the same enzyme in binary complex with NAD.

Crystal structure of the malic enzyme from Ascaris suum complexed with nicotinamide adenine dinucleotide at 2.3 A resolution.

The structure of the Ascaris suum mitochondrial NAD-malic enzyme in binary complex with NAD has been solved to a resolution of 2.3 A by X-ray crystallography. The structure resembles that of the human mitochondrial enzyme determined in complex with NAD [Xu, Y.