Aspartate kinase reaction

The aspartate kinase reaction is not dependent upon the presence of hydroxylamine during the assay, only at the end of the assay for the development of the color complex (Davies and Miflin, 1977). 

Aspartate kinase [EC 2.T.2.4], also known as asparto-kinase, catalyzes the reaction of aspartate with ATP to produce 4-phosphoaspartate and ADP. The enzyme isolated from E. coli is a multifunctional protein, also exhibiting the ability to catalyze the reaction of homoserine with NAD(P) to produce aspartate 4-semialdehyde and NAD(P)H (that is, the activity of homoserine dehydrogenase, EC 1.1.1.3). 

The L-threonine biosynthetic pathway consists of five enzymatic steps from L-aspartate. E. coli has three aspartate kinase isoenzymes, key enzymes which catalyze the first reaction of the L-threonine biosynthetic pathway. The aspartate kinase isoenzymes I, II, and III encoded by the thrA, metL, and lysC genes, respectively, are affected by feedback inhibition by L-threonine, L-methionine, and L-lysine, respectively. C. glutamicum has only one aspartate kinase encoded by the lysC gene, which is subjected to feedback inhibition by L-lysine and... 

The synthesis of the other members of the aspartate family (Figure 14.8) is initiated by aspartate kinase (often referred to as aspartokinase) in an ATP-requiring reaction in which the side chain carboxyl group is phosphorylated. Aspartate... 

Fig. 1. Bio thetic pathways for the essential aspartate-family amino adds. The numbers represent enzymes catalyzing the reaction 1, aspartate kinase 2, homoserine dehydrogenase 3, homoserine kinase 4, threonine thase 5, threonine dehydrogenase 6, acetolactate thase 7, dihydrodipicolinate thase 8, diaminopimelate decarboiylase.

So, the biosynthesis of methionine (Met, M), the first of the essential amino adds to be considered (Scheme 12.13), begins by the conversion of aspartate (Asp, D) to aspartate semialdehyde in the same way glutamate (Glu, E) was converted to glutamate semialdehyde (vide supra. Scheme 12.6). Phosphorylation on the terminal carboxylate of aspartate (Asp, D) by ATP in the presence of aspartate kinase (EC 2.7.2.4) and subsequent reduction of the aspart-4 yl phosphate by NADPH in the presence of aspartate semialdehyde dehydrogenase (EC 1.2.1.11) yields the aspartate semialdehyde. The aspartate semialdehyde is further reduced to homoserine (homoserine oxoreductase, EC 1.1.1.3) and the latter is succinylated by succinyl-CoA with the liberation of coenzyme A (CoA-SH) in the presence of homoserine O-succinyl-transferase (EC 2.3.1.46). Then, reaction with cysteine (Cys, C) in the presence of cystathionine y-synthase (EC 2.5.1.48) produces cystathionine and succinate. In the presence of the pyridoxal phosphate protein cystathionine P-lyase (EC 4.4.1.8), both ammonia and pyruvate are lost from cystathionine and homocysteine is produced. Finally, methylation on sulfur to generate methionine (Met, M) occurs by the donation of the methyl from 5-methyltetrahydrofolate in the presence of methonine synthase (EC 2.1.1.13). 

Fig. 14.1 The biosynthesis pathway of L-threonine. The pathway consists of centeral metabolic pathways and the threonine terminal pathways. The centeral metabolic pathways involve glycolysis, phosphate pentose pathway, TCA cycle and anaplerotic pathways. The threonine terminal pathway consists of five enzymetic steps. The first, third, and fourth reactions are catalyzed by the three key enzymes aspartate kinase, homoserine dehydrogenase, tmd homoserine kinase, respectively. There are four competing pathways that affect the biosynthesis of L-threonine, leading to formation of L-lysine, L-metMonine, L-isoleucdne, and glycine...

Starting from the building block of L-aspartate, the biosynthesis of L-threonine comprises five successive reactions sequencially catalyzed by aspartate kinase, aspartyl semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine synthase. 

The kinase reaction is reversible and the disappearance of aspartyl phosphate in the presence of adenosine diphosphate (ADP) is associated with the stoichiometric formation of ATP. On the assumption that the appearance of ADP and the disappearance of ATP and aspartate are equivalent to aspartyl phosphate formation, the equilibrium constant of the reaction was calculated from the experimental data to be 3.5 X 10 at pH 8.0 and a temperature of 15°. It is to be noted that the /3-aspartokinase reaction is quite analogous to that involving ATP and 3-phosphoglyceric acid. 

Unlike glycolysis, which occurs strictly in the cell cytosol, gluconeogen-esis involves a complex interaction between the mitochondrion and the cytosol. This interaction is necessitated by the irreversibility of the pyruvate kinase reaction, by the relative impermeability of the inner mitochondrial membrane to oxaloacetate, and by the specific mitochondrial location of pyruvate carboxylase. Compartmentation within the cell has led to the distribution of a number of enzymes (aspartate and alanine aminotransferases, and NAD -malate dehydrogenase) in both the mitochondria and the cytosol. In the classical situation represented by the rat, mouse, or hamster hepatocyte, the indirect "translocation" of oxaloacetate—the product of the pyruvate carboxylase reaction—into the cytosol is effected by the concerted action of these enzymes. Within the mitochondria oxaloacetate is converted either to malate or aspartate, or both. Following the exit of these metabolites from the mitochondria, oxaloacetate is regenerated by essentially similar reactions in the cytosol and is subsequently decarboxylated to P-enolpyruvate by P-enol-pyruvate carboxykinase. Thus the presence of a membrane barrier to oxaloacetate leads to the functioning of the malate-aspartate shuttle as an important element in gluconeogenesis. 

Enzymes, measured in clinical laboratories, for which kits are available include y-glutamyl transferase (GGT), alanine transferase [9000-86-6] (ALT), aldolase, a-amylase [9000-90-2] aspartate aminotransferase [9000-97-9], creatine kinase and its isoenzymes, galactose-l-phosphate uridyl transferase, Hpase, malate dehydrogenase [9001 -64-3], 5 -nucleotidase, phosphohexose isomerase, and pymvate kinase [9001-59-6]. One example is the measurement of aspartate aminotransferase, where the reaction is followed by monitoring the loss of NADH ... 

The answer is C. Pyruvate kinase deficiency is ruled out by the elevated serum lactate levels. The coma is associated with a fasting hypoglycemia, which is indicative of pyruvate carboxylase deficiency. The elevated citrulline and lysine in the serum are due to a reduction of aspartic acid levels, which are caused by the reduced levels of oxaloacetate, the product of the pymvate carboxylase reaction. 

In addition to the enzyme the reaction mixture contained Hepes buffer, IMP, GTP, MgCl, and creatine phosphate and phosphocreatine kinase (a regeneration system for GTP). The reaction was initiated by adding aspartic acid. Samples were removed at intervals, and the reactions were terminated by direct injection onto the HPLC column. Figure 9.113 shows chromatograms of samples removed at 0,5, and 10 minutes of incubation. The disappearance of IMP and GTP and the appearance of GTP and sAMP can be noted. 

Much structural biology analysis has been performed on protein kinase A (PKA), and its catalytic residues are conserved across the family (6, 7). In PKA, lysine 72 and glutamate 91 orient the y-phosphate toward the protein substrate (Fig. 2b). Aspartate 166 acts as a catalytic base to accept the proton from the hydroxyl nucleophile, and Lys 168 acts as an electrostatic catalyst to stabilize the y-phosphate during the reaction. Asparagine 171 positions a magnesium ion that coordinates the a/fi phosphates (Fig. 2b). 

Figure 2 Snapshots of the overall structure and catalytic machinery of protein kinase A. (a) The overall fold of the catalytic domain is formed by two subdomains, a beta sheet N-terminus (gray) and a C-terminal helical domain (green). ATP binds a cleft between the two lobes, and the phosphoacceptor substrate binds the C-terminal lobe, (b) N-terminal residues Lys 72 and Glu 91 orient the phosphates toward the phosphoacceptor peptide (pink/yellow) in concert with one of two magnesium ions, (c) C-terminal residue Lys 168 acts as an electrostatic catalyst to stabilize the y-phosphate during the reaction while asparagine 171 and aspartate 184 position the phosphates within the active site.

The first step in the formation of urea from ammonia is its combination with bicarbonate to form carbamyl phosphate (Fig. 1). This contributes only one nitrogen atom to urea, the other being donated by aspartic acid in the third step of the pathway. A -Acetylglutamate is required as cofactor, and the presence of Mg is essential, ATP being converted to ADP in the process. The reaction is catalyzed by carbamyl phosphate synthetase (carbamate kinase EC 2.7.2.2). It has been shown that there are probably two forms of this enzyme, at least in rat liver. One is ammonia dependent, is primarily associated with mitochondria, and may be the enzyme responsible for the formation of carbamyl phosphate in the synthesis of urea. The other, which is glutamine dependent, is probably mainly extramitochondrial and may supply the carbamyl phosphate used... 

The answer is e. (Murray, pp 375-401. Scriver, pp 2663-2704. Sack, pp 121-138. Wilson, pp 287—320.) Orotic aciduria is the buildup of orotic acid due to a deficiency in one or both of the enzymes that convert it to UMP Either orotate phosphoribosyltransferase and orotidylate decarboxylase are both defective, or the decarboxylase alone is defective. UMP is the precursor of UTP, CTP, and TMP All of these end products normally act in some way to feedback-inhibit the initial reactions of pyrimidine synthesis. Specifically, the lack of CTP inhibition allows aspartate transcarbamoylase to remain highly active and ultimately results in a buildup of orotic acid and the resultant orotic aciduria. The lack of CTP, TMP, and UTP leads to a decreased erythrocyte formation and megaloblastic anemia. Uridine treatment is effective because uridine can easily be converted to UMP by omnipresent tissue kinases, thus allowing UTP, CTP, and TMP to be synthesized and feedback-inhibit further orotic acid production. 

Fig. 59. Analytically utilized reactions involving pyruvate. PEP phosphenolpyruvate, PK pyruvate kinase, ASAT aspartate aminotransferase, ALAT alanine aminotransferase.

Fig. 86. Coupling of enzyme reactions for the design of a sensor family based on LMO. CK = creatine kinase, PK = pyruvate kinase, MDH = malate dehydrogenase, PEP = phosphoenolpyruvate, ALAT = alanine aminotransferase, ASAT = aspartate aminotransferase.

Bypassing the pyruvate kinase step requires oxaloacetate. The oxaloacetate can come from either of two sources. First, various reactions can build up TCA cycle intermediates, among them oxaloacetate. For example, aspartic acid has the same carbon skeleton as oxaloacetate and ammonia can be removed by several means to yield oxaloacetate ... 

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