Phosphate, enzymatic reactions

A non-linear regression analysis is employed using die Solver in Microsoft Excel spreadsheet to determine die values of and in die following examples. Example 1-5 (Chapter 1) involves the enzymatic reaction in the conversion of urea to ammonia and carbon dioxide and Example 11-1 deals with the interconversion of D-glyceraldehyde 3-Phosphate and dihydroxyacetone phosphate. The Solver (EXAMPLEll-l.xls and EXAMPLEll-3.xls) uses the Michaehs-Menten (MM) formula to compute v i- The residual sums of squares between Vg(,j, and v j is then calculated. Using guessed values of and the Solver uses a search optimization technique to determine MM parameters. The values of and in Example 11-1 are ... 

Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues, 97.44 kD). Each subunit contains a pyridoxal phosphate cofactor, covalently linked as a Schiff base to Lys °. Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface (Eigure 15.15). In addition, a regulatory phosphorylation site is located at Ser on each subunit. A glycogen-binding site on each subunit facilitates prior association of glycogen phosphorylase with its substrate and also exerts regulatory control on the enzymatic reaction. 

Pyridoxal phosphate mainly serves as coenzyme in the amino acid metabolism and is covalently bound to its enzyme via a Schiff base. In the enzymatic reaction, the amino group of the substrate and the aldehyde group of PLP form a Schiff base, too. The subsequent reactions can take place at the a-, (3-, or y-carbon of the respective substrate. Common types of reactions are decarboxylations (formation of biogenic amines), transaminations (transfer of the amino nitrogen of one amino acid to the keto analog of another amino acid), and eliminations. 

CK catalyzes the reversible phosphorylation of creatine in the presence of ATP and magnesium. When creatine phosphate is the substrate, the resulting creatine can be measured as the ninhydrin fluorescent compound, as in the continuous flow Auto Analyzer method. Kinetic methods based on coupled enzymatic reactions are also popular. Tanzer and Gilvarg (40) developed a kinetic method using the two exogenous enzymes pyruvate kinase and lactate dehydrogenase to measure the CK rate by following the oxidation of NADH. In this procedure the main reaction is run in a less favorable direction. 

Lequea et al. used the activity of tyrosine apodecarboxylase to determine the concentration of the enzyme cofactor pyridoxal 5 -phosphate (vitamin B6). The inactive apoenzyme is converted to the active enzyme by pyridoxal 5 -phosphate. By keeping the cofactor the limiting reagent in the reaction by adding excess apoenzyme and substrate, the enzyme activity is a direct measure of cofactor concentration. The enzymatic reaction was followed by detecting tyramine formation by LCEC. The authors used this method to determine vitamin B6 concentrations in plasma samples. 

The experiments were performed in a CINC V-02 separator also known as the CS-50 (15). Two Verder VL 500 control peristaltic tube pumps equipped with a double pump head (3,2 x 1,6 x 8R) were used to feed the CCS. In case of the enzymatic reaction, the low mix bottom plate was applied. To operate the reactor at a desired temperature, it was equipped with a jacket which was coimected to a temperature controlled water bath with an accuracy of 0.01°C. The CCS was fed with pure heptane and pure water, both with a flow rate of 6 mL/min. Subsequently, the centrifuge was started (40 Hz, which corresponds to 2400 rpm) and the set-up was allowed to equilibrate for a period of 1 h. At this point, the heptane feed stream was replaced by the organic feed stream (oleic acid (0.6M) and 1-bntanol (0.9M) in heptane). After equilibration for 10 minutes, the reaction in the CCS was started by replacing the water stream with the aqueous feed stream (0.1 M phosphate buffer pH 5.6 containing 1 g/1 of the lipase form Rhizomucor miehei). Samples were taken at regular intervals and analysed by GC. 

In the processes that require regeneration of cofactors such as nicotinamide adenine dinucleotide phosphate (NAD(P)H) and adenosine triphosphate (ATP), whole-cell biotransformations are more advantageous than enzymatic systems [12,15]. Whole cells also have a competitive edge over the isolated enzymes in complex conversions involving multiple enzymatic reactions [14]. 

The role of Schiff bases formed between pyridoxal phosphate and amino acid residues as intermediate products in many enzymatic reactions is well known and documented. NMR is an excellent tool for studies of the enzymatic processes involving Schiff bases formation. 

A similar study has also been conducted to determine the suitability of ascorbic acid 2-phosphate (AAP) as an alternative substrate to 4-AP for AP under identical conditions [48], Although 4-APP and AAP were suitable substrates for amperometric immunosensors, 4-APP was superior owing to its sixfold faster enzymatic reaction and lower detection potential (approximately 200-400mV). Notably, the lower detection potential for the hydrolysis product of 4-APP minimizes interferences from other species and hence improves the sensitivity of the immunosensor. 

A FIA system has been proposed for the CL detection of phosphate based on an enzymatic reaction and the application of a subsequent luminol reaction [41], The system consists of an immobilized pyruvate oxidase column, a mixing chamber for the CL reaction, and a PMT. H202 is generated by the reaction of phosphate and pyruvate oxidase and then reacts with luminol and HRP, producing... 

In order to test this hypothesis, enzymatic reactions were monitored in NMR tubes. ATP (100 p.M-3 mM) and the aminoglycosides (100 tLM-1.5 mM) were dissolved in phosphate buffer (10 mM phosphate, 2.5-5.0mM MgCl2,... 

The enzymatic reaction was performed at 30 °C for 2 hours in a volume of 1 ml of 250 mM phosphate buffer (pH 6.5) containing 50 mM of KOH, 32 U/ml of the enzyme, and [1- C]-substrate. The product was isolated as the methyl ester. When the (S)-enantiomer was employed as the substrate, C remained completely in the product, as confirmed by C NMR and HRMS. In addition, spin-spin coupling between and was observed in the product, and the frequency of the C-O bond-stretching vibration was down-shifted to 1690 cm" (cf. 1740 cm for C-O). On the contrary, reaction of the (R)-enantiomer resulted in the formation of (R)-monoacid containing C only within natural abundance. These results clearly indicate that the pro-R carboxyl group of malonic acid is ehminated to form (R)-phenylpropionate with inversion of configuration [28]. This is in sharp contrast to the known decarboxylation reaction by malonyl CoA decarboxylase [1] and serine hydroxymethyl transferase [2], which proceeds with retention of configuration. 

An enzymatic reaction intermediate formed by phospho-ryl transfer to a carboxyl group on an enzyme. Acyl-phosphates are structurally analogous to acid anhydrides (R—CO —O —CO—R ), and they are thermodynamically less stable than either of the two phosphoanhydride bonds in ATP. This is evident by the fact that the acetate kinase reaction (ADP + acetyl-phosphate = ATP + acetate) favors ATP formation with an equilibrium constant of about 3,000. Acetyl-phosphate can be chemically synthesized by reacting orthophosphate with acetic anhydride. 

Thiolester intermediates are formed in the glyceralde-hyde-3-phosphate dehydrogenase and many CoA-linked enzymatic reactions. 

The main drawback of the DHAP-dependent aldolases is their strict specificity for the donor substrate. Apart from the scope limitation that this fact represents, DHAP is expensive to be used stoichiometrically in high-scale synthesis, and labile at neutral and basic pH, and therefore its effective concentration decreases over time in enzymatic reaction media, hindering the overall yield of the aldol reaction. In addition, due to the presence of a phosphate group in both DHAP and the... 

Building blocks are amphiphiles, which have a delicate balance between the hydrophilic and hydrophobic group crucial to facilitate self-assembly. The peptide component serves to precisely control this balance, and the enzymatic reaction serves to alter it in favour of self-assembly. As illustrated in Fig. 3, the molecular switch may involve (1) phosphatase-catalysed removal of a (phosphate) group from the precursor to control the electrostatic balance (reaction (i) in Fig. 3) (2) hydrolysis of alkyl esters by hydrolases to change the amphiphilic balance (reaction (ii) in Fig. 3) or (3) condensation between two non-self-assembling precursors via a condensation reaction, e.g. involving protease-catalysed amide synthesis to alter the hydrophilic/hydrophobic balance (reaction (iii) in Fig. 3). A number of examples of each type are summarised in Table 1. 

Figure 9.28 Non-enzymatic reactions leading to membranogenic polyprenyl or dipolyprenyl phosphates. (Modified from Ourisson and Nakatani, 1999.)...

Perhaps one of the very first examples of enzymatic reactions carried out in liposomes with the aim of building a minimal cell is the work by Schmidli et al. (1991), as already mentioned in the previous chapter (Fig. 10.5). The general idea is illustrated in Figure 11.6, whereas the biochemical pathway is illustrated in Figure 11.7. The basic idea is to have inside the liposomes the series of reactions that, starting from a relatively simple product (G3P, glycerol-3-phosphate)... 

Protein phosphorylation is a specific enzymatic reaction in which one protein serves as a substrate for a protein kinase. Protein kinases are phophotransferases. They catalyze the transfer of a phosphate group from ATP to an acceptor amino acid in the substrate protein (fig. 2.10). A detailed discussion of protein kinases can be found in chapter 7. 

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