Pyruvate kinase preparation

Kdo was prepared on the 38-mmol scale starting from D-arabinose, by the simultaneous operation of three enzymes in the same vessel. One is Kdo synthetase 56 hexokinase catalyzes the phosphorylation57 of D-arabinose by ATP (catalytic), and pyruvate kinase catalyzes58 the regeneration of ATP with enolpyruvate phosphate. Such systems are described in more detail in Section IV. In this preparation, enolpyruvate phosphate serves two very different purposes, acting as a source of high-energy phosphate, and as a three-carbon donor.59... 

Phosphorolysis of ribonucleic acid with polynucleotide phosphorylase gives a mixture of the diphosphates of the four common nucleosides, which are transformed into triphosphates with enolpyruvate phosphate and pyruvate kinase. This mixture may be used as such as a source of uridine triphosphate in the preparation of the nucleotide-sugar uridine 5 -(a-D-glucopy-ranosyl diphosphate) ( uridine-diphosphate-glucose, UDP-Glc), or as a... 

Behm, C.A. and Bryant, C. (1 980) Regulatory properties of a partially purified preparation of pyruvate kinase from Fasciola hepatica. International Journal for Parasitology 1 0, 107-114. 

Some of the reactions of PO3- parallel enzymatic reactions promoted by adenosine triphosphate (ATP). Pyruvate kinase catalyzes the equilibration of ATP and pyruvate with adenosine diphosphate (ADP) and phosphoenol pyruvate (11,12). In a formal sense, this reaction resembles the preparations of enol phosphate (eqs. 6 and 7). Cytidine triphosphate synthetase catalyzes the reaction of uridine triphosphate with ammonia to yield cytidine triphosphate (13). In a formal sense, this reaction resembles the replacement of the ester carbonyl group of ethyl acetate by the nitrogen of aniline (eq. 8). 

There is now abundant evidence that the activity of pyruvate kinase is inhibited by glucagon in intact liver preparations [58,108] (Fig. 6), leading to reduced flux of... 

The chiral y-[l80]phosphorothioate of ATP, (Pp)-ATPyS, yl80(/3, y)lsO, was synthesized by the procedure outlined in Fig. 8 [25]. (Sp)-ADPaS, alsO(a,/3)l80 was prepared by stereospecific phosphorylation of AMPaS, al802 using the adenylate and pyruvate kinase system followed by dephosphorylation with glucose and hexokinase. This was the starting material for the synthesis, with the chiral a-... 

Scheme 5.40. Preparative scale production of DAHP by multi-enzyme synthesis. Ei = hexoki-nase, E2 = pyruvate kinase, E3 = transketolase + D-ribose 5-P, E4 = DHAP synthetase. P, = inorganic phosphate, P = PC>32. ...

The high ATPase activity of broken cell preparations from many tissues makes the use of an ATP-regenerating system mandatory. This may consist of 20 mM phosphoenol pyruvate and 130/ig/0.15 ml of pyruvate kinase [20], which serves to recycle the ADP formed by ATPase back to ATP. 

Because of the very high price of ATP, reaction (5.7) must be coupled with a regenerating system, the transfer of phosphate to ADP starting from the enol phosphate of pyruvic acid (an easily accessible and inexpensive phosphate), catalysed by the enzyme pyruvate kinase (reaction (5.8). In the same flask are mixed glucose, phosphoenolpyruvate, hexokinase, pyruvate kinase, and a catalytic quantity of ATP (about 1% mol) and the system produces D-glucose 6-phosphate until the phosphoenolpyruvate runs out. The kinases are easily accessible and, if they are immobilized on an insoluble support (see Section 10.4.1), they are reusable a certain number of times. In this way glucose 6-phosphate can be easily prepared on a 250 g scale (Poliak et al. 1977). 

Creighton and Rose (45) have explored the route shown in Scheme 7 to synthesize chiral pyruvate. The method takes advantage of the fact that pyruvate kinase decarboxylates oxalacetate with retention of configuration. The requisite labeled oxalacetates were prepared as shown from the appropriately labeled L-malates, available from equilibration with fumarase in either deuterated or tritiated water. This method is convenient for the preparation of small amounts of chiral acetate but suffers from the low oxalacetate decarboxylase activity of pyruvate kinase. 

When chemical and enzymatic methods for NTP synthesis are compared[941, enzymatic techniques provide the most convenient route to CTP and GTP, whereas chemical deamination of CTP is the best method for preparing UTP 94L ATP is relatively inexpensive from commercial sources, although it has been synthesized enzymatically from AMP on 50 mmol scale. Mixtures of NTPs can be prepared from RNA by sequential nuclease Pi, polynucleotide phosphorylase, and pyruvate kinase-catalyzed reactions11101. This mixture can be selectively converted to a sugar nucleotide using a particular sugar nucleoside diphosphate pyrophosphorylase11101. 

CMP-N-acetylneuraminic acid (CMP-NeuAc). CMP-N-acetylneuraminic acid has been prepared enzymatically on small scales (> 0.5 mmol) from CTP and NeuAc, under catalysis by CMP-NeuAc synthetase (EC 2.7.7.43) [131l An improvement in this procedure, involving in situ production of CTP from CMP under adenylate kinase and pyruvate kinase catalysis, is suitable for multigram-scale synthesis11321. Adenylate kinase catalyzes the equilibration of CTP and CMP to CDP, which is subsequently phosphorylated by pyruvate kinase to provide CTP. A one-pot synthesis of CMP-NeuAc based on this procedure involves the in situ synthesis of NeuAc from N-acetylmannosamine and pyruvate, catalyzed by sialic acid aldolase (Fig. 11.3-12)[10S1. Chemical syntheses of CMP-NeuAc have also been reported11421. 

In practice, for most synthetic applications, either acetyl phosphate/acetate kinase or phosphoenolpyruvate/pyruvate kinase are used to regenerate ATP. Because of the ease of preparing AcP, AcP/AcK is the most economical method for large-scale work. Its application is, however, limited to fast phosphorylation reactions where the hydrolysis of AcP is not important. The PEP/pyruvate kinase system is used in instances where the requirement for a strong, stable phosphorylating reagent outweighs the relative inconvenience of preparation of PEP. 

Phosphoenolpyruvate/pyruvate kinase. Phosphoenolpyruvate (PEP 2)/pyruvate kinase (PK E.C. 2.7.1.40) is the most efficient system for the regeneration of ATP from ADP. The phosphorylating agent PEP can be prepared in a mole scale [47L Starting from crude pyruvic acid, the crystalline monopotassium salt PEP-K is synthesized in a three-step procedure. For transformations on a scale <1 mol, PEP can be prepared from commercially available 3-phosphoglyceric acid in an enzyme-catalyzed reaction1641. This method is more expensive than the chemical preparation, but is more convenient because it requires less time and produces less organic waste (see Section 13.2.1.2 Fig. 13-5). 

The high stability of the ( a)g barrel is emphasized in the statement that you can do almost anything and still get an a/jS-barrel [186], For example, a protein expected to contain a ( a)io barrel has been prepared, but it really forms a (j9a)g barrel and the other two )Sa-portions of the molecule form an additional dimer [187]. If the amino and carboxy ends of iV-(5 -phosphoribosyl)anthranilate isomerase are moved to different loops between j3-strands and a helices, there is not a large effect on enzymic activity [183], Since this ( )8 folding pattern is so common, its evolution has been the subject of much discussion, but no firm conclusions can yet be made [186], It is found that (jSa)g barrel enzymes can be recruited for other purposes , such as the use of enolase in the x-crystallin in the duck lens [186]. It has been noted that enolase and pyruvate kinase, both (jSa)g barrels, are consecutive enzymes in glycolysis [186, 188]. 

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