Pyruvate kinase and

Transfer of the phosphoryl group to ADP in step 10 then generates ATP and gives enolpyruvate, which undergoes tautomerization to pyruvate. The reaction is catalyzed by pyruvate kinase and requires that a molecule of fructose 1,6-bis-phosphate also be present, as well as 2 equivalents of Mg2+. One Mg2+ ion coordinates to ADP, and the other increases the acidity of a water molecule necessary for protonation of the enolate ion. 

Let us consider Figure 5.3 again. Both pyruvate kinase and dtrate synthase (enzymes III and V) are inhibited by elevated ATP concentrations. During citric acid production ATP concentrations are likely to arise (ATP produced in glycolysis) and either of these enzymes could, if inhibited, slow down the process. In fact all of the evidence suggests that both enzymes are modified or controlled in some way such that they are insensitive to other cellular metabolites during citric add production. 

Decrease of cAMP, the second messenger of glucagon. Induction of pyruvate kinase and glycerinaldehyde dehydrogenase... 

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. 

A tri-enzymatic sensing layer based on kinase-oxidase activities for the detection of acetate was also described. A reaction sequence using acetate kinase, pyruvate kinase and pyruvate oxidase enabled the production of H2O2 in response to acetate injection in the range 10 pM - 100 mM59. 

In biological systems, therefore, the behavior of Li+ is predicted to be similar to that of Na+ and K+ in some cases, and to that of Mg2+ and Ca2+ in others [12]. Indeed, research has demonstrated numerous systems in which one or more of these cations is normally intrinsically involved, including ion transport pathways and enzyme activities, in which Li+ has mimicked the actions of these cations, sometimes producing inhibitory or stimulatory effects. For example, Li+ can replace Na+ in the ATP-dependent system which controls the transport of Na+ through the endoplasmic reticulum Li+ inhibits the activity of some Mg2+-dependent enzymes in vitro, such as pyruvate kinase and inositol monophosphate phosphatase Li+ affects the activity of some Ca2+-dependent enzymes— it increases the levels of activated Ca2+-ATPase in human erythrocyte membranes ex vivo and inhibits tryptophan hydroxylase. 

Facilitated diffusion within organisms takes place when carriers or proteins residing within membranes—ion channels, for instance—organize the movement of ions from one location to another. This diffusion type is a kinetic, not thermodynamic, effect in which a for the transfer is lowered and the rate of diffusion is accelerated. Facilitated diffusion channels organize ion movements in both directions, and the process can be inhibited both competitively and noncompetitively. It is known that most cells maintain open channels for K+ most of the time and closed channels for other ions. Potassium-ion-dependent enzymes include NaVK+ ATPases (to be discussed in Section 5.4.1), pyruvate kinases, and dioldehydratases (not to be discussed further). 

The pathway for gluconeogenesis is shown in Figures 6.23 and 6.24. Some of the reactions are catalysed by the glycolytic enzymes i.e. they are the near-equilibrium. The non-equilibrium reactions of glycolysis are those catalysed by hexokinase (or glucokinase, in the liver), phosphofructokinase and pyruvate kinase and, in order to reverse these steps, separate and distinct non-equilibrium reactions are required in the gluconeogenic pathway. These reactions are ... 

The hormones glucagon, cortisol and insulin regulate the concentrations of some enzymes and hence their activities. These include glucokinase, pyruvate kinase and phospho-enolpyruvate carboxykinase. Most work has been carried out on the carboxykinase enzyme, for which it is known that glucagon and cortisol increase the concentration whereas insulin decreases it. These changes are brought about at the transcriptional level by changing the activity of transcription factors (Chapter 20). Since the hormones... 

Hydroxycyclopropanecarboxylic acid phosphate HCP 34 is an analogue of phosphoenolpyruvate (PEP) 35 which is metabolized by various enzymes. HCP 34 is a potent competitive inhibitor of enzymes utilizing PEP 35, such as PEP carboxylase, enolase, pyruvate kinase, and probably other enzymes. It is a substantially better inhibitor than phospholactate 36 or phosphoglycolate 37, presumably because of the similarity of its geometric and electronic structures with phosphoenol pyruvate,Eq. 12 [28]. 

ADP NADH Disappearance Pyruvate Kinase and Lactate Dehydrogenase Fructokinase, y-Glutamylcysteine Synthetase, Mannokinase, ATPase ... 

GMP NADH Disappearance GMP Kinase, Pyruvate Kinase, and Lactate Dehydrogenase Hypoxanthine-Guanine Phosphoribosyl-transferase ... 

Figure 6-1. The steps of glycolysis. Feedback inhibition of glucose phosphorylation by hexokinase, inhibition of pyruvate kinase, and the main regulatory, rate-limiting step catalyzed by phosphofructoki-nase (PFK-I) are indicated, pyruvate formation and substrate-level phosphorylation are the main outcomes of these reactions. Regeneration of NAD occurs by reduction of pyruvate to lactate during anaerobic glycolysis.

Insulin stimulates the synthesis of hexokinases II and IV, PFK-1, pyruvate kinase, and several enzymes involved in lipid synthesis. Insulin stimulates glycogen synthesis in muscle and liver. 

In this one-pot procedure NeuAc 16 is generated from ManNAc 15 and pyruvic acid in situ with sialic acid aldolase and then converted irreversibly to CMP-NeuAc 17. CMP is converted to CDP with myokinase and ATP. The released ADP is converted to ATP with pyruvate kinase and PEP. CDP is then converted to CTP also with pyruvate kinase and phosphoenolpyruvate (PEP). The formed CTP reacts with NeuAc catalyzed by NeuAc synthetase to give 17. 

Oxaloacetate is also decarboxylated without phosphorylation of the enolate anion formed but with release of free pyruvate. Both pyruvate kinase and PEPCK can act as oxaloacetate decarboxylases.261... 

In this technique, the enzyme solution is put inside a dialysis bag which is then immersed in a solution of substrate, or cofactors. Small molecules can diffuse through the wall of the bag and react in the presence of the enzyme, while products, if also small molecules, diffuse into the outside solution, where they may be recovered. This technique has been used in syntheses with sialyl aldolase, Kdo-synthetase, the common aldolase, a mixture of hexokinase and pyruvate kinase, a-(2— 6) sialyl transferase,26 a mixture of pyruvate kinase and adenylate kinase,27 and CMP-Neu5Ac synthetase.28... 

Other types of enzymes When no oxidase or dehydrogenase is available for a target analyte, other types of enzymes have been used for biospedfic recognition e.g. for citric acid detection, citrate lyase, and amperometric detection was possible by coupling to two more enzymatic reactions oxaloacetate decarboxylase and pyruvate oxidase, which convert citric add into H2O2 with the latter being monitored amperometrically with an H202 probe. For detection of acetic add, acetate kinase is used, coupled to pyruvate kinase and pyruvate oxidase [34,35]. 

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