Substrate inhibition pyruvate

Pyruvate oxidase requires the presence of thiamine pyrophosphate (0.1 mmol/1) and Ca2+ (2.5 mmol/1) for maximum activity. It should be used in 40 mmol/1 Tris buffer, pH 6.5-7.5, containing 0.5 mmol/1 phosphate. At higher phosphate concentrations substrate inhibition occurs this effect has been utilized in a phosphate sensor based on immobilized PyOD (Tabata and Murachi, 1983). Since PyOD is relatively unstable, for biosensors the enzyme has been immobilized by physical entrapment in, e.g., collagen (Mizutani et al., 1980), poly(vinyl chloride) and acetylcellulose (Kihara et al., 1984a,b). 

ALT catalyzes the reaction (reaction [VII]), and the pyruvate formed is reduced by NADH in a reaction catalyzed by LDH (indicator reaction, reaction [VIII]). The activity is measured by monitoring the decrease in absorbance of 340 nm due to the oxidation of NADH. The substrate concentrations are optimized theoretically, based on the kinetics of a two-substrate inhibited reaction because the ALT is subject to both substrate and product inhibitions. 

Excess substrate inhibited the oxidation of the p-hydroxyphenyl-pyruvate as did also benzoquinone acetic acid. This could be prevented by adding large amounts of ascorbic acid or small amounts of reduced 2,6-dichlorophenolindophenol. The latter was seven hundred times as effective as ascorbic acid with the purified enzyme 214), Zannoni and LaDu speculate that a product formed from p-hydroxyphenylpyruvate is the true inhibiting agent of the reaction. 

The data showed that Vj ax almost remained constant, and Kf increased in the presence of IC-22. The inhibition could be decreased with the increase in the concentration of sodium pyruvate as a substrate. When the concentration of substrate sodium pyruvate increased to 0.5 mM, the inhibition almost disappeared. This indicated that the inhibition of PDHc by IC-22 could be overcome by increasing the concentration of substrate. was increased in the presence of IC-22, which showed that the apparent affinity of enzyme for its substrate was decreased in the presence of IC-22. IC-22 could diminish the rate of catalysis by reducing the proportion of enzyme molecules bound to the substrate. However, the Cj ax were almost the same in the presence and absence of IC-22. IC-22 was thus recognized to act as a competitive inhibitor of PDHc [16]. 

In addition the transaminase from O. anthropi showed no substrate inhibition up to 500 mM of (S)-a-methylbenzylamine in any cases. For the kinetic resolution with 500 mM (S)-a-methylbenzylamine, 300mM pyruvate, and 75U/ml of O. anthropi transaminase, reaction yield of 95.3% and ee values of >99% were obtained after 3h compared to the transaminase of P. denitrificans vith no detectable conversion and the ee values of <22% obtained after 10 h. 

Bringer-Meier and Sahm [28] demonstrated that pyruvate decarboxylase from Sac-charomyces carlsbergensis efficiently synthesizes phenylacetyl carbinol from pyruvate and benzaldehyde, whereas pyruvate decarboxylase from Z. mobilis is unsuitable for the biotransformations due to its low affinity for benzaldehyde and remarkable substrate inhibition. 

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the a-amino acid counterpart of the a-keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Flormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phos-phorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the pathway (to be described in Chapter 23), instead... 

The rate of mitochondrial oxidations and ATP synthesis is continually adjusted to the needs of the cell (see reviews by Brand and Murphy 1987 Brown, 1992). Physical activity and the nutritional and endocrine states determine which substrates are oxidized by skeletal muscle. Insulin increases the utilization of glucose by promoting its uptake by muscle and by decreasing the availability of free long-chain fatty acids, and of acetoacetate and 3-hydroxybutyrate formed by fatty acid oxidation in the liver, secondary to decreased lipolysis in adipose tissue. Product inhibition of pyruvate dehydrogenase by NADH and acetyl-CoA formed by fatty acid oxidation decreases glucose oxidation in muscle. 

Figure 30. A medium complexity model of yeast glycolysis [342], The model consists of nine metabolites and nine reactions. The main regulatory step is the phosphofructokinase (PFK), combined with the hexokinase (HK) reaction into a single reaction vi. As in the minimal model, we only consider the inhibition by its substrate ATP, although PFK is known to have several effectors. External glucose (Glc ) and ethanol (EtOH) are assumed to be constant. Additional abbreviations Glucose (Glc), fructose 1,6 biphosphate (FBP), pool of triosephosphates (TP), 1,3 biphosphogly cerate (BPG), and the pool of pyruvate and acetaldehyde (Pyr).

NAM is produced by base-catalysed epimerization of N-acetyl-o-glucosamine (NAG), generating an unfavourable 1 4 mixture of NAM NAG. NAG, although not a substrate for the aldolase, inhibits the reaction. In addition, excess pyruvate is required to push the equilibrium in favour of product formation (Scheme 1.31). Although 90% yields can be obtained at laboratory scale using E. coli NANA aldolase using a NAG NAM mixture, the NANA product is difficult to separate from the excess pyruvate required to achieve this. 

Pyruvate kinase, the activity of which is inhibited by ATP and phosphocreatine but is activated by ADP, which is its substrate (Figure 6.16(a)). 

Interconversion processes (see p. 120) also play an important role. They are shown here in detail using the example of the PDH complex (see p. 134). The inactivating protein kinase [la] is inhibited by the substrate pyruvate and is activated by the products acetyl-CoA and NADH+H. The protein phosphatase [Ibj—like isodtrate dehydrogenase [3] and the ODH complex [4j-is activated by Ca. This is particularly important during muscle contraction, when large amounts of ATP are needed. Insulin also activates the PDH complex (through inhibition of phosphorylation) and thereby promotes the breakdown of glucose and its conversion into fatty acids. 

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.

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