Intermediates accumulation

Leppik R A (1989) Steroid catechol degradation disecoandrostane intermediates accumulated by Pseudomonas transposon mutant strains. J Gen Microbiol 135 1979-1988. 

The work by Scott and Lee 165) on the isolation of a crude enzyme system from a callus tissue culture of C. roseus was followed by studies of Zenk et al. on an enzyme preparation from a cell suspension system which produced indole alkaloids 166). The cell-free preparation was incubated with tryptamine and secologanin (34) in the presence of NADPH to afford ajmalicine (39), 19-epiajmalicine (92), and tetrahydroalstonine (55) in the ratio 1 2 0.5. No geissoschizine (35) was detected. In the absence of NADPH, an intermediate accumulated which could be reduced with a crude homogenate of C. roseus cells in the presence of NADPH to ajmalicine (39). Thus, the reaction for the formation of ajmalicine is critically dependent on the availability of a reduced pyridine nucleotide. 

A kinetic and computational study of the hydrolysis of a-R-a-SMe-methylene Meldrum s acid (R = H, Me, Et, r-Bu, /-Bu) has been published <2006JOC4795> and confirmed that crowding at the transition state is an important factor on the rate of nucleophilic attack no intermediate accumulated to detectable level. 

Respiration Studies in Isolated Mitochondria Cellular respiration can be studied in isolated mitochondria by measuring oxygen consumption under different conditions. If 0.01 m sodium malonate is added to actively respiring mitochondria that are using pyruvate as fuel source, respiration soon stops and a metabolic intermediate accumulates. 

T Given that many amino acids are either neurotransmitters or precursors or antagonists of neutrotransmitters, genetic defects of amino acid metabolism can cause defective neural development and mental retardation. In most such diseases specific intermediates accumulate. For example, a genetic defect in phenylalanine hydroxylase, the first enzyme in the catabolic pathway for phenylalanine (Fig. 18-23), is responsible for the disease phenylketonuria (PKU), the most common cause of elevated levels of phenylalanine (hyperphenylalaninemia). 

As we discussed in Chapter 3, the KM for an enzymatic reaction is not always equal to the dissociation constant of the enzyme-substrate complex, but may be lower or higher depending on whether or not intermediates accumulate or Briggs-Haldane kinetics hold. Enzyme-substrate dissociation constants cannot be derived from steady state kinetics unless mechanistic assumptions are made or there is corroborative evidence. Pre-steady state kinetics are more powerful, since the chemical steps may often be separated from those for binding. 

Neither the occurrence of a constant value of Vmax or a constant product ratio is sufficient proof of the presence of an intermediate. It was seen for alkaline phosphatase that a constant value for Vmax is an artifact, and also that there is no a priori reason why the attack of acceptors on a Michaelis complex should not also give constant product ratios. In order for partitioning experiments to provide a satisfactory proof of the presence of an intermediate, they must be linked with rate measurements. When the rate measurements are restricted to steady state kinetics, the most favorable situation is when the intermediate accumulates. If the kinetics of equations 7.5 to 7.7 hold, it may be concluded beyond a reasonable doubt that an intermediate occurs. The ideal situation is a combination of partitioning experiments with pre-steady state studies, as described for chymotrypsin and amides. 

The calculation of rate constants from steady state kinetics and the determination of binding stoichiometries requires a knowledge of the concentration of active sites in the enzyme. It is not sufficient to calculate this specific concentration value from the relative molecular mass of the protein and its concentration, since isolated enzymes are not always 100% pure. This problem has been overcome by the introduction of the technique of active-site titration, a combination of steady state and pre-steady state kinetics whereby the concentration of active enzyme is related to an initial burst of product formation. This type of situation occurs when an enzyme-bound intermediate accumulates during the reaction. The first mole of substrate rapidly reacts with the enzyme to form stoichiometric amounts of the enzyme-bound intermediate and product, but then the subsequent reaction is slow since it depends on the slow breakdown of the intermediate to release free enzyme. 

The theory predicts that unless there is a change of rate-determining step with pH, the pH dependence of kcJKM for all non-ionizing substrates should give the same pKa that for the free enzyme. With one exception, this is found (Table 5.2). At 25°C and ionic strength 0.1 M, the pKa of the active site is 6.80 0.03. The most accurate data available fit very precisely the theoretical ionization curves between pH 5 and 8, after allowance has been made for the fraction of the enzyme in the inactive conformation. The relationship holds for amides with which no intermediate accumulates and the Michaelis-Menten mechanism holds, and also for esters with which the acylenzyme accumulates. 

The accumulation problem is most severe for enzymes such as the digestive enzymes, which have to cope with pulses of high substrate concentrations. If the concentration of the substrate is below the KM for the reaction under physiological concentrations, no intermediate accumulates in vivo in any case, since the enzyme is unbound. But in a test tube experiment in which the experimenter can use artificially high concentrations of substrate, an intermediate can sometimes be made to accumulate. An example occurs with glyceraldehyde 3-phosphate dehydrogenase. As Table 12.4 shows, the concentration of the aldehyde is below the Km in vivo. But in the laboratory, the acylenzyme accumulates at saturating substrate concentrations. 

The equations so far apply to a simple two-state equilibrium of D N. That is, protein denaturation is a cooperative (or all-or-none) transition in which denatu-ration occurs in a single step without any intermediates accumulating. How can we tell that this is so ... 

The classic example of competitive inhibition is inhibition of succinate dehydrogenase, an enzyme, by the compound malonate. Hans Krebs first elucidated the details of the citric acid cycle by adding malonate to minced pigeon muscle tissue and observing which intermediates accumulated after incubation of the mixture with various substrates. The structure of malonate is very similar to that of succinate (see Figure 1). The enzyme will bind malonate but cannot act further on it. That is, the enzyme and inhibitor form a nonproductive complex. We call this competitive inhibition, as succinate and malonate appear to compete for the same site on the enzyme. With competitive inhibition, the percent of inhibition is a function of the ratio between inhibitor and substrate, not the absolute concentration of inhibitor. 

It has been known that cyclohexanones are formed as intermediates in the hydrogenation of phenols.83,88,123 124 However, the amounts of the ketone intermediates accumulated during the hydrogenation depend greatly on the nature of catalysts and reaction conditions, as well as on the structure of phenols. 

Conclusively establishing the role of potential intermediates in a biosynthetic pathway is a difficult aspect of biosynthesis. Typically, intermediates accumulate because subsequent enzymatic reactions are slow. Organisms also produce shunt metabolites that are off the main pathway and may not be further metabolized these will also accumulate. Isolation of an intermediate does not, therefore, establish intermediacy. Trapping experiments are sometimes used to overcome these problems. In the pathway A B C, where A is a known precursor of C, labeled A and non-labeled B are fed at the same time. The latter is metabolized to C and labeled B is produced from A Bis then temporarily available for isolation. An alternative approach for microbial metabolites is to mutate the organism or add specific enzyme inhibitors. This may allow intermediates to accumulate. Incorporation of a labeled, potential intermediate into a product does not prove that the intermediate lies on the main biosynthetic pathway. It may simply serve as a substrate for the enzymes involved. Only when each of the enzymes in a pathway has been isolated and characterized, and the substrate specificity determined, can the intermediates in a biosynthetic route be characterized. 

After alcohol ingestion, which of the. following intermediates accumulates in liver that is not typical of glycolysis or the citric acid cycle ... 

Although not a subject of this chapter, Toney and coworkers have quantitated the reaction coordinate of a PLP-dependent L-alanrne racemase [15]. Despite the expectation that the cofactor provides resonance stabilization of the carbanion/enolate anion (quinonoid) intermediate derived by abstraction of the a-proton, the spectroscopic and kinetic analyses for the wild type racemase at steady-state provided no evidence for the intermediate in the reaction catalyzed by the wild type enzyme. Indeed, Toney had previously demonstrated that a kinetically competent quinonoid intermediate accumulates in the impaired R219E mutant [16] Arg 219 is hydrogen-bonded to the pyridine nitrogen of the cofactor. For the wild type racemase, the derived transition state energies for conversion of the bound enantiomers of alanine,... 

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