Rate-limiting enzyme reaction

D. Elimination involves a rate-limiting enzymic reaction operating at its maximal velocity (Vm). 

Phiosphoffuctokinases (PFK-1 and PFK-2) PFK-1 is the rate-limiting enzyme and main control point in glycolysis. In this reaction, fructose 6-phosphate is phosphorylated to fructose 1,6-bisphosphate using ATP. 

The anticholesterolemic action of simvastatin is based on its effectiveness as a competitive inhibitor of the rate-limiting enzyme in cholesterol biosynthesis. The reaction product normally produced by this enzyme is... 

Figure 1.1 illustrates a condensed version of the classical pathway of bile-acid synthesis, a series of 12 enzymatic reactions that convert cholesterol, which is insoluble, into BAs, which are water soluble. The cholesterol is first converted to 7 alpha-hydroxy cholesterol, followed by the series of enzymatic transformations, eventually producing cholic and chenodeoxycholic acids (not all steps shown). The rate-limiting enzyme in this pathway is cholesterol 7 alpha-hydroxylase (CYP 7A1), which originates from microsomal cytochrome P-450 enzymes, expressed only in the liver hepatocytes. 

The best-known exception to exponential kinetics is the elimination of alcohol (ethanol), which obeys a linear time course (zero-order kinetics), at least at blood concentrations > 0.02 %. It does so because the rate-limiting enzyme, alcohol dehydrogenase, achieves half-saturation at very low substrate concentrations, i.e at about 80 mg/L (0.008 %). Thus, reaction velocity reaches a plateau at blood ethanol concentrations of about 0.02 %, and the amount of drug eliminated per unit of time remains constant at concentrations above this level. 

Achromobacter xylosoxydans has been used to cany out the selective hydroxylation in high yield, using the enzyme which catalyses the first step in nicotinic acid degradation. The whole-cell biotransformation process has been scaled-up to 12 m, which is sufficient to produce high purity 6-hydroxynicotinic acid for the subsequent chemical reactions. The hydroxylation is oxygen requiring, so that oxygen transfer rate-limits the reaction. 

One of the many important consequences of these summation theorems is that, in the case where all flux control coefficients are positive, all coefficients have values between 0 and 1. In this case, the reaction for which C,1 is greatest represents the reaction to which the flux Jj is most sensitive. In the limit that one flux control coefficient has a value close to 1 and all others have values close to 0, we can say that there exists a rate-limiting step a change in activity of the rate-limiting enzyme would be expected to elicit a proportional change in the flux Jj. 

Activity assays of enzymes bound to solid phases in EIA systems have previously been limited to fixed-time spectrophotometric methods following incubation of substrate and solid phase for extended periods of time. Kinetic assays of enzyme activity have not been used to date because of the difficulty in directly monitoring initial rates of enzyme reactions in a turbid solid phase suspension. With urease as the label, an ammonia gas sensing electrode can be used to directly quantitate the amount of urease-labeled antigen or hapten bound to a double-antibody solid phase by continuously measuring the initial rate of ammonia produced from urea as a substrate. 

The first step in heme formation is the rate-limiting condensation reaction between succinyl-CoA and glycine to form 5-aminolevulinic acid (ALA). This reaction is catalyzed by a mitochondrial matrix enzyme, ALA synthase (ALAS). 

Aside from the inordinately dominant light of molecular genetics, the new wave in biochemistry today is, what has come to be called, metabolic control analysis (MCA) (Comish-Bowden and Cardenas, 1990). The impetus behind this wave is the desire to achieve a holistic view of the control of metabolic systems, with emphasis on the notion of system. The classical, singular focus on individual, feedback-modulated (e.g., allosteric), rate-limiting enzymes entails a naive and myopic view of metabolic regulation. It has become increasingly evident that control of metabolic pathways is distributive, rather than localized to one reaction. MCA places a given enzyme reaction into the kinetic context of the network of substrate-product connections, effector relationships, etc., as supposedly exist in situ, it shows that control of fluxes, metabolite concentrations, inter alia, is a systemic function and not an inherent property of individual enzymes. Such... 

The frequency with which two reactive species encounter one another in solution represents an upper bound on the bimolecular reaction rate. When this encounter frequency is rate limiting, the reaction is said to be diffusion controlled. Diffusion controlled reactions play an important role in a number of areas of chemistry, including nucleation, polymer and colloid growth, ionic and free radical reactions, DNA recognition and binding, and enzyme catalysis. 

The major advantages unique to cryoenzymology stem from the potential to accumulate essentially all of the enzyme in the form of a particular intermediate. The large rate reductions allow the most specific substrates to be used and hence provide the most accurate model for the in vivo catalyzed reactions. Virtually all the standard chemical and biophysical techniques used in studying proteins and enzymes under normal conditions may be used at subzero temperatures. The main limitations of the technique are the necessity to use aqueous organic cryosolvent systems to prevent the inherent rate-limiting enzyme-substrate diffusion of frozen solutions, and the possibility that the potential-energy surface for the reaction may be such that conditions in which an intermediate accumulates cannot be attained. 

Bremer and Gloor [40] concluded that enzymes for both reactions were present in hepatic microsomes, but recent studies with microsomes of rat [40-42] and human liver [43] have confirmed the presence of only one enzyme, CoA ligase. The assay system, essentially that for long-chain acyl-CoA ligase [42,43], includes 50 mM NaF, a phosphate buffer (pH 7.5), the enzyme preparation, and constituents of Eqn. 1. Product formation was linear up to 12 min with added protein (between 0.1 and 1.2 mg) from a crude microsomal fraction. Sterol carrier protein [44], cysteine or nicotinamide [38,40] were without effect. This rate-limiting enzyme in the two-step sequence catalyzing conjugation of bile acids exhibits a diurnal variation such that the time of maximum enzyme activity coincides with predicted maximum activity of cholesterol 7a-hydroxylase and the time of maximal biosynthesis of bile acids [45]. The enzyme has not been purified. 

In any particular metabolic pathway in a steady state, the reaction carried out by the rate-limiting enzyme is slower than the nonrate-limiting enzyme reactions in that pathway. 

Xanthine oxidase and xanthine dehydrogenase represent different forms of the same gene product. Xanthine dehydrogenase and xanthine oxidase are interconvertable thus, these two enzyme forms and their reactions often are referred to as xanthine oxidoreduotase. Xanthine oxidase is the rate-limiting enzyme in purine catabolism of hypoxanthine to uric acid via xanthine. Both xanthine oxidase and xanthine dehydrogenase play important roles in the ... 

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