Enzymatic cycling

Figure C1.5.17.(A) Enzymatic cycle of cholesterol oxidase, which catalyses tire oxidation of cholesterol by molecular oxygen. The enzyme s naturally fluorescent FAD active site is first reduced by a cholesterol substrate,...

Figure 6. The enzymatic cycle for cytochrome P-450, detailing possible electronic structure of intermediates. (P represents protoporphyrin.)...

In the other subdivision, water activation occurs in the first step of the enzymatic cycle. This activation is achieved by a carboxylate group in aspartic hydrolases (Fig. 3.10), Zn2+ and a carboxy group in metallopep-tidases (Fig. 3.12 ), a histidine side chain in calcium-dependent hydrolases (Fig. 3.14), or a Zn2+ in carbonic anhydrase (Fig. 3.15). The substrate, on the other hand, is polarized (activated) by a carboxy group in aspartic hydrolases or by a cation in metallopeptidases and calcium-dependent hydrolases. In this manner, the reactivity of both the water molecule and the substrate is enhanced and fine-tuned to drive formation of a tetrahedral intermediate that will break down to form the hydrolysis products. 

L. E. Kopp and R. P. Miech, Nonlinear enzymatic cycling systems the exponential cycling system, / Biol. Chem., 247(11), 3558-3563 (1972). 

Hydroperoxo-ferric intermediate, termed also Compound 0, is the immediate precursor of the main catalytic intermediate Compound I in peroxidase enzymatic cycle. Attempts to study this intermediate directly in reactions of hydrogen peroxide with HRP using fast kinetic methods have been inconclusive, possibly because it is not accumulated in sufficient concentrations.90,91 However, Compound 0 could be prepared and studied by EPR and optical absorption spectroscopy via cryoreduction of... 

Horseradish peroxidase (and urease) played an important role in the development of the modern concept of the nature of an enzyme and the role of metal ions (Sumner and Somers, 1943 Willstatter, 1965). The species now known as compound II (HRP-II) formed as a result of the reaction of HRP with H202, was discovered in 1937 (Keilen and Mann, 1937). Later compound I (HRP-I), formed prior to HRP-II was identified (Theorell, 1941). The spectra of HRP-I and HRP-II in the 400 nm (Soret band) region have been determined (Chance, 1949 a, b) and measurements have also been extended to the visible region (Chance, 1952). Formation of HRP-I is first order in H202 and HRP (Chance, 1943) and the -OOH group is essential for the oxidation of HRP by peroxide. The enzymatic cycle can be summarised by the following equations (George, 1952),... 

The hydridorhodium(III) species HRh(NH3)52+ and HRh(CN)4-(H20)2 are known to react with 02 to give hydroperoxide species, Rh—OOH (102, 103, 104) the sulfoxide oxidation via such an intermediate could be accommodated by several mechanisms akin to those discussed already for olefinic substrates (cf. Reactions 17 and 18). The oxidation also could occur via liberated hydrogen peroxide, which oxidizes sulfoxide to sulfone and can be formed by treatment of Rh—OOH species with acid (104). Such a mechanism would amount to a novel catalytic conversion of H2/02 mixtures to in situ H202. The part of the enzymatic cycle of P 450 that utilizes 02 and two electrons (from a hydride source) can be bypassed by using hydrogen peroxide (16, 18). 

Assay of cyclic nucleotides by enzymatic cycling techniques Protein binding assay Protein kinase assay Radioimmunoassay... 

Only two other assays allow the determination of amounts as small as 0.1 pmol of cyclic GMP per tube the cycling system [141,142] and the radioimmunoassay [84,143]. The assay described above is faster and less laborious than these methods involving enzymatic cycling. 

ASSAY OF CYCLIC NUCLEOTIDES BY ENZYMATIC CYCLING TECHNIQUES... 

Fig. 22.1. (A) Enzymatic cycle of cholesterol oxidase which catalyzes the oxidation of cholesterol by oxygen. The enzyme s naturally fluorescent FAD active site is first reduced by a cholesterol substrate molecule, generating a non-fluorescent FADH2, which is then oxidized by oxygen. (B) Structure of FAD, the active site of cholesterol oxidase. (C) A portion of the fluorescence intensity time trace of a single cholesterol oxidase molecule. Each on-off cycle of emission corresponds to an enzymatic turnover. (D) Distribution of emission on-times derived from (C). The solid line is the convolution of two exponential functions with rate constants fci[S] = 2.5 s and fc2 = 15.3 s, reflecting the existence of an intermediate, ES, the enzyme-substrate complex, as shown in the kinetic scheme in the inset. From ref. [15]...

Fig 19. The NIR MCD spectra at room temperature of (a) oxidized and (b) semire-duced diheme cytochrome c peroxidase Pseudomorms aeruginosa). The assignments of the various features of the spectra are indicated in terms of a simple model of heme ligation, (c) The complete enzymatic cycle established hy MCD and EPR spectroscopy in terms of the oxidation levels of the two hemes. 

We have extended our studies on Ni complexes [17] in order to ascertain if Ni itself is able to catalyze the formation of a thioester, reproducing it alone the full enzymatic cycle that uses CO2 for the formation of the thioester group. 

Eyer, P. and Podhradsky, D. (1986) Evaluation of the micromethod for determination of glutathione using enzymatic cycling and Ellman s reagent. Anal. Biochem. 153, 57-66. 

Kishi K, Ochiai K, Ohta Y, Uemura Y, Kanatani K, Nakajima K, Nakamura M. Highly sensitive cholesterol assay with enzymatic cycling applied to measurement of remnant lipoprotein-cholesterol in serum. Clin Chem 2002 48 737-41. 

Yamaguchi F, Etoh T, Takahashi M, Misaki H, Sakuraba H, Ohshiraa T. A new enzymatic cycling method for ammonia assay using NAD synthetase. Clin Chim Acta 2005 352 165-73. 

The enzymatic cycling systems are characterized by a combination of enzymes (mostly oxidases and dehydrogenases) that are capable of a multiple regeneration of a substrate. Here, a repeated regeneration of the substrate causes an accumulation of heat and amplifies the signals. Enzymatic cycling systems have an important requisite, namely the analyte concentration has to be well below its Michaelis-Menten-value KM. Otherwise, the reaction speed is not proportional to the analyte concentration (Bergmeyer, 1983). 

Determination of low metabolic concentrations with enzymatic cycling In some cases where small amounts of substrate have... 

Sakakibara T, S.Murakami, Eisaki N, Nakajima M, Imai K. An enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase for the simultaneous determination of ATP and AMP(RNA). Anal Biochem 1999 268 94-101. 

Jansson V, Jansson K. An enzymatic cycling assay for AMP using adenylate kinase, nucleoside-diphosphate kinase and firefly luciferase. Anal Biochem. 2003 321 263-5. 

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