Cyanides, enzyme activation

Ibrahim MZ, Briscoe PB, Bayliss OB, et al. 1963. The relationship between enzyme activity and neuroglia in the prodromal and demyelinating stages of cyanide encephalopathy in the rat. J Neurol Neurosurg Psychiatry 26 479-486. 

The enzyme activity was assayed by measuring the production of optically active mandelonitrile synthesized from benzaldehyde and cyanide. The standard assay solution contained 300 gmo citrate buffer (pH 3.5-6.0), 50 /rmol of benzaldehyde, 100 /rmol potassium cyanide and 100 jA of the enzyme in a final volume of 1.0 mL. The reaction was started by an addition of 100 fx of the enzyme solution and incubated at 25 °C for 1-120 min. Aliquots (100 jiY) were withdrawn at various reaction times and the reaction was stopped by the addition of 0.9 mL of organic solvent (9 1 hexane iso-propanol by volume). The mandelonitrile formed was extracted and the supernatant, obtained by centrifugation (15,000 x g, 1.0 min at 4 °C), was assayed by HPLC. A blank reaction was also performed without enzyme and the amount of mandelonitrile obtained was deducted from the biocatalyzed reaction product. One unit of the enzyme is defined as the amount of the enzyme that produces 1 /imol of (R)-mandelonitrile under reaction conditions in 1 min. 

Xanthine oxidase (XO) was the first enzyme studied from the family of enzymes now known as the molybdenum hydroxylases (HiUe 1999). XO, which catalyzes the hydroxylation of xanthine to uric acid is abundant in cow s milk and contains several cofactors, including FAD, two Fe-S centers, and a molybdenum cofactor, all of which are required for activity (Massey and Harris 1997). Purified XO has been shown to use xanthine, hypoxan-thine, and several aldehydes as substrates in the reduction of methylene blue (Booth 1938), used as an electron acceptor. Early studies also noted that cyanide was inhibitory but could only inactivate XO during preincubation, not during the reaction with xanthine (Dixon 1927). The target of cyanide inactivation was identified to be a labile sulfur atom, termed the cyanolyzable sulfur (Wahl and Rajagopalan 1982), which is also required for enzyme activity. 

An alternative approach, adopted by Albery et al. [59-61], is to determine the mechanism giving rise to the sensor response and to use this information together with the measured data at short times to calculate the final response. This was used for an electrochemical sensor system incorporating cytochrome oxidase where the steady-state responses of the measurement system were insufficiently fast for useful measurement of respiratory inhibitors such as cyanide, hydrogen sulphide, etc. By using mechanistic information, it was possible to successfully calculate the concentration in a test sample by real-time analysis of the sensor signals at short times after exposure to the test sample. The analysis could cope with the gradual loss of enzyme activity commonly found in these biosensor devices. 

Cobalt has recently been used as an ESR active substitute in zinc metalloenzymes. Whilst liquid helium temperatures may be needed and theoretical aspects of the spectra are not yet as well understood, cobalt has two important advantages over copper as a metal substitute, namely that many cobalt derivatives show some enzymic activity (e.g. cobalt in carbonic anhydrase, alkaline phosphatase and superoxide dismutase) and that g values and hyperfine splitting are more sensitive to ligand environment, particularly when low spin. ESR data have been reported for cobalt substituted thermolysin, carboxypeptidase A, procarboxypeptidase A and alkaline phosphatase [51]. These are all high spin complexes. Cobalt carbonic anhydrase has been prepared and reacted with cyanide [52]. In... 

The enzyme preparation from Aerobacier aerogenes was found by Hogness and Battley to be inactivated by oxygen, iodoacetamide, p-(chloromercuri)-benzoate, and A -ethylmaleimide. After inactivation with oxygen, activity could be recovered on treatment with mercaptoacetate, cysteine, sodium sulfide, sodium cyanide, or 2-mercaptoethanol." The authors, however, have not drawn any conclusion as to whether these observations should be taken to indicate that sulfhydryl groups are involved in the enzyme activity. 

Ethylene production in plants is driven by two enzyme activities, 1-aminocyelopropane-l-carboxylate synthase (ACC synthase EC 4.4.1.14) that produces 1-aminocyclopropane acid from 5 -adenosylmethionine followed by aminocyclopropanecarboxylate oxidase (ACC oxidase EC 1.14.17.4) decomposing 5 -adenosylmethionine to ethylene, cyanide and... 

Some divalent ions, such as Mg > Co and Mn A are activators of the enzyme, and is a constituent metal ion. The correct ratio of Mg VZn ions is necessary to avoid displacement of Mg and to obtain optimal activity. Phosphate, borate, oxalate, and cyanide ions are inhibitors of ALP activity. Variations in and substrate concentrations change the pH optimum. The type of buffer present (except at low concentrations) affects the rate of enzyme activity. Buffers can be classified as inert (carbonate and barbital), inhibiting (glycine and propylamine), or activating (2-amino-2 methyl-l-propanol [AMP], tris (hydroxymethyl) aminomethane [TRIS], and diethanolamine [DBA]). 

The results of enzymatic determinations of ceruloplasmin are often expressed in arbitrary units, and the values judged in the light of a series of results obtained in normal subjects by the same method. Expression of the enzyme activity in milligrams of ceruloplasmin per unit volume of serum is also possible. The relation between oxidase activity and the amount of ceruloplasmin in serum can be determined by measuring in parallel samples of sera both the oxidase activity and the change of optical density at 610 mix before and after the addition of ascorbic acid or cyanide. On the basis of the known absorbancy index, the ceruloplasmin concentration can be calculated (see Section 2.2.1) and the relation between it and the enzyme activity determined. Alternatively, purified human ceruloplasmin can be used for standardization of the enzymatic method. The ceruloplasmin content of the purified preparation can be determined colorimetrically or, in the case of a highly purified preparation, by nitrogen analysis. Predetermined increments of ceruloplasmin can then be added to aliquots of a selected serum. It is convenient to select a serum with relatively low ceruloplasmin level to start with. Serum of a patient with Wilson s disease, some of whom have no measurable amount of enzyme activity, would be ideal for the purpose however, Walshe (W5) has recently found an inhibitor in these sera. 

The basic reaction involves transfer of sulfane sulfur from the donor (SCN) to the enzyme, forming a persulhde intermediate. The persulfide sulfur is transferred from the enzyme to a nucleophilic receptor (CN) to yield SCN. For most species, this enzyme activity is high in liver, kidney, brain, muscle, and olfactory mucosa (Himwich and Saunders, 1948 Dahl, 1989 Aminlari et al., 1994). The nasal metabolism of CN may have relevance to the toxicity of inhaled HCN. (3-Mercaptopymvate-cyanide transulfurases are present in blood, liver, and kidney, and catalyze the reaction ... 

Alkaline phosphatase exists in two forms in animal tissues, one activated by added Mg(II) or Mn(II) and not inhibited by F or CN ions, the other independent of added divalent ions but inhibitable by cyanide . Yeast phosphatase, active at neutral pH, is activated by divalent metal ions in the order Mg > Mn > Ni, Co, Fe ". Metal analysis of purified kidney phosphatase reveals a mixture of bound ions Zn, Cu, Mn, Mg, and Fe . This variety of responses illustrates a classical problem in studies of enzyme activation by metal ions defining the species that is of greatest importance in vivo. 

The pH value has an influence not only on the activity of enzymes but also on chemical reactions. The chemical cyanohydrin reaction is base-catalysed, as, compared to HCN, the cyanide ion more easily attacks the carbonyl group. As a result, a distinct decrease of the reaction rate for the non-enzymatic synthesis of mandeloni-trile occurs at lower pH values. Also, the enzyme activity decreases but not to the same extent therefore, the enantioselective enzymatic reaction becomes dominant at lower pH (see Fig. 7-11). 

Acetyl DL-methionine which is used as a substrate for amino acylase activity determination was prepared by acetylation of DL-methionine with acetic anhydride in acetic acid [5]. The rate of enzymatic hydrolysis was determined by measuring the liberated amino acid by ninhydrin method [6] where ascorbic acid was used as oxidizing agent instead of sodium cyanide. The activity curve of pure amino acylase enzyme is shown in Fig. 1 as a continuous line. For determining the effect of metal ions on the activity of amino acylase the following procedure was adopted. 

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