Intermediate states in the catalytic mechanism of lentil copper amine oxidase have been investigated by ESR and optical spectroscopy. Using highly purified apo- and holoenzyme in combination with a poor substrate and a range of inhibitors, under both aerobic and anaerobic conditions, the single steps of the reaction mechanism can be slowed down or 'frozen' completely. In this way, a sequence of six intermediate species in the catalytic cycle has been established. Oxidative deamination of p-(dimethylamino)benzylamine is 5 x 10(5) times slower than for putrescine; the rate-limiting step is shown to be release of the aldehyde product. This process is not affected in the apoenzyme, but subsequent intramolecular electron transfer to form the characteristic free radical intermediate is completely blocked, and the apoenzyme is trapped as an aminoresorcinol species. Classic hydrazine and hydrazide inhibitors bind to the 6-hydroxydopa cofactor in the same way as active substrates, but rearrangements lead to formation of stable intermediate adducts at the step preceding release of aldehyde. The semicarbazide-6-hydroxydopa adduct is shown to bind simultaneously to Cu(II), providing the first direct evidence for localization of 6-hydroxydopa close to the copper site.
Spectrophotometry and rapid-scanning stopped-flow spectroscopy have been used to investigate the visible absorbance changes that occur in the course of the reduction of lentil (Lens esculenta) seedling amine oxidase by substrate. The catalytic cycle of the enzyme employs several intermediates but, owing to kinetic limitations, some of them were not identified in previous studies. In this study we have examined several substrates, either rapidly reacting (e.g. putrescine) or slowly reacting (e.g. gamma-aminobutanoic acid). Two forms of the enzyme, namely the Cu(I)-aminoresorcinol and quinone ketimine derivatives, whose characterization was elusive in previous studies, have been identified and assigned an optical spectrum. Moreover the reduced form of the enzyme is shown to be an equilibrium mixture of two species, the Cu(I)-semiquinolamine radical and Cu(II)-aminoresorcinol; these have been resolved by pH dependence and assigned spectra as well as a second-order rate constant for the reaction with oxygen. Thus the results presented here identify all the catalytic intermediates suggested by the chemical nature of the coenzyme and define their spectroscopic and reactivity properties.
A cationic peroxidase was isolated and characterized from the latex of the perennial Mediterranean plant Euphorbia characias. The purified enzyme contained one heme prosthetic group identified as ferric iron-protoporphyrin IX. In addition, the purified peroxidase contained 1 mol of endogenous calcium per mol of enzyme; removal of this calcium ion resulted in almost complete loss of the enzyme activity. However, when excess Ca(2+) was added to the native enzyme the catalytic efficiency was enhanced by 3 orders of magnitude. The mechanism of activation was studied using a wide range of spectroscopic and analytic techniques. Analysis of the steady state by stopped-flow measurements suggests that the main effect of calcium ions is to favor the oxidation of the ferric enzyme by hydrogen peroxide to form compound I, whereas the other steps of the catalytic cycle seem to be affected to a lesser extent. UV/vis absorption spectra and CD measurements show that the heme iron is pentacoordinated high-spin in native enzyme and remains so after the binding of Ca(2+). Only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ca(2+) ions, except for a significant perturbation of the Fe(3+) inner sphere geometry, as detected by EPR measurements. We propose that Ca(2+) binding to a low affinity site induces a reorientation of the distal histidine changing the almost inactive form of Euphorbia peroxidase to a high activity form. This is the first example of a peroxidase that responds as an on/off switch to variations in the external Ca(2+) level.
Alzheimer's disease is characterized by a tendency to malnutrition, present even in the mild-moderate stages, and a tendency to dehydration that appears in the severe stage. The BIVA technique is a promising tool for the screening and monitoring of nutrition and hydration status in Alzheimer's disease.
Summary
The HIV-1 reverse transcriptase (RT) has two associated activities, DNA polymerase and Ribonuclease H (RNase H), both essential for viral replication and validated drug targets. While all RT inhibitors approved for therapy target the DNA polymerase activity, the search of new RT inhibitors which target the RNase H function and are possibly active on RTs resistant to the known non-nucleoside inhibitors (NNRTI) is a viable approach for anti-HIV drug development. In this report several alizarine derivatives were synthesized and tested on both HIV-1 RT-associated activities. Alizarine analogues K-49 and KNA-53 showed IC50 values for both RT-associated functions around 10 μM. When tested on the K103N RT both derivatives equally inhibited the RT-associated functions, while when tested on the Y181C RT, only KNA-53 inhibited the RNase H function but was inactive on the polymerase function. Mechanism of action studies showed that these derivatives do not intercalate into DNA and do not chelate the divalent cofactor Mg2+. Kinetic studies demonstrated that they are non-competitive inhibitors, they do not bind to the RNase H active site or to the classical NNRTI binding pocket, even though efavirenz binding negatively influenced K-49/KNA-53 binding and vice versa. This behavior suggested that the alizarine derivatives binding site could be close to the NNRTI binding pocket. Docking experiments and molecular dynamic simulation confirmed the experimental data and the ability of these compounds to occupy a binding pocket close to the NNRTI site.
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