Enzymes cycle

A minimal mechanism for Na, K -ATPase postulates that the enzyme cycles between two principal conformations, denoted Ej and Eg (Figure 10.11). El has a high affinity for Na and ATP and is rapidly phosphorylated in the presence of Mg to form Ei-P, a state which contains three oeeluded Na ions (occluded in the sense that they are tightly bound and not easily dissociated from the enzyme in this conformation). A conformation change yields Eg-P, a form of the enzyme with relatively low affinity for Na, but a high affinity for K. This state presumably releases 3 Na ions and binds 2 ions on the outside of the cell. Dephosphorylation leaves EgKg, a form of the enzyme with two... 

P-site ligands inhibit adenylyl cyclases by a noncompetitive, dead-end- (post-transition-state) mechanism (cf. Fig. 6). Typically this is observed when reactions are conducted with Mn2+ or Mg2+ on forskolin- or hormone-activated adenylyl cyclases. However, under- some circumstances, uncompetitive inhibition has been noted. This is typically observed with enzyme that has been stably activated with GTPyS, with Mg2+ as cation. That this is the mechanism of P-site inhibition was most clearly demonstrated with expressed chimeric adenylyl cyclase studied by the reverse reaction. Under these conditions, inhibition by 2 -d-3 -AMP was competitive with cAMP. That is, the P-site is not a site per se, but rather an enzyme configuration and these ligands bind to the post-transition-state configuration from which product has left, but before the enzyme cycles to accept new substrate. Consequently, as post-transition-state inhibitors, P-site ligands are remarkably potent and specific inhibitors of adenylyl cyclases and have been used in many studies of tissue and cell function to suppress cAMP formation. 

Very recently, a sandwich assay for prostatic acid phosphatase antigen was carried out using two cascaded enzyme reactions to provide amplification of the immunochemical event. In one format, an optical readout was used whereby a forma-zan dye was generated by reaction of a dye precursor and NADH generated from the second enzyme cycle. In the electrochemical format, the NADH generated in the second enzyme cycle was used to reduce Fe(CN) to FeCCN) " which was then detected amperometrically. While the use of Fe(CN) in ECIA has appeared in the... 

Catalases catalyze the conversion of hydrogen peroxide to dioxygen and water. Two families of catalases are known, one having a heme cofactor and the second a structurally distinct family, found in thermophilic and lactic acid bacteria. The manganese enzymes contain a binuclear active site and the functional form of the enzyme cycles between the (Mn )2 and the (Mn )2 oxidation states. When isolated, the enzyme is in a mixture of oxidation states including the Mn /Mn superoxidized state and this form of the enzyme has been extensively studied using XAS, UV-visible, EPR, and ESEEM spectroscopies. Multifrequency EPR and microwave polarization studies of the (Mn )2 catalytically active enzyme from L. plantarum have also been reported. ... 

Peroxidases are haem proteins that are activated from the ferric state to one-electron oxidants by H202. They play a significant role in the generation of radicals from xenobiotics. The compound I state contains one oxidising equivalent as an oxoferryl-haem entity and the second as a porphyrin -radical cation. Upon the oxidation of a substrate the porphyrin radical is repaired, giving the compound II. Reduction of the oxoferryl haem back to the ferric state by a second substrate molecule completes the enzyme cycle. In addition to the classical peroxidases, several other haem proteins display pseudo-peroxidase activity. The plant enzyme horseradish peroxidase (HRP) is often employed in model systems. 

Various aspects of the (Ca2+, Mg2+)-ATPase have been reviewed.126 133 It can be isolated in vesicular form from homogenized skeletal muscle. Since it is the major protein component of SR membranes, it can also be isolated fairly easily in relatively pure form. The enzyme has a molecular weight in the range 100 000-120 000, and forms oligomers in the SR membranes, probably tetramers.133 Each subunit is associated with about 30 molecules of phospholipid, which are suggested to form a shell or annulus around the protein. The monomeric subunit has ATPase activity, and remains monomeric throughout the enzyme cycle,136 although the behaviour of the monomer is dependent on the solubilization procedure. 37... 

There is much evidence, usually based upon ESR studies with spin labels, for conformational changes in the protein during the enzyme cycle. Enzyme activity shows a discontinuity in the Arrhenius plot, once attributed to phase transitions in the membrane phospholipid, but which is similar to discontinuities in the Arrhenius plot for rotational mobility and which have been rationalized in terms of a conformational change in the ATPase.142... 

Figure 3 Speculative model for the hydrogenase enzyme cycle such as that from D. gigas. The highest oxidation states of the enzyme are at the top, and each step down corresponds to a one-electron reduction. Some hydrons that are transferred to sites in the protein are not shown. Redox states of the iron-sulfur clusters are omitted.

Here, +i[S] serves as the apparent mass-action rate constant for the conversion E — ES. Each time an enzyme cycles from state E to ES and back to E again, one molecule of S is converted to P. If the rate of turnover of the catalytic cycle is significantly greater than the rate of change of reactant (S and P) concentrations, then the apparent mass-action constant +i[S] in Equation (4.2) remains effectively constant over the timescale of the catalytic cycle. This is true, for example, when the enzyme concentration is small compared to reactant concentrations, many catalytic cycles are required to produce a significant change in reactant concentrations. 

Fig. 1. A schematic representation of the twin-site ELISA for fos and myc proteins. The signal from the alkaline phosphatase label is amplified via the AMPAK enzyme cycle to generate the red formazan dye.

Two molecules of the active intermediate of omeprazole bind to one active site of gastric H /K -ATPase [63, 64], This binding is a disulphide linkage and can be prevented and reversed by the addition of mercaptan [65-67]. Detailed investigations of three reactions of H /K -ATPase enzyme cycle have shown that the K -stimulated ATPase-activity, / -nitrophenol-phosphatase(pNPPase)-activity and formation of phosphoenzyme are also inhibited [63, 68]... 

C (66). If electron transfer from type 1 to type 3 copper couples the two halves of the enzyme cycle, as proposed for laccase, then this intramolecular redox reaction must be extremely rapid to account for the effects of trace dioxygen on the reduction of the type 1 copper. Consequently, despite the fact that an ambiguous assignment of a type 1 to type 3 transfer is not possible in this example, facile intramolecular electron transfer processes probably ensure a rapid distribution of electrons among the type 1 and type 3 copper centers, at least in some of the enzyme molecules. The equilibrium distribution, and quite conceivably the relative rates of approach to this state, should be influenced by the oxidation-reduction potentials, which, as described earlier in this chapter (Figure 5), favor electron occupancy of the type 3 copper pairs at 10.0°C. 

Since the catalytic core of MerA contains four active site thiols, there are four possible redox states of the enzyme (Scheme 17) EH4, in which both disulfides and the flavin are reduced EH2, in which both disulfides are reduced but the flavin is oxidized E x, where the C-terminal disulfide is reduced and the flavin is oxidized and nonactivated E x, which has two active site disulfides and an oxidized flavin. Nonactivated E x is not active in assays, but can be converted to an active form of the enzyme by incubation with NADPH.Titrations of MerA show that four electrons are required to reach the EH2 state of the enzyme. EH2 can bind Hg, but the reaction is not completed until another molecule of NADPH is consumed, showing that the enzyme cycles between EH2 and EH4 during turnover. 

The XANES of the Mn catalase provided the first definitive proof that this enzyme cycles between the MnJ and Mn n oxidation levels (137, 352) The extent of catalase activity correlated with the proportion of MnJ1 or Mn enzyme however, samples with Mnlv quantitatively showed reduced activity. The EXAFS of the Mn catalases have been less informative because of the Mn-Mn separations in the reduced, active enzymes (135). Nevertheless, EXAFS of the superoxi-dized enzyme demonstrated that the Mn,uMnlv enzyme has a Mn-Mn separation of —2.7 A, which is consistent with a di-yu.2-oxo core (135). Subsequent spectroscopic analysis confirmed that a diamond core with a bridging syn,syn acetate formed the enzyme active site (9). 

Golgi apparatus <7> (<7> enzymes cycles between trans-Golgi network and late endosomes, facing the cytosol [20]) [20] cytoplasm <1, 6> [3] cytosol <7> [10]... 

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