Cofactor dissociation

PDF as a zinc-dependent enzyme is attributed to the aerobic instability of Fe, with the oxidation of Fe Fe leading to cofactor dissociation and replacement by Zn " ". Interestingly, PDF from Borrelia burgdorferi, an Fe -limited organism, has been shown to be a zinc-dependent enzyme.This reiterates the fact that enzymes from different organisms can have different preferences for metal ion cofactors, and may allow organisms to adapt to their specific environments. 

A rapid activity loss (a few days) was observed with whole cells. Immobilisation increased the stability and continuous production of L-phenylalanine was possible using alginate bead immobilised cells of P. fluorescerts for 60 days. However, to achieve this the cofactor pyridoxalphosphate had to be continuously added to the beads to correct for the dissociation of the cofactor and loss from the cells. 

Cofactors serve functions similar to those of prosthetic groups but bind in a transient, dissociable manner either to the enzyme or to a substrate such as ATP. Unlike the stably associated prosthetic groups, cofactors therefore must be present in the medium surrounding the enzyme for catalysis to occur. The most common cofactors also are metal ions. Enzymes that require a metal ion cofactor are termed metal-activated enzymes to distinguish them from the metalloenzymes for which metal ions serve as prosthetic groups. 

It has been shown that the activity of NO synthases is regulated by cofactors calcium binding protein calmodulin and tetrahydrobiopterin (H4B). Abu-Soud et al. [149] have studied the effect of H4B on the activity of neuronal nNOS I, using the isolated heme-containing oxygenase domain nNOSoxy. It was found that nNOSoxy rapidly formed an oxygenated complex in the reaction with dioxygen, which dissociated to produce superoxide (Reaction (6)) ... 

The reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F) has been analyzed extensively14 26-30 and a kinetic scheme for E. Coli DHFR was proposed in which the steady-state kinetic parameters as well as the full time course kinetics under a variety of substrate concentrations and pHs were determined. From these studies, the pKa of Asp27 is 6.5 in the ternary complex between the enzyme, the cofactor NADPH and the substrate dihydrofolate. The second observation is that, contrary to earlier results,27 the rate determining step involves dissociation of the product from the enzyme, rather than hydride ion transfer from the cofactor to the substrate. 

As mentioned before, the main problem for the application of such oxidizing enzymes in organic synthesis is the availability of an effective reactivation method for the prosthetic groups or the freely dissociated cofactors. Especially, the long-term stability of the whole system is the limiting factor [25]. All oxidases mentioned previously can be reactivated by an aerobic... 

At the end of the dialysis procedure, the bag is blotted dry and enzyme solution is removed prior to assessment of activity. It may be necessary to include cofactor in the assay mixture if it is possible that a dissociable cofactor was lost from the enzyme during dialysis. As the volume containing enzyme inside the dialysis bag changes to some degree during the dialysis procedure, it is usually necessary to correct measured enzyme activity to reflect this change in volume, and a correction based on protein concentration is often done in this regard. It is normal for activity thus measured to be expressed as a fraction of that in a parallel (dialyzed) control experiment. 

BH4 is essential for the AAHs to carry out their respective catalytic reactions and, at least for PAH, the prereductive activation, which appears to produce dihydrobiopterin quinonoid (g-BH2) directly (20). After the PAH catalytic cycle an oxygen atom is incorporated into the cofactor, providing 4a-OH-BH4 which dissociates from the active site. In order to regenerate the functional tetrahydro form of BH4 pterin carbinolamine dehydratase catalyzes the dehydration of 4-OH-BH4 to g-BH2, which is reduced back to by dihydropteridine reductase (Scheme 2). g-BH2 can also be converted to 7,8-dihydropterin (BH2) which can be regenerated to BH4 by dihydrofolate reductase (DHFR). 

Fig. 9. The most stable optimized structure after dissociation of the second water molecule with a cofactor coordination via the carbonyl oxygen atom 04 (3a) and a less stable structure in which the cofactor coordinates via N5 (3c). Distances are given in angstroms.

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