Mechanism Of Action In Processes Research Articles

The bioremediator glycerophosphodiesterase employs a non-processive mechanism for hydrolysis

Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase that catalyzes the breakdown of a broad range of phosphate ester substrates, and it is of interest for its potential application in the destruction of organophosphate nerve agents and pesticides. The reaction mechanism of GpdQ has been proposed to involve a nucleophilic attack by a terminally bound hydroxide molecule. The hydroxide species bridging the two metal ions is suggested to activate the nucleophile, thus favoring a sequential rather than a processive mechanism of action. Here, the hydrolysis of the two ester bonds in the substrate bis(para-nitrophenyl) phosphate (bpNPP) is probed using (31)P NMR. The kinetic rates measured compare well with those determined spectrophotometrically. Furthermore, the data indicate that the diester bonds are cleaved in two separate (non-processive) reactions, indicating that only a single nucleophile (the terminal hydroxide molecule) is likely to be employed as a nucleophile for GpdQ.

Xeroderma Pigmentosum Complementation Group A Protein Acts as a Processivity Factor

We have previously shown that endonucleases present in a protein complex, which has specificity for cyclobutane pyrimidine dimers, locate sites of damage in DNA by a processive mechanism of action in normal human lymphoblastoid cells. In contrast, the endonucleases present in this complex from xeroderma pigmentosum complementation group A (XPA) cells locate damage sites by a distributive or significantly less processive mechanism. Since the XPA protein has been shown to be responsible for the DNA repair defect in XPA cells, this protein was examined for involvement in the mechanism of target site location of these endonucleases. A recombinant XPA protein, produced by expression of the normal XPA cDNA in E. coli, was isolated and purified. The results show that the recombinant XPA protein was able to correct the defect in ability of the XPA endonucleases to act by a processive mechanism of action on UVC irradiated DNA. These studies indicate that the XPA protein, in addition to a role in damage recognition or damage verification, may function as a processivity factor.

A processive versus a distributive mechanism of action correlates with differences in ability of normal and xeroderma pigmentosum group A endonucleases to incise damaged nucleosomal DNA

A DNA endonuclease, isolated from the nuclei of normal human and xeroderma pigmentosum complementation group A (XPA) cells, which recognizes predominately pyrimidine dimers, was examined for the mechanism by which it locates sites of damage on UVC-irradiated DNA. In reaction mixtures with low ionic strengths (i.e. lacking KCl), the normal and XPA endonuclease locate sites of UV damage on both naked and reconstituted nucleosomal DNA by different mechanisms. On both of these substrates, the normal endonuclease acts by a processive mechanism, meaning that it binds non-specifically to DNA and scans the DNA for sites of damage, whereas the XPA endonuclease acts by a distributive one, meaning that it randomly locates sites of damage on DNA. However, while both the normal and XPA endonucleases can incise UVC irradiated naked DNA, they differ in ability to incise damaged nucleosomal DNA. The normal endonuclease showed increased activity on UVC treated nucleosomal DNA compared with naked DNA, whereas the XPA endonuclease showed decreased activity on the damaged nucleosomal substrate. Since a processive mechanism of action is sensitive to the ionic strength of the micro-environment, the KCl concentration of the reaction was increased. At 70 mM KCI, the normal endonuclease switched to a distributive mechanism of action and its ability to incise damaged nucleosomal DNA also decreased. These studies show that there is a correlation between the ability of these endonucleases to act by a processive mechanism and their ability to incise damaged nucleosomal DNA; the normal endonuclease, which acts processively, can incise damaged nucleosomal DNA, whereas the XPA endonuclease, which acts distributively, is defective in ability to incise this substrate.