Study of metal binding site of methyl parathion hydrolase

Abstract

Metal ions are an essential component of numerous proteins. In some cases they have a structural role, but their more important roles are redox proteins and enzymes. Metals interact quite differently with proteins depending upon the function of the protein. In enzymes metals are coordinated to the protein, but maintain vacant coordination sites to allow them to interact with substrates or to activate nucleophiles for attack. In binuclear metalloenzymes, metals have the potential to function in both capacities where they can bind substrate and can also activate water for attack. Most studies of binuclear metalloenzymes focus on the native enzyme how it might interact with the substrate. Clearly, the interaction between protein and substrate depends critically upon the metal centers. This study sets out to study the metal centers, to identify the role of particular residues in binding metals and the effect of perturbing the metal centers on catalytic activity. This study was never intended to make a detailed study of catalytic activity, but rather to provide background information for such studies. It was also hoped that data collected during the course of this study might shed some light on previous mechanistic studies. To achieve the objected outlined above a suitable enzyme was selected for study, which is methyl-parathion hydrolase (MPH). Its active site residues were mutated and the resulting protein variants were subjected to a variety of experiments. The effects of mutations on metal binding affinity, catalytic activity as well as overall protein stability were determined. Very early in the study, it became clear that metals ions had a significant effect on protein stability and that the apo-protein was much less stable that the fully metal bound form. Some mutations had a profound effect on activity yet caused minor changes in stability while other mutants exhibited the opposite effect. Surprisingly, in some cases removing the side-chain of a coordinating group had little effect on the affinity of the protein for the metal or on its catalytic activity. However, it was also clear that two metal ions were essential for activity; little activity was observed with a single metal ion bound to the protein. MPH has evolved to accommodate two metal ions in its active site such that minor alterations to the coordinating residues results in a loss of either stability or activity. These observation suggest that MPH has evolved to optimize activity as well as stability; the latter being important for the long-term ability of the enzyme to be an efficient catalyst.