Inositol Signaling Project
Biochemistry Chemistry Research at UCF

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Purpose

Mammalian phosphatidylinositol-specific phospholipases C (PI-PLC) are key effectors of the action of growth factors, neurotransmitters, and hormones. These enzymes respond to stimulation of extracellular receptors by increasing hydrolysis of phosphatidylinositols and generation of intracellular second messengers, diacylglycerol and inositol phosphates. The flow of biochemical molecules that facilitate the cellular response is shown in Figure 1. Note the release of CA++ from the ER upon IP3 release. While bacterial PI-PLCs have no confirmed physiological function, their small size, good stability and analogous chemical mechanism to mammalian enzymes make them an excellent model to begin to understand the more complex mammalian enzymes.

The mammalian active site is known to contain a Ca++ ion to facilitate the hydrolysis reaction. The catalytic domain of this enzyme has been determined by x-ray crystallography and the active site interactions are shown in Figure 2. The reaction mechanism follows two steps as shown in Figure 3. The first step involves a transesterification reaction to form the cyclic intermediate and the second step is driven by hydrolysis of the cyclic inositol phosphate (cIP).

Figure 1. Biochemical information flow following hormone binding and release of inositol triphosphate (IP3) from the lipid membrane via hydrolysis by the PLC enzyme.

Figure 2. Interactions of phosphoinositol with the Ca++ ion in the mammalian active site as determined by the x-ray structure of the catalytic domain

Figure 3. Reaction of the membrane bound inositol compound (1) involving formation of the intermediate cyclic form (2) and hydrolysis to form the product (3). Note that the R group on compound (1) is a large hydrophobic functional group which keeps the compound bound to the membrane until compound 2 is formed. It is also known that compound 2 can be released from the enzyme before hydrolysis occurs to function as an additional signaling molecule.

Goals

We are currently collaborating with Professor Karol Bruzik at The University of Illinois at Chicago on this project to understand the complete catalytic mechanism for this class of enzymes beginning with the smaller bacterial enzymes as model and comparing the active sites of these enzymes to the larger mammalian systems as well as the other bacterial enzymes to understand their active site architecture and small molecule interactions for the design of inhibitors. The results of this study will provide a better understanding of the differences between proteins in this enzyme class and how each one utilizes its active site amino acids to catalyze the different steps in the release of inositol signaling molecules.

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Current Focus (1)

We have recently determined the X-ray crystal structure of the PI-PLC enzyme from Bacillus thurigiensis (Bt) that has been altered in manner that makes the enzyme metal dependent. The bacterial enzymes are known to be smaller molecular weight metal-independent enzymes, so this change has produced a "Mammalian" type active site on a "Bacterial" type enzyme frame. These changes have provided insight into the role of Ca++ on the interaction with active site amino acids, and also provided information on the possible role of these changes in the process of protein evolution/adaptation. Our x-ray structure is shown in Figure 4.

Figure 4. X-ray structure of the R69D enzyme showing the location of the two Ca++ ions and the catalytic general acid/general base histidine residues.

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Current Focus (2)

We have also performed computational docking studies on various members of the PI-PLC family to demonstrate the different types of interactions that contribute to binding the inositol compound(s) and carrying out the reaction. An example of these different types of interactions is shown in Figure 5.

Figure 5. Structural representation of the various interactions of inositol rings in the active sites of two different PLC enzymes.

Next Phase

The next phase of the project is to determine the structure of a bacterial enzyme that naturally used Ca++ as a cofactor during the reaction. We have collected data sets for this enzyme and we are currently working to complete the structure. This enzyme's structure will be determined in complex with various compounds from the Bruzik lab to understand the specific active site interactions for a Ca++ dependent bacterial enzyme.

For More Information

Thomas L. Selby
Department of Chemistry, CH 117
University of Central Florida
Orlando, FL 32816