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computer - BC367 Experiment 4B Computer Modeling of Acid...

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Unformatted text preview: BC367 Experiment 4B Computer Modeling of Acid Phosphatase Introduction In this exercise, you will examine the crystal structures of acid phosphatase alone and complexed with various inhibitors. The questions that you should address include the following: What interactions contribute to substrate binding? Which groups on the enzyme participate in catalysis? What effect does the inhibitor have on the structure (locally and globally)? What would be the effect of specific mutations on the structure and function of the enzyme? In the laboratory, we are studying acid phosphatase from wheat germ. The function of the wheat germ phosphatase has not been definitively established, but it has been proposed that it is involved in the mobilization of the phosphate reserves of the seed. However, there are currently no high-resolution crystal structures of the wheat germ enzyme available. Therefore, we will examine crystal structures of the human prostatic acid phosphatase (PAP) instead. PAP has been shown to possess tyrosine phosphatase activity in vitro , but its physiological substrate remains unknown. It has been demonstrated that human prostrate cancer cells show a decreased level of expression of PAP and the intracellular level of PAP is inversely correlated with cellular growth rate. As such, it has been suggested that PAP may be involved in progression of tumor growth and metastasis in prostatic cancer. The catalytic mechanism of the enzyme has been studied extensively. PAP acts like a histidine phosphatase and involves the formation of a phosphohistidine enzyme-substrate intermediate. Site- specific mutagenesis experiments have shown that Arg11 and His12 are essential for catalysis. Further, catalytic activity is also severely impaired when any of the other active site residues are mutated (Arg15, Arg79, His257, Asp258). Your pre-lab assignment is to look up the mechanism of PAP before arriving and record the details in your lab notebook. Use the reported mechanism to facilitate your understanding of the structure during the course of the exercise. Procedure You will use the Molecular Operating Environment (MOE) software package from the Chemical Computing Group. MOE is comprised of a variety of applications used to construct, model, analyze, and manipulate molecular structures. The steps outlined below are not meant to be all-inclusive, but they should provide a good start to guide you through an analysis of a protein structure. You should examine the following this afternoon: BC 367, Experiment 4, Fall 2009 2 The structure of human prostatic acid phosphatase ( 1CVI in the PDB) and one of the following inhibitor-bound structures: human PAP + phosphate ion ( 1ND6 ) human PAP + alpha-benzyl-aminobenzyl-phosphonic acid ( 1ND5 ) rat PAP + vanadate ion ( 1RPT ) For each structure, follow these general steps: a. download the coordinates from the PDB b. open and view the structure in MOE c. use the visualization tools of MOE to understand the structural basis of inhibition...
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This note was uploaded on 02/09/2010 for the course CH CH242 taught by Professor Katz during the Spring '10 term at Colby.

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computer - BC367 Experiment 4B Computer Modeling of Acid...

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