MCB 9406644 Speckhard This project will provide detailed information about kinase active sites, specifically about the contacts between the enzyme surface and the coordinated waters in inert bidentate chromium adenosine triphosphate (ATP) and phosphoenolpyruvate (PEP) complexes which are analogs of the cellular magnesium complexes. In the absence of detailed crystallographic information, the best way to determine if the metal ion coordination sphere is involved in the recognition of the substrate by the enzyme is to construct a series of chromium complexes with a single ammonia in all possible locations. These complexes are then used as competitive inhibitors of the magnesium complexes. Comparison of the inhibitions allows a map of the active site in the region around the metal to be constructed. This micromapping technique, recently demonstrated for hexokinase, will be applied to glycerokinase, adenylate kinase, pyruvate kinase and others. The potential for even more detailed information about the active site will result from the combination of micromapping and the study of x-ray crystal structures like that of adenylate kinase. Analysis of enzymes modified by site directed mutagenesis will complement the micromaps. A new micromapping tool, inert monoamminechromium complexes of phosphoenol pyruvate, will be developed and used to map the PEP site in pyruvate kinase. Inert chromium PEP itself will be useful to scientists seeking to use EPR and NMR relaxation techniques to study enzymes that use PEP. An important feature of this proposal is that it will primarily involve work by undergraduate students at an undergraduate institution. %%% Many enzymes are remarkably specific catalysts. Understanding the ability of enzymes to selectively interact with substrates is one of the top priorities in biochemistry. This understanding can lead to advances in agriculture and medicine, by allowing construction of potent inhibitors with high selectivity. This is the basis of r ational pesticide and herbicide design and rational drug design. This project will help to identify and carefully 'map' specific areas in a family of enzymes called kinases, which transfer energy within the cell. There are certain sites on the kinases where special chemical compounds can be attached. If these chemicals, in the form of stable molecules, can be attached firmly, they will interfere with the way the cell normally functions. These molecules effectively scramble the cell's internal communications and compromise their ability to function. This can create a new substance or it can change the way a cell behaves. As an example, some cells that may have been destructive can now be rendered harmless. Undergraduate students will learn about the chemicals used to identify these sites and conduct 'mapping' experiments. ***
