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The difficulties in formulating guidelines for the above aspects are mainly due to the fact that the problems themselves have a very large number of degrees of freedom.The objective of this proposal is to characterize the mechanism of the inhibition of guanylate cyclase by the hydrophobic cations La3+ and Gd3+. These ions are known to mimic GTP in inhibiting the enzyme; however, the mechanism of this inhibition is not known. The hypothesis to be tested is that these ions affect the catalytic activity of guanylate cyclase by interfering with the equilibrium between the two catalytic nucleotide complexes and their corresponding ground states. Experiments proposed will involve measurements of the Michaelis constants for GMP, the steady state kinetics, and isotope effects on kinetic constants. Enzymological measurements of the equilibrium between GMP and GMP.GMP. La3+ will be carried out and these experiments will provide data which will establish if this inhibition mechanism is due to the steric disruption of the reaction, or whether it is due to the formation of a ternary complex. If it is due to the former, the crystal structure of guanylate cyclase with GMP.GMP bound will be solved and the crystal structure of the ternary complex will be worked out to identify the solvent accessible surface of the enzyme and any steric interactions which may be affected by these complexes. To establish if the inclusion of hydrophobic cations causes similar effects in other guanylate cyclases, two approaches will be taken. One is to study the effect of hydrophobic ions on the biochemical properties of guanylate cyclases from several sources. The other is to study the crystal structure of guanylate cyclase with its substrate (GMP) bound to the enzyme. An attempt will be made to alter the equilibrium between the enzyme
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