W0112

Structures of Argininosuccinate Synthetase in Enzyme- ATP-Substrates and Enzyme-AMP-Product Forms. Ikuko Miyahara^1,2, Masaru Goto^1,2, and Ken Hirotsu^1,2,¹Dept. of Chemistry, Graduate School of Science, Osaka City Univ., Osaka 558-8585, Japan, ²Harima Institute/SPring-8, The Institute of Physical and Chemical Research (RIKEN), Sayo-gun, Hyogo, 679-5148, Japan.

We now determined the reaction intermediate model structures of argininosuccinate synthetase from Thermus thermophilus HB8 (tAsS), elucidated enzymatic reaction mechanism. The enzyme reversibly catalyzes the ATP-dependent condensation of a citrulline with an aspartate to give argininosuccinate. The magnesium ion is required for maximal activity of AsS. The structures of tAsS complexed with an ATP and substrate (citrulline and asparatate), with AMP and product (argininosuccinate), and with AMP-PNP, substrate analogues (arginine and aspartate) and Mg²⁺. The tAsS is shown to have the same overall structure regardless of whether the enzyme is in the native or the complexed form and to have a structure quite similar to that of AsS from Escherichia coli in the complex with ATP and citrulline. The reaction sites of ATP and the bound substrates are adjacent and might converge sufficiently for the reaction to proceed without the conformational change at the domain level. The mobility of the triphosphate group in ATP and the side chain of citrulline plays an important role in the catalytic action. The protonated amino group of the bound aspartate interacts with α-phosphate of ATP and the ureido group of citrulline thus stimulating the adenylation of citrulline. The enzyme-product complex explains how citrullyl-AMP intermediate is bound to the active site. The detailed stereochemistry of the catalysis of the enzyme is clarified on the basis of the structures of tAsS complexes.