Role of the Hinge Loop Linking the N- and C-Terminal Domains of the Amidotransferase Subunit of Carbamoyl Phosphate Synthetase Xinyi Huang and Frank M. Raushel 1 Department of Chemistry, Texas A&M University, College Station, Texas 77843 Received March 16, 2000, and in revised form May 2, 2000 Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme consists of a large synthetase sub- unit and a small amidotransferase subunit. The small subunit is structurally bilobal. The N-terminal domain is unique compared to the sequences of other known proteins. The C-terminal domain, which contains the direct catalytic residues for the amidotransferase ac- tivity of CPS, is homologous to other members of the Triad glutamine amidotransferases. The two domains are linked by a hinge-like loop, which contains a type II  turn. The role of this loop in the hydrolysis of glutamine and the formation of carbamoyl phosphate was probed by site-directed mutagenesis. Based upon the observed kinetic properties of the mutants, the modifications to the small subunit can be separated into two groups. The first group consists of G152I, G155I, and 155. Attempts to disrupt the turn confor- mation were made by the deletion of Gly-155 or sub- stitution of the two glycine residues with isoleucine. However, these mutations only have minor effects on the kinetic properties of the enzyme. The second group includes L153W, L153G/N154G, and a ternary complex consisting of the intact large subunit plus the separate N- and C-terminal domains of the small sub- unit. Although the ability to synthesize carbamoyl phosphate is retained in these enzymes, the hydrolysis of glutamine is partially uncoupled from the syn- thetase reaction. It is concluded that the hinge loop, but not the type-II turn structure of the loop per se, is important for maintaining the proper interface inter- actions between the two subunits and the catalytic coupling of the partial reactions occurring within the separate subunits of CPS. © 2000 Academic Press Key Words: carbamoyl phosphate synthetase; Triad glutamine amidotransferase; site-directed mutagene- sis; type-II  turn. Carbamoyl phosphate synthetase (CPS) 2 is a mem- ber of the Triad class of glutamine amidotransferases, which also includes anthranilate synthase, GMP syn- thetase, CTP synthetase, PABA synthetase, and ami- nodeoxychorismate synthase, among others (1). The glutamine-binding site of this family of amidotrans- ferases contains a strictly conserved Cys–His–Glu triad (1). This class of enzymes initiates the hydrolysis of glutamine at one active site and then transfers the ammonia product to another active site within the same protein (1). The CPS from Escherichia coli is a heterodimer. The small subunit (42 kDa) hydrolyzes glutamine though the intermediacy of a thioester with the catalytic Cys-269 (2, 3). The large subunit (118 kDa) assembles carbamoyl phosphate from ammonia, bicarbonate, and two molecules of ATP (4 – 6). Based upon isotopic labeling studies and the discovery of three partial reactions, Anderson and Meister pro- posed that carbamoyl phosphate is constructed within the active site(s) of CPS via four distinct chemical steps as illustrated in Scheme 1 (7). The three-dimensional structure of the CPS from E. coli has confirmed that the large subunit contains separate sites for the phos- phorylation of bicarbonate and carbamate while the small subunit contains the active site for the hydrolysis 1 To whom correspondence should be addressed. Fax: (979) 845- 9452. E-mail: raushel@tamu.edu. 2 Abbreviations used: CPS, carbamoyl phosphate synthetase; CAD, the trifunctional mammalian enzyme consisting of carbamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroo- rotase; PABA, para-aminobenzoate; PCR, polymerase chain reac- tion; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel elec- trophoresis. 174 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 380, No. 1, August 1, pp. 174 –180, 2000 doi:10.1006/abbi.2000.1913, available online at http://www.idealibrary.com on