N-Acetil-L-Glutamato quinasa de escherichia coli: Estructura tridimensional e implicaciones funcionales


Abstract (Summary)
ABSTRACT: N-acetyl-L-glutamate kinase (NAGK) catalyzes the second step of microbial arginine biosynthesis. The gene for Escherichia coli NAGK was cloned and expressed in E. coli, allowing enzyme purification and crystallization with and without substrates. The E. coli NAGK crystal structure to 1.5 Å resolution reveals a 258-residue subunit homodimer nucleated by a central 16-stranded molecular open _-sheet sandwiched between _-helices. In each subunit MgAMPPNP binds along the sheet C-edge, and N-acetyl-L-glutamate (NAG) binds near the dyadic axis with its _-carboxilate aligned at short distance from the _-phosphoryl. The structural resemblance with carbamate kinase and sequence alignment suggest that NAGK is the prototype for the amino acid kinase family. Moreover, a large volume of unexplained electron density in this crystal is interpreted as an external, very extended, metal-free AMPPNP molecule that occupies two alternative positions and that makes contacts with the protein exclusively through its _-imidophosphate. We determine here also the crystal structures of NAGK complexes with MgADP and NAG alone or with the transition-state analog AlF4- and with ADP and sulphate. Comparison of these structures allows to delineate three successive steps during phosphoryl transfer. The transfer occurs in line and is strongly associative, with Lys8 and Lys217 stabilizing the transition state and the leaving group, respectively. Three water molecules play, together with Asp162 and the Mg, crucial structural roles. Two glycine-rich loops are also very important, moving in concert with the ligands. The active site is too narrow to accommodate the substrates without compressing the reacting groups, and this compressive strain appears a crucial component of the catalytic mechanism of NAGK. Initial binding of the two substrates would require a different enzyme conformation with a wider active site, and the energy of substrate binding would be used to change the active center conformation. In many species, NAGK is the pathway-controlling enzyme and is subject to feedback inhibition by arginine. The arginine-inhibitable NAGK from Pseudomonas aeruginosa has been cloned, overexpressed, purified and crystallized in presence of MgADP and NAG. Prismatic crystals diffract to 2.75 Å resolution with space group P1. Self-rotation function suggest the presence of 3-7 dimers in the unit cell.
This document abstract is also available in Spanish.
Bibliographical Information:

Advisor:Rubio Zamora Vicente

School:Universitat de València

School Location:Spain

Source Type:Master's Thesis



Date of Publication:07/04/2003

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