Substrate Induced Conformational Changes in Argininosuccinate Synthetase

Abstract

Argininosuccinate synthetase (AS) is the rate-limiting enzyme of both the urea and arginine-citrulline cycles. In mammals, deficiency of AS leads to citrullinemia, a debilitating and often fatal autosomal recessive urea cycle disorder, whereas its overexpression for sustained nitric oxide production via the arginine-citrulline cycle leads to the potentially fatal hypotension associated with septic and cytokine-induced circulatory shock. The crystal structures of Escherichia coli argininosuccinate synthetase (EAS) in complex with ATP and with ATP and citrulline have been determined at 2.0-Å resolution. These are the first EAS structures to be solved in the presence of a nucleotide substrate and clearly identify the residues that interact with both ATP and citrulline. Two distinct conformations are revealed for ATP, both of which are believed to be catalytically relevant. In addition, comparisons of these EAS structures with those of the apoenzyme and EAS complexed with aspartate and citrulline (Lemke, C. T., and Howell, P. L. (2001) Structure (Lond.) 9, 1153-1164) provide structural evidence of ATP-induced conformational changes in the nucleotide binding domain. Combined, these structures also provide structural explanations of some of the observed kinetic properties of the enzyme and have enabled a detailed enzymatic mechanism of AS catalysis to be proposed.Argininosuccinate synthetase (AS 1 ; EC 6.3.4.5) catalyzes the reversible conversion of citrulline, aspartate, and ATP to argininosuccinate, AMP, and inorganic pyrophosphate (Fig. 1). There are three important metabolic processes that require this catalysis. First, in all organisms AS catalyzes the penultimate step in the biosynthesis of arginine, one of the 20 amino acid building blocks of life, and a precursor for the synthesis of several other biomolecules. Second, AS participates in the urea cycle, a five-enzyme cycle that employs four of the enzymes of arginine biosynthesis to detoxify ammonia through the production of urea. Ammonia detoxification is critical for the survival of higher organisms. In humans, failure to produce functional AS leads to the buildup of citrulline, ammonia, and orotic acid. If untreated, the neurotoxic ammonia can cause brain damage and coma, and in cases where urea cycle function is significantly compromised (less than 5% activity) the condition is typically fatal (1). Finally, AS and a second urea cycle enzyme, argininosuccinate lyase, together with the flavoprotein nitricoxide synthase form the arginine-citrulline cycle, an abbreviated urea cycle that provides de novo arginine biosynthesis for sustainable overproduction of nitric oxide (NO (2, 3)). NO is a small, membrane-permeable, highly reactive molecule that plays key roles in a wide range of mammalian processes including blood pressure control, neurotransmission, apoptosis, immune system function, and wound healing (for reviews, see . AS is the rate-limiting enzyme in both the urea and the arginine-citrulline cycles (11,12) and is therefore a key participant in all thes...

Structures of Argininosuccinate Synthetase in Enzyme-ATP Substrates and Enzyme-AMP Product Forms

“…3A and 4A). The ␣-amino group and ␣-carboxylate of citrulline and both carboxylates of aspartate are bound to the sites specific for citrulline and aspartate, respectively, as observed in eAsS⅐citrulline⅐aspartate, eAsS⅐ATP⅐citrulline (12), and tAsS⅐ AMP-PNP⅐arginine⅐succinate (13). The ureido group of citrulline, the amino group of aspartate, and the ␣-phosphate group of ATP, which are directly involved in the catalytic reaction of AsS, are close to one another.…”

“…deviations of 0.68 and 0.67 Å, respectively. The native eAsS presents a wider surface of the active site than native tAsS or tAsS complex and shows the induced fit for the ATP binding domain to move and bind ATP (12). The tAsS does not show its overall conformational change upon binding of ATP or ATP analogue and has a common structure, i.e.…”

“…The eAsS shows the ATP-induced conformational change in the ATP binding domain, which is approximated to be a 3°( eAsS⅐ATP) and a 5°(eAsS⅐ATP⅐citrulline) rotation of the ATP binding domain toward the synthetase domain compared with that of native eAsS (12). The overall subunit structure of native tAsS or tAsS⅐ATP⅐citrulline⅐aspartate overlaps well with that of eAsS⅐ATP⅐citrulline because the subunit ␣-carbon atoms of native tAsS and tAsS complex are fitted to those of the eAsS complex with average r.m.s.…”

“…The ␥-phosphate of U-shaped ATP approaches the consensus PP-loop and is coordinated to the loop with a ␤-phosphate free from direct hydrogen bonds, as was observed in x-ray structures of eAsS⅐ATP⅐citrulline (12), tAsS⅐ATP, and tAsS⅐AMP-PNP⅐arginine⅐succinate (13). The ␥-phosphate of S-shaped ATP leaves the PP-loop, loses interactions, approaches the citrulline and aspartate binding sites of the synthetase domain, and forms a hydrogen bond with the synthetase domain residue, Ser-173.…”

“…A corollary of the ATP binding model to the active site is that a conformational change in the enzyme is necessary for the catalytic reaction. To confirm this proposal, the structures of eAsS⅐ATP and eAsS⅐ATP⅐citrulline have been determined by x-ray methods (12). Comparisons of these two complexes with eAsS and eAsS⅐citrulline⅐aspartate revealed that ATP binding induces a rotation of the ATP binding domain toward the synthetase domain.…”