Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site

Weaver, Todd; Lees, M.; Banaszak, Leonard J.

doi:10.1002/pro.5560060410

Cited by 47 publications

(60 citation statements)

References 17 publications

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“…The location of the active site cleft in the ASL superfamily was first suggested from the wild type t␦c1 structure (15) and later confirmed by structural studies of fumarase C (20,21,44) and H162N d␦c2 (19). In the present structure, argininosuccinate was bound in all four active sites of S283A d␦c2.…”

Section: Figsupporting

confidence: 76%

Mutational Analysis of Duck δ2 Crystallin and the Structure of an Inactive Mutant with Bound Substrate Provide Insight into the Enzymatic Mechanism of Argininosuccinate Lyase

Sampaleanu¹,

Yu²,

Howell³

2002

Journal of Biological Chemistry

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The major soluble avian eye lens protein, ␦ crystallin, is highly homologous to the housekeeping enzyme argininosuccinate lyase (ASL). ASL is part of the urea and arginine-citrulline cycles and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate. In duck lenses, there are two ␦ crystallin isoforms that are 94% identical in amino acid sequence. Only the ␦2 isoform has maintained ASL activity and has been used to investigate the enzymatic mechanism of ASL. The role of the active site residues Ser-29, Asp-33, Asp-89, Asn-116, Thr-161, His-162, Arg-238, Thr-281, Ser-283, Asn-291, Asp-293, Glu-296, Lys-325, Asp-330, and Lys-331 have been investigated by site-directed mutagenesis, and the structure of the inactive duck ␦2 crystallin (d␦c2) mutant S283A with bound argininosuccinate was determined at 1.96 Å resolution. The S283A mutation does not interfere with substrate binding, because the 280's loop (residues 270 -290) is in the open conformation and Ala-283 is more than 7 Å from the substrate. The substrate is bound in a different conformation to that observed previously indicating a large degree of conformational flexibility in the fumarate moiety when the 280's loop is in the open conformation. The structure of the S283A d␦c2 mutant and mutagenesis results reveal that a complex network of interactions of both protein residues and water molecules are involved in substrate binding and specificity. Small changes even to residues not involved directly in anchoring the argininosuccinate have a significant effect on catalysis. The results suggest that either His-162 or Thr-161 are responsible for proton abstraction and reinforce the putative role of Ser-283 as the catalytic acid, although we cannot eliminate the possibility that arginine is released in an uncharged form, with the solvent providing the required proton. A detailed enzymatic mechanism of ASL/d␦c2 is presented.␦ Crystallin is a taxon-specific crystallin that represents the major soluble protein in the eye lenses of birds and terrestrial reptiles. This crystallin was recruited from the housekeeping enzyme argininosuccinate lyase (ASL 1 ) (1, 2) through a process called gene sharing (3,4). ASL is a cytosolic enzyme that catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate. Gene recruitment of ASL was followed by gene duplication and resulted in two proteins, ␦1 and ␦2 crystallin. In ducks, there is 94% amino acid sequence identity between the ␦1 and ␦2 isoforms (d␦c1 and d␦c2), and 69 and 71% identity to human ASL, respectively (2, 5). During evolution, the ␦1 isoform has presumably become a more specialized lens protein and has lost ASL activity (6 -8), while the ␦2 isoform has retained enzymatic activity and is the duck orthologue of ASL in non-lens tissues (9, 10).ASL and ␦ crystallin are members of a superfamily of enzymes that are active as homotetramers and catalyze similar ␤-elimination reactions in which a C ␣ -N or C ␣ -O bond is cleaved with the subsequent release of fumarate as one of the pro...

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Section: Figsupporting

confidence: 76%

Mutational Analysis of Duck δ2 Crystallin and the Structure of an Inactive Mutant with Bound Substrate Provide Insight into the Enzymatic Mechanism of Argininosuccinate Lyase

Sampaleanu¹,

Yu²,

Howell³

2002

Journal of Biological Chemistry

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“…Residues in these conserved regions have been implicated in catalysis. [10][11][12][13][14] ADL catalyzes the cleavage of ADS through a general acid-base mechanism involving the β-elimination of fumarate (Figure 1(a)). The reaction is initiated by abstraction of the C β -proton from the substrate by the general base, resulting in the formation of a carbanion intermediate.…”

Section: Introductionmentioning

confidence: 99%

Substrate and Product Complexes of Escherichia coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism

Tsai

Koo

et al. 2007

Journal of Molecular Biology

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Adenylosuccinate lyase (ADL) catalyzes the breakdown of 5-aminoimida-zole-(Nsuccinylocarboxamide) ribotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribotide (AICAR) and fumarate, and of adenylosuccinate (ADS) to adenosine monophosphate (AMP) and fumarate in the de novo purine biosynthetic pathway. ADL belongs to the argininosuccinate lyase (ASL)/ fumarase C superfamily of enzymes. Members of this family share several common features including: a mainly α-helical, homotetrameric structure; three regions of highly conserved amino acid residues; and a general acid-base catalytic mechanism with the overall β-elimination of fumarate as a product. The crystal structures of wild-type Escherichia coli ADL (ec-ADL), and mutant-substrate (H171A-ADS) and -product (H171N-AMP•FUM) complexes have been determined to 2.0, 1.85, and 2.0 Å resolution, respectively. The H171A-ADS and H171N-AMP•FUM structures provide the first detailed picture of the ADL active site, and have enabled the precise identification of substrate binding and putative catalytic residues. Contrary to previous suggestions, the ec-ADL structures implicate S295 and H171 in base and acid catalysis, respectively. Furthermore, structural alignments of ec-ADL with other superfamily members suggest for the first time a large conformational movement of the flexible C3 loop (residues 287-303) in ec-ADL upon substrate binding and catalysis, resulting in its closure over the active site. This loop movement has been observed in other superfamily enzymes, and has been proposed to be essential for catalysis. The ADL catalytic mechanism is re-examined in light of the results presented here.

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“…In prokaryotes on the other hand, there are two distinct classes of fumarase molecules: class I fumarases are heat-labile and Fe 2ϩ -dependent, dimeric enzymes with subunits of 60 kDa each, having no obvious sequence homology to the eukaryotic enzymes, whereas class II fumarases are heat-stable, Fe 2ϩ -independent tetrameric enzymes with subunits of 50 kDa and showing extensive homology to the eukaryotic fumarases. Recently the three-dimensional structure of Escherichia coli class II fumarase has been unraveled (5)(6)(7). According to crystallographic data, each subunit is composed of three domains, of which the central one (D2) forms a 5-helix bundle.…”

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confidence: 99%

“…Residues Thr-100, Ser-139, Ser-140, and Asn-141 from the b-subunit, Thr-187, His-188 from the d-subunit, and Lys-324, Asn-326 from the c-subunit were supposed to form direct hydrogen bonds with substrates and inhibitors (5,6). Moreover a highly coordinated buried water molecule was found at the A site and supposed to be associated to residues Ser-98, Thr-100, Asn-141 from the b-subunit and His-188 from the d-subunit (6,7). An additional binding site (site B) is supposed to be nearby and is built up of residues belonging to one single subunit (the bsubunit), i.e.…”

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confidence: 99%

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Pig Heart Fumarase Contains Two Distinct Substrate-binding Sites Differing in Affinity

Beeckmans

Driessche

1998

Journal of Biological Chemistry

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A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substrates L-malate and fumarate, the active sites (sites A) also bind their analogs D-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogs D-malate and oxaloacetate, as well as L-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (K d being in the millimolar range) is 1-2 orders of magnitude lower than the affinity of sites A (of which K d is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time. Four molecules of pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (K d ‫؍‬ 0.6 to 2.2 M, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating the low affinity binding sites (B).Fumarase (fumarate hydratase, EC 4.2.1.2) catalyzes the reversible, stereospecific addition of water to fumarate to form L-malate (1). Being an enzyme of the citric acid cycle, it serves both catabolic and anabolic purposes and, as such, it is found within all living organisms. In eukaryotes, both mitochondrial fumarase (which is involved in the citric acid cycle) and the cytoplasmic enzyme are encoded by the same gene (2-4). These fumarase molecules are tetramers consisting of identical subunits of 50 kDa each, and their activity does not depend upon the presence of metal cations (1). In prokaryotes on the other hand, there are two distinct classes of fumarase molecules: class I fumarases are heat-labile and Fe 2ϩ -dependent, dimeric enzymes with subunits of 60 kDa each, having no obvious sequence homology to the eukaryotic enzymes, whereas class II fumarases are heat-stable, Fe 2ϩ -independent tetrameric enzymes with subunits of 50 kDa and showing extensive homology to the eukaryotic fumarases. Recently the three-dimensional structure of Escherichia coli class II fumarase has been unraveled (5-7). According to crystallograph...

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Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site

Cited by 47 publications

References 17 publications

Mutational Analysis of Duck δ2 Crystallin and the Structure of an Inactive Mutant with Bound Substrate Provide Insight into the Enzymatic Mechanism of Argininosuccinate Lyase

Mutational Analysis of Duck δ2 Crystallin and the Structure of an Inactive Mutant with Bound Substrate Provide Insight into the Enzymatic Mechanism of Argininosuccinate Lyase

Substrate and Product Complexes of Escherichia coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism

Pig Heart Fumarase Contains Two Distinct Substrate-binding Sites Differing in Affinity

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