Kinetic mechanism and location of rate-determining steps for aspartase from Hafnia alvei

Nuiry, Ileana I.; Hermes, Jeffrey D.; Weiss, Paul M.; Chen, Cheau Yun; Cook, Paul F.

doi:10.1021/bi00317a013

Cited by 45 publications

(30 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Each reaction is considered as a “uni-bi” reaction, in which fumarate is released from the enzyme before AMP or AICAR (2). The rate limiting step of the reaction catalyzed by adenylosuccinate lyase and other members of the fumarase family is thought to be the cleavage of the C(γ)-N bond (3-5)…”

mentioning

confidence: 99%

Biochemical and Biophysical Analysis of Five Disease-Associated Human Adenylosuccinate Lyase Mutants

et al. 2009

View full text Add to dashboard Cite

Adenylosuccinate lyase (ASL), a catalyst of key reactions in purine biosynthesis, is normally a homotetramer in which three subunits contribute to each of four active sites. Human ASL deficiency is an inherited metabolic disease associated with autism and mental retardation. We have characterized five disease-associated ASL mutants: R194C and K246E are located at subunit interfaces, L311V is in the central helical region away from the active site, and R396C and R396H are at the entrance to the active site. The Vmax (at 25 °C) for R194C is comparable to that of WT; while those of L311V, R396C, R396H and K246E are considerably reduced and affinity for adenylosuccinate is retained. The mutant enzymes have decreased positive cooperativity as compared to WT. K246E exists mainly as dimer or monomer, accounting for its negligible activity; whereas the other mutant enzymes are similar to WT in the predominance of tetramer. At 37 °C, the specific activity of WT and these mutant enzymes slowly decreases 30-40% with time and reaches a limiting specific activity without changing significantly the amount of tetramer. Mutant R194C is unique in being rapidly inactivated at the harsher temperature of 60°C, indicating that it is the least stable enzyme in vitro. Conformational changes in the mutant enzymes are evident from protein fluorescence intensity at 25 °C and after incubation at 37 °C, which correlates with the loss of enzymatic activity. Thus, these disease-associated single mutations can yield enzyme with reduced activity either by affecting the active site or by perturbing the enzyme’s structure and/or native conformation which are required for catalytic function.

show abstract

mentioning

confidence: 99%

Biochemical and Biophysical Analysis of Five Disease-Associated Human Adenylosuccinate Lyase Mutants

et al. 2009

View full text Add to dashboard Cite

show abstract

“…These enzymes have been purified and characterized from a number of Gram‐positive and Gram‐negative bacteria, including Escherichia coli , Hafnia alvei , Pseudomonas fluorescens , Bacillus subtilis and Bacillus sp. YM55‐1 [1–11]. The best studied example is the aspartase (AspA) from E. coli , for which the crystal structure has been elucidated [12].…”

mentioning

confidence: 99%

“…Stereochemical, kinetic isotope and pH‐rate studies indicate that AspA catalyzes an anti ‐elimination reaction, where an active site base (with a p K a of approximately 6.5) abstracts the proton from C3 of 1 to form an enzyme‐stabilized enediolate intermediate ( 4 ; Scheme 2) [5,6,16–18]. The proposed enediolate intermediate (i.e.…”

mentioning

confidence: 99%

Site‐directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp. YM55‐1

Veetil¹,

Raj²,

Quax³

et al. 2009

The FEBS Journal

View full text Add to dashboard Cite

Aspartate ammonia lyases (also referred to as aspartases) are microbial enzymes that catalyze the reversible deamination of l-aspartate (1) to yield fumarate (2) and ammonia (3) (Scheme 1). These enzymes have been purified and characterized from a number of Gram-positive and Gram-negative Aspartate ammonia lyases (also referred to as aspartases) catalyze the reversible deamination of l-aspartate to yield fumarate and ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of l-aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of this intermediate eliminates ammonia and yields the product, fumarate. Although two crystal structures of aspartases have been determined, details of the catalytic mechanism have not yet been elucidated. In the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) were mutated in the structurally characterized aspartase (AspB) from Bacillus sp. YM55-1. On the basis of a model of the complex in which l-aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of l-aspartate were predicted to be Thr101, Asn142 and His188. This postulate is supported by the mutagenesis studies: mutations at these positions resulted in mutant enzymes with reduced activity and significant increases in the K m for l-aspartate. Studies of the pH dependence of the kinetic parameters of AspB revealed that a basic group with a pK a of approximately 7 and an acidic group with a pK a of approximately 10 are essential for catalysis. His188 does not play the typical role of active site base or acid because the H188A mutant retained significant activity and displayed an unchanged pHrate profile compared to that of wild-type AspB. Mutation of Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these residues are most likely involved in substrate binding and in stabilizing the enediolate intermediate. Mutagenesis studies corroborate the essential role of Lys324 because all mutations at this position resulted in mutant enzymes that were completely inactive. The substrate-binding model and kinetic analysis of mutant enzymes suggest that Thr187 and Asn326 assist Lys324 in binding the C1 carboxylate group of the substrate. A catalytic mechanism for AspB is presented that accounts for the observed properties of the mutant enzymes. Several features of the mechanism that are also found in related enzymes are discussed in detail and may help to define a common substrate binding mode for the lyases in the aspartase ⁄ fumarase superfamily.Abbreviations Ap, ampicillin; AspA, aspartase from E. coli; AspB, aspartase from Bacillus sp. YM55-1; FumC, fumarase C from E. coli; RCSB, Research Collaboratory for Structural Bioinformatics; PDB, Protein Data Bank.

show abstract

“…All well-characterized enzyme-catalyzed j3-eliminations are suggestive of proceeding through a carbanion intermediate (Anderson, 1991). The enzyme systems studied by both primary and secondary isotope effects, fumarase (Blanchard & Cleland, 1980), enolase (Anderson, 198 1;Stubbe & Abeles, 1980), aspartase (Nuiry et al, 1984), and arginine succinate lyase (Kim & Raushel, 1986) all catalyze anti eliminations that proceed through discrete carbanion intermediates. However, precedent for a concerted elimination may be taken from the concerted a-0 dehydrogenation of acyl-CoAs catalyzed by acyl-CoA dehydrogenase (Muffin, 1974;Reinsh et al, 1980;Pohl et al, 1986).…”

mentioning

confidence: 99%

Crotonase-catalyzed .beta.-elimination is concerted: a double isotope effect study

Bahnson¹,

Anderson²

1991

Biochemistry

View full text Add to dashboard Cite

Determining the sequence of bond cleavages, and consequently the nature of intermediates, in enzyme-catalyzed reactions is a major goal of mechanistic enzymology. When significant primary isotope effects on V/K are observed for two different bond cleavages, both bonds may be broken in the same transition state or they can reflect two different transition states that are of nearly identical energy and consequently both are partially rate limiting. For the crotonase-catalyzed dehydration of 3-hydroxybutyrylpantetheine, the primary D(V/K) and 18(V/K) are 1.60 and 1.053 [Bahnson, B. J., & Anderson, V. E. (1989) Biochemistry 28, 4173-4181], respectively. In this case, double isotope effects can discriminate between the two possibilities [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114; Belasco, J. G., Albery, W. J., & Knowles, J. R. (1983) J. Am. Chem. Soc. 105, 2475-2477]. The ratio of the alpha-secondary D(V/K) for the hydration of crotonylpantetheine catalyzed by crotonase in H2O and D2O has been determined to be 1.003 +/- 0.006. The invariance of the alpha-secondary effect where the chemical reaction is completely rate determining requires that both bond cleavages be concerted or that the substitution of 2H at the primary position not significantly alter the partitioning of a hypothetical carbanion. The observation of a solvent discrimination isotope effect determined from the relative incorporation of 2H from 50% D2O of 1.60 +/- 0.03, identical with the primary D(V/K), and the determination that the rate of exchange of the abstracted proton with solvent proceeds at less than 3% of the overall reaction rate also fail to provide evidence for a carbanion intermediate and are consistent with a concerted reaction. Identical primary D(V/K)s determined in H2O and D2O indicate that there is not a significant solvent isotope effect on C-O bond cleavage. The isotope ratios determined in these studies were performed by negative ion chemical ionization whole molecule mass spectrometry of the pentafluorobenzyl esters, a new method whose validity is established by comparison with previously determined kinetic and equilibrium isotope effects.

show abstract

Kinetic mechanism and location of rate-determining steps for aspartase from Hafnia alvei

Cited by 45 publications

References 24 publications

Biochemical and Biophysical Analysis of Five Disease-Associated Human Adenylosuccinate Lyase Mutants

Biochemical and Biophysical Analysis of Five Disease-Associated Human Adenylosuccinate Lyase Mutants

Site‐directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp. YM55‐1

Crotonase-catalyzed .beta.-elimination is concerted: a double isotope effect study

Contact Info

Product

Resources

About