Mechanism of the Reaction Catalyzed by Mandelate Racemase: Importance of Electrophilic Catalysis by Glutamic Acid 317

Mitra, Bharati; Kallarakal, Abraham T.; Kozarich, John W.; Gerlt, J.A.; Clifton, James R; Petsko, Gregory A.; Kenyon, George L.

doi:10.1021/bi00009a006

Cited by 78 publications

(127 citation statements)

References 26 publications

(58 reference statements)

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“…Lys166 and His297 have been identified as the general bases responsible for abstracting an R-proton from S-and Rmandelic acid, respectively (8). The enolate anion thus produced is stabilized by the metal ion and Glu317 (37).…”

Section: Resultsmentioning

confidence: 99%

Virtual Screening against Highly Charged Active Sites: Identifying Substrates of Alpha−Beta Barrel Enzymes

2005

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We have developed a virtual ligand screening method designed to help assign enzymatic function for alpha-beta barrel proteins. We dock a library of ∼19,000 known metabolites against the active site and attempt to identify the relevant substrate based on predicted relative binding free energies. These energies are computed using a physics-based energy function based on an all-atom force field (OPLS-AA) and a generalized Born implicit solvent model. We evaluate the ability of this method to identify the known substrates of several members of the enolase superfamily of enzymes, including both holo and apo structures (11 total). The active sites of these enzymes contain numerous charged groups (lysines, carboxylates, histidines, and one or more metal ions) and thus provide a challenge for most docking scoring functions, which treat electrostatics and solvation in a highly approximate manner. Using the physics-based scoring procedure, the known substrate is ranked within the top 6% of the database in all cases, and in 8 of 11 cases, it is ranked within the top 1%. Moreover, the top-ranked ligands are strongly enriched in compounds with high chemical similarity to the substrate (e.g., different substitution patterns on a similar scaffold). These results suggest that our method can be used, in conjunction with other information including genomic context and known metabolic pathways, to suggest possible substrates or classes of substrates for experimental testing. More broadly, the physics-based scoring method performs well on highly charged binding sites and is likely to be useful in inhibitor docking against polar binding sites as well. The method is fast (<1 min per ligand), due largely to an efficient minimization algorithm based on the truncated Newton method, and thus, it can be applied to thousands of ligands within a few hours on a small Linux cluster.Computational ligand screening ("virtual screening" or "docking") is widely used in structure-based drug design projects to rapidly and inexpensively identify lead compounds (1-3). Here, we consider a different application of docking methods, namely to assist in the identification of possible substrates of an enzyme, when the function of the enzyme is unknown. We expect this capability to become increasingly important as the "production phase" of structural genomics efforts gets under way. These projects are expected to generate thousands of protein structures, with the ultimate goal of providing structural representatives of the majority of protein families. However, knowing the structure of a protein does not always uniquely or unambiguously suggest its function. Although the term "function" can encompass a broad range of meanings, here we refer specifically to the reactions that an enzyme can catalyze in vivo.The alpha-beta barrel enzymes, a subset of which is considered in this paper, pose a particular challenge for functional annotation. The basic alpha-beta barrel scaffold of eight parallel strands forming a barrel, flanked by eight helices, is know...

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Section: Resultsmentioning

confidence: 99%

Virtual Screening against Highly Charged Active Sites: Identifying Substrates of Alpha−Beta Barrel Enzymes

2005

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“…Additional details are provided under "Experimental Procedures." enzyme (11). This observation of stereospecific exchange activity was indeed instrumental in assigning the role of His 297 .…”

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confidence: 86%

“…In Fig. 1, we invoke a general acid to facilitate ␣-proton abstraction and to stabilize the enediolate intermediate based merely on the prevalence of such groups among mechanistically characterized enzymes, which also abstract ␣-protons of carbon acids, inclusive of triosephosphate isomerase (4 -6), 3-ketosteroid isomerase (7)(8)(9), and mandelate racemase (10,11).…”

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confidence: 99%

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Identification of a Catalytic Aspartyl Residue ofd-Ribulose 5-Phosphate 3-Epimerase by Site-directed Mutagenesis

Chen

Larimer

Serpersu

et al. 1999

Journal of Biological Chemistry

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Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp 186 of D-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants. The wild-type k cat of 7.1 ؋ 10 3 s ؊1 is lowered to 3.3 ؋ 10 ؊4 s ؊1 in D186A, 0.13 s ؊1 in D186N, and 1.1 s ؊1 in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the ␤-carboxyl of Asp 186 facilitates catalysis by >10 7 -fold. Relative to the wildtype enzyme, the K m for D-ribulose 5-phosphate is essentially unaltered with D186N and D186E but increased 10-fold with D186A. Apart from their impairments in epimerase activity, the mutants are unable to catalyze exchange between solvent protons and the C3 proton of substrates. This deficiency and the differential alterations of kinetic parameters among the mutants are consistent with Asp 186 serving as an electrophile to facilitate ␣-proton abstraction. This study is the first to identify a catalytic group of the epimerase.Despite its participation in the ubiquitous oxidative pentose phosphate pathway and its concurrent role in the reductive pentose phosphate pathway (i.e. the Calvin cycle) of photosynthetic organisms, D-ribulose 5-phosphate 3-epimerase (EC 5.1.3.1), which catalyzes the interconversion of Ru5P 1 and Xu5P, has not been well-characterized with respect to structure-function relationships. This relative neglect is probably a consequence of low natural abundance of the epimerase and, in the case of the plant enzyme, pronounced instability. We have recently removed these barriers to detailed structural and mechanistic studies by designing a heterologous overexpression system for the gene that encodes spinach chloroplast Ru5P epimerase and by devising a facile purification scheme for the recombinant enzyme that is compatible with its lability (1).The epimerase-catalyzed reaction probably entails a twobase mechanism with an enediolate intermediate (Fig. 1). This supposition is based on the observations that Ru5P formed from [3-2 H]Xu5P was completely free of deuterium, even though deuterium was completely retained in the remaining Xu5P (2). Later findings (3) that isomerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate by Ru5P epimerase proceeded by intramolecular proton transfer, whereas the C2 proton of D-threose-4-phosphate formed concurrently by epimerization of D-erythrose-4-phosphate was derived entirely from water, lends additional credence to a two-base mechanism. In Fig. 1, we invoke a general acid to facilitate ␣-proton abstraction and to stabilize the enediolate intermediate based merely on the prevalence of such group...

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“…Thus, as the enol(ate) intermediate is formed, the pK of the hydrogen-bonded carboxylate oxygen will increase toward that of Glu-317. The k cat of the E317Q racemase is decreased by a factor of ϳ10 5 , suggesting that the increased strength of the hydrogen bond between the enolate anion and Glu-317 relative to that involving the bound substrate contributes at least 7 kcal/mol to the stabilization of the intermediate (48). Because the kinetic evidence suggests that a transiently stable intermediate is also formed in the reaction catalyzed by E317Q and a hydrogen bond is observed between Gln-317 and (S)-atrolactate, these observations support the proposal that significant increases in hydrogen bond strength are possible when the pKs of the active site donors and substrate/intermediate acceptors become more closely matched.…”

Section: Mandelate Racemasementioning

confidence: 99%

The Low Barrier Hydrogen Bond in Enzymatic Catalysis

Cleland¹,

Frey²,

Gerlt³

1998

Journal of Biological Chemistry

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540

503

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The proposal that low barrier (i.e. short, very strong) hydrogen bonds (LBHBs) 1 play a role in enzymatic catalysis was first put forth in 1993 and 1994 (1-4). The proposal was accepted by some but rejected by others (5-8). Initial rejection on theoretical grounds has been followed by increasing experimental support, and recent improvements in theory have been able to account for the experimental observations of LBHBs in enzymes (9 -15). In this minireview we will explain the original proposal, summarize the experimental data from the past few years, and argue that LBHBs do play important roles in enzymatic reactions. Properties of Hydrogen BondsThe strength of a hydrogen bond depends on its length and linearity, the nature of its microenvironment, and the degree to which the pK values of the conjugate acids of the heavy atoms sharing the proton are matched. In water, the hydrogen-bonded oxygens are separated by ϳ2.8 Å, and the ⌬H of formation is ϳ5 kcal mol Ϫ1 . The hydrogen bonds in water are, however, weak because of the poor pK match between the participating oxygen atoms. Because the pKs of H 3 O ϩ and H 2 O are Ϫ1.7 and 15.7, respectively, the proton in the structure H 2 O⅐⅐⅐H-OH is tightly associated with the OH Ϫ group as a water molecule. In the gas phase, where the dielectric constant is low, hydrogen bonds between heteroatoms with matched pKs can be very short and strong, and experimental as well as calculated values of ⌬H of formation can approach 25 or 30 kcal mol Ϫ1 (16, 17). Likewise, in crystals hydrogen bonds can be very strong. The O-O distance in the ion [H- O⅐⅐⅐H⅐⅐⅐O-H] -in a crystal of a chromium complex is only 2.29 Å (18,19). In organic solvents, strong hydrogen bonds can also form, although the ⌬H of formation probably never exceeds 20 kcal mol Ϫ1. Recent calculations suggest that once the dielectric constant is at least 6 the strength of a strong hydrogen bond levels off at a level about half that in the gas phase (13,17). Because the active site of an enzyme is no longer aqueous once it has closed around a substrate, the properties of hydrogen bonds in organic solvents are highly pertinent to enzymatic catalysis.What happens energetically as hydrogen bonds become shortened can be seen in Fig. 1. Structure A represents the situation in water, where the hydrogen is firmly attached to either the lefthand or right-hand oxygen and is more loosely bonded to the other one, with an O-O distance of Ն2.8 Å. There is an energy barrier between the two possible positions of the hydrogen, with the zero point energy levels shown in Fig. 1. Such a hydrogen bond is essentially electrostatic, and the covalent O-H bond is the usual 0.9 -1.0 Å in length.As the overall O-O distance is shortened, the energy barrier drops until it reaches the zero point energy level at an O-O distance of ϳ2.5 Å (Fig. 1B); this is a LBHB. The ⌬H of formation has increased to 15-20 kcal/mol, and the hydrogen can now move freely between the two oxygens. In crystals containing LBHBs, neutron diffraction shows the hydrogen dif...

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Mechanism of the Reaction Catalyzed by Mandelate Racemase: Importance of Electrophilic Catalysis by Glutamic Acid 317

Cited by 78 publications

References 26 publications

Virtual Screening against Highly Charged Active Sites: Identifying Substrates of Alpha−Beta Barrel Enzymes

Virtual Screening against Highly Charged Active Sites: Identifying Substrates of Alpha−Beta Barrel Enzymes

Identification of a Catalytic Aspartyl Residue ofd-Ribulose 5-Phosphate 3-Epimerase by Site-directed Mutagenesis

The Low Barrier Hydrogen Bond in Enzymatic Catalysis

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