The role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: Mechanistic and crystallographic evidence for stereospecific alkylation by (R)-.alpha.-phenylglycidate

Landro, James A.; Gerlt, J.A.; Kozarich, John W.; Koo, Carolyn W.; Shah, Vibhakar J.; Kenyon, George L.; Neidhart, David J.; Fujita, Shigeo; Petsko, Gregory A.

doi:10.1021/bi00169a003

Cited by 123 publications

(178 citation statements)

References 15 publications

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“…Thus, kinetic competence of the intermediate requires that it be stabilized. The x-ray structure of a complex with (S)-atrolactate ((S)-␣-methylmandelate) reveals that the carboxylate group of the bound inhibitor is both coordinated to an essential Mg 2ϩ and hydrogen-bonded to Glu-317 (as evidenced by the 2.7-Å O-O distance) (47).…”

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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Section: Mandelate Racemasementioning

confidence: 99%

The Low Barrier Hydrogen Bond in Enzymatic Catalysis

Cleland¹,

Frey²,

Gerlt³

1998

Journal of Biological Chemistry

Self Cite

540

503

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“…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).…”

mentioning

confidence: 99%

“…(10,11,26,27). Whereas the side chain of Asn could engage the substrate and intermediate through hydrogen bonding, partial proton transfer is unlikely because of the much higher pK a of an amide.…”

mentioning

confidence: 99%

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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“…Further structural and kinetic experiments on MRase have bolstered this view. [27][28][29] The mechanism of lactonization is the same in MCIases and CMCIases but, as shown by extensive studies of catalysis of various substituted CCMs, 4,5,8,30 the proteobacterial CMCIases such as ReCMCIase have evolved four new catalytic features. First, they have increased specificity for chloromuconates over CCM, and they bind them in a specific orientation: 2-Cl-CCM binds predominantly ''2-down'' (Fig.…”

Section: Introductionmentioning

confidence: 99%

Structural basis for the activity of two muconate cycloisomerase variants toward substituted muconates

Schell¹,

Helin²,

Kajander³

et al. 1999

Proteins

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We have refined to 2.3 Å resolution two muconate cycloisomerase (MCIase) variant structures, F329I and I54V, that differ from each other and from wild-type in their activity toward cis,cis-muconate (CCM) and substituted CCMs. The working and free R-factors for F329I are 17.4/21.6% and for I54V, 17.6/22.3% with good stereochemistry. Except for the mutated residue, there are no significant changes in structure. To understand the differences in enzymatic properties we docked substituted CCMs and CCM into the active sites of the variants and wild type. The extra space the mutations create appears to account for most of the enzymatic differences. The lack of other structural changes explains why, although structurally equivalent changes occur in chloromuconate cycloisomer-ase (CMCIase), the changes in themselves do not convert a MCIase into a dehalogenating CMCIase. Reanalysis of the CMCIase structure revealed only one general acid/base, K169. The structural implication is that, in 2-chloro-CCM conversion by CMCIase, the lactone ring of 5-chloromuconolactone rotates before dehalogenation to bring the acidic C4 proton next to K169. Therefore, K169 alone performs both required protonation and deprotonation steps, the first at C5 as in MCIase, and the second, after ring rotation, at C4. This distinguishes CMCIase from / barrel isomerases and racemases, which use two different bases. Proteins 1999;34:125-136. 1999 Wiley-Liss, Inc.

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The role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: Mechanistic and crystallographic evidence for stereospecific alkylation by (R)-.alpha.-phenylglycidate

Cited by 123 publications

References 15 publications

The Low Barrier Hydrogen Bond in Enzymatic Catalysis

The Low Barrier Hydrogen Bond in Enzymatic Catalysis

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

Structural basis for the activity of two muconate cycloisomerase variants toward substituted muconates

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